搜档网
当前位置:搜档网 › Caffeine induces apoptosis by enhancement of

Caffeine induces apoptosis by enhancement of

Caffeine induces apoptosis by enhancement of
Caffeine induces apoptosis by enhancement of

?2011 L a n d e s B i o s c i e n c e .

D o n o t d i s t r i b u t e .

Autophagy 7:2, 176-187; February 2011; ? 2011 Landes Bioscience

BAsic ReseARch PAPeR

*Correspondence to: Nobutaka Hattori; Email: nhattori@juntendo.ac.jp Submitted: 06/22/10; Revised: 10/27/10; Accepted: 11/02/10

Previously published online: https://www.sodocs.net/doc/449398986.html,/journals/autophagy/article/14074DOI: 10.4161/auto.7.2.14074Introduction

Caffeine has a diverse range of pharmacological effects.1 In addi-tion to its various effects on the cell cycle and growth arrest,

higher (4–10 mM) concentrations of caffeine can induce apop-tosis in several cell lines, such as 10 mM caffeine in human neuroblastoma cells,2 4 mM caffeine in human pancreatic adenocarcinoma cells 3 and 5 mM caffeine in human A549 lung adenocarcinoma cells.4 Although caffeine has been reported to modulate cell cycle checkpoints and perturb molecular targets of the cell cycle, the exact mechanism of caffeine-induced apoptosis remains unclear.1

Autophagy is a key mechanism in various physiopathological processes, including tumorigenesis, development, cell death and survival.5,6 I t has also been shown to have a complex relation-ship with apoptosis, especially in tumor cell lines.7 Several reports

caffeine is one of the most frequently ingested neuroactive compounds. All known mechanisms of apoptosis induced by caffeine act through cell cycle modulation or p53 induction. it is currently unknown whether caffeine-induced apoptosis is associated with other cell death mechanisms, such as autophagy. herein we show that caffeine increases both the levels of microtubule-associated protein 1 light chain 3-ii and the number of autophagosomes, through the use of western blotting, electron microscopy and immunocytochemistry techniques. Phosphorylated p70 ribosomal protein s6 kinase (Thr389), s6 ribosomal protein (ser235/236), 4e-BP1 (Thr37/46) and Akt (ser473) were significantly decreased by caffeine. in contrast, eRK1/2 (Thr202/204) was increased by caffeine, suggesting an inhibition of the Akt/mTOR/p70s6K pathway and activation of the eRK1/2 pathway. Although insulin treatment phosphorylated Akt (ser473) and led to autophagy suppression, the effect of insulin treatment was completely abolished by caffeine addition. caffeine-induced autophagy was not completely blocked by inhibition of eRK1/2 by U0126. caffeine induced reduction of mitochondrial membrane potentials and apoptosis in a dose-dependent manner, which was further attenuated by the inhibition of autophagy with 3-methyladenine or Atg7 siRNA knockdown. Furthermore, there was a reduced number of early apoptotic cells (annexin V positive, propidium iodide negative) among autophagy-deficient mouse embryonic fibroblasts treated with caffeine than in their wild-type counterparts. These results support previous studies on the use of caffeine in the treatment of human tumors and indicate a potential new target in the regulation of apoptosis.

Caffeine induces apoptosis by enhancement of autophagy via PI3K/Akt/mTOR/p70S6K inhibition

shinji saiki,1 Yukiko sasazawa,2 Yoko imamichi,1 sumihiro Kawajiri,1 Takahiro Fujimaki,2 isei Tanida,3 hiroki Kobayashi,2

Fumiaki sato,4 shigeto sato,1 Kei-ichi ishikawa,1 Masaya imoto 2 and Nobutaka hattori 1,*

1

Department of Neurology; Juntendo University school of Medicine; Bunkyo, Tokyo; 2Department of Biosciences and informatics; Faculty of science and Technology;

Keio University; Kohoku, Yokohama; 3Department of Biochemistry and cell Biology; National institute of infectious Diseases; shinjyuku, Tokyo;

4

Research institute for Disease of Old Age; Juntendo University school of Medicine; Tokyo, Japan

Key words: apoptosis, autophagy, PI3K/Akt/mTOR/p70S6K, ERK1/2, caffeine

Abbreviations: PI3K, phosphoinositide-3 kinase; 4E-BP1, eukaryotic initiation factor 4-binding protein 1; ERK, extracellular signal-regulated kinase; mTOR, mammalian target of rapamycin; 3-MA, 3-methyladenine; MEFs, mouse embryonic fibroblasts;

p70S6K, 70-kDa ribosomal protein S6 kinase; PI, propidium iodide; MPP +, 1-methyl-4-phenylpyridinium

have shown that autophagy not only enhances caspase-dependent cell death, but is also required for it.8 I n contrast, it has also been shown that autophagy plays an important role in promot-ing cell survival against apoptosis.7 Caffeine has been reported to inhibit some kinase activities, including various forms of phosphoinositol-3 kinase and mammalian target of rapamycin (mTOR).9,10 Recently, in food spoilage studies involving yeast, caffeine has been shown to induce a starvation response,11 which is a key regulator of autophagy causing its induction. However, the exact mechanism by which caffeine induces autophagy is still unknown.

Here we report that higher concentrations of caffeine enhance autophagic flux in a dose-dependent manner in various cell lines. Furthermore, we show that caffeine-induced autophagy is mainly dependent on PI3K/Akt/mTOR/p70S6 signaling and eventually results in apoptosis.

?2011 L a n d e s B i o s c i e n c e .

D o n o t d i s t r i b u t e .

BAsic ReseARch PAPeR

BAsic ReseARch PAPeR

increase of the ratio (Fig. 2F ). These results strongly indicate that

high concentration of caffeine treatment enhances autophagic flux.

The class I phosphatidylinositol 3-phosphate kinase (PI3K)/Akt/mTOR/p70ribosomal protein S6 kinase (p70S6K) signaling pathway and the Ras/Raf-1/mitogen-activated protein kinase 1/2 (MEK1/2)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway are two well-known pathways involved in the regulation of autophagy. Both are associated with tumorigenesis and often activated in numerous types of tumors.17 Therefore, we examined the effect of caffeine on both of these pathways, using western blotting, according to the protocol by I noki and colleagues.18 After a 24 hour treatment with caffeine, there was a significant decrease in the levels of phosphorylated p70 S6 kinase, S6 ribo-somal protein and 4E-BP1, compared with total normal levels in SH-SY5Y (Fig. 3A ), HeLa and PC12D cells (data not shown). Consistent with these results, nonphosphorylated 4E-BP1 pro-teins were increased by caffeine treatment (Fig. 3A ). To further investigate the upstream inhibition of mTOR by caffeine, we examined Ser473 phosphorylation of Akt, which measures both Akt/mTOR and mTORC2 activity. As shown in Figure 3B , treatment with caffeine also decreased the level of phosphory-lated Akt in SH-SY5Y cells, which was consistent with a previ-ous report.19 Similar findings were obtained in HeLa (Suppl. Fig. S2A ) and PC12D cells (data not shown). Subsequently, we exam-ined whether caffeine increases the phosphorylation of ERK1/2, a key regulator of autophagy downstream of Akt. As shown in Figure 3C , treatment with caffeine increased phosphorylated ERK1/2. The effects of caffeine on mTOR inhibition were ini-tially detected 3 hours after the addition of caffeine and reached a maximal level after 6 hours in SH-SY5Y (Fig. 3D ) and 9 hours in HeLa cells (Suppl. Fig. S2B and C ).

Caffeine has been shown to inhibit PI 3K and components of the PI 3K/Akt pathway.9,20 Next, we performed experiments to confirm whether caffeine-induced autophagy is activated through the PI3K/Akt pathway. Insulin or insulin-like growth factor upregulates PI 3K and its downstream targets including Akt and mTOR, resulting in the inactivation of autophagy.21-23 As shown in Figure 4A and B , insulin treatment for 30 minutes significantly phosphorylated Akt at Ser473, whereas the phos-phorylation was completely abolished by additional treatment with caffeine. No significant differences of the LC3-II/actin ratio between caffeine treatment and caffeine treatment with insulin were observed. Also, caffeine and Akt1/2 inhibitors did not have additive effects on the levels of LC3-II/actin ratio compared to the single treatment of caffeine or Akt inhibitors (Fig. 4C and D ). To further confirm the caffeine effects on this pathway, cells were transiently transfected with myristoylated Akt (myr-Akt), a constitutively active form of Akt.24 Caffeine treatment of both cells transfected with control vector and myr-Akt markedly decreased the levels of the phosphorylated Akt (Fig. 3E ), indi-cating that caffeine directly inhibits the Akt phosphorylation. If caffeine facilitates autophagy through PI 3K/Akt and ERK1/2 signalings, the autophagy should be partially blocked by ERK1/2 inhibition using the mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor, U0126. U0126 significantly but mildly

Results and Discussion

Caffeine (Fig. 1A ) is a widely used psychoactive drug that has been used for centuries to increase alertness and energy. It has been reported that caffeine induces autophagy in Zygosaccharomyces bailii in association with a starvation response, caused by a unknown mechanism.11 However, it remains unknown whether caffeine affects autophagy in mammalian cells. To determine if caffeine regulates autophagy at a steady state, we first exam-ined levels of the microtubule-associated protein 1 light chain 3 (LC3)-I I , which is an LC3-phosphatidyl-ethanolamine con-jugate and a promising autophagosomal marker.12 LC3-I I lev-els (compared to actin loading controls) increased with 5–25 mM caffeine treatment over 48 hours in SH-SY5Y (Fig. 1B and C ), PC12D and HeLa cells (Suppl. Fig. S1A and B ). The LC3-I I /actin ratio also increased in a time-dependent manner in SH-SY5Y (Fig. 1D and E ) and HeLa cells (data not shown). Using an electron microscopy technique, the numbers of auto-phagic vacuoles (AVs) were markedly increased in SH-SY5Y cells treated with 10 or 25 mM caffeine, but not in the control (Fig. 1F and G ). Morphometric analysis revealed that the number of AVs per 100 μm 2 of SH-SY5Y cytoplasm in control (Mean ± standard deviation: 1.3 ± 0.50), whereas that in caffeine-treated cells (10 mM: 8.0 ± 0.82; 25 mM: 15 ± 1.9) for 24 hours. Expression levels of p62, a well-known autophagic substrate, were also decreased by caffeine treatment in SH-SY5Y (Fig. 1H and I ) and HeLa cells (Suppl. Fig. S1C and D ). Furthermore, 10 mM caffeine treatment markedly increased the number of EGFP-LC3-positive vesicles in SH-SY5Y cells transiently transfected with EGFP-LC3 (data not shown) and HeLa cells stably expressing EGFP-LC3 (Figs. 1J and K ).12,13 This effect was confirmed by the observa-tion that caffeine administration also increased the number of vesicles positive to endogenous LC3 (Suppl. Fig. S1E ).

Endogenous LC3 is post-transcriptionally processed into LC3-I, which is found in the cytosol. LC3-I is in turn lipidated to LC3-I I , which then associates with autophagosome mem-branes.14 LC3-I can accumulate due to increased upstream autophagosome formation or impaired downstream autophago-some-lysosome fusion. To distinguish between these two possi-bilities, we assayed LC3-II in the presence of E64D plus pepstatin A or bafilomycin A1, which inhibits lysosomal proteases or blocks downstream autophagosome-lysosome fusion and lysosomal pro-teases, respectively.15,16 Caffeine significantly increased LC3-I I levels in the presence of E64d plus pepstatin A or bafilomycin compared to E64d plus pepstatin A or bafilomycin alone in (Fig. 2A and B ; Suppl. Fig. S1F and G ) and HeLa cells (Fig. 2C and D; Suppl. Fig. S1H and I ). A saturating dosage of bafilomycin A1 was used in this assay and no further increases in LC3-II lev-els were observed when cells were treated with higher concentra-tions. Similar results were observed in PC12D cell lines (data not shown). To confirm the caffeine effect on autophagic flux, we assessed the numbers of autolysosomes and autophagosomes in HeLa cells. The ratio of the numbers of autolysosomes (positive to both LC3 and LAMP2) to autophagosomes (positive to LC3) was increased by 10 mM caffeine treatment for 48 hours (Fig. 2E ). Quantification data using I mageJ also showed significant

?2011 L a n d e s B i o s c i e n c e .

D o n o t d i s t r i b u t e .

hairpin RNAs (shRNAs) to specifically and stably knock down all three Akt isoforms sufficiently increases autophagic flux.25 Therefore, we concluded that the caffeine-induced autophagy is mainly dependent on the PI3K/Akt/mTOR pathway.

reversed the levels of LC3-II /actin ratio (Fig. 4F and G ). The

failure of U1026 to reverse completely the caffeine effect can be explained by the autophagy induction through Akt/mTOR sig-naling. In addition, only Akt knockdown with inducible short

?2011 L a n d e s B i o s c i e n c e .

D o n o t d i s t r i b u t e .

Therefore, we decided to investigate whether caffeine-induced

autophagy rescues or induces cell death. Using PC12D cells treated with 1-methyl-4-phenylpyridinium (MPP +), a well-estab-lished Parkinson disease model,34 we determined that 1 mM caf-feine treatment was not sufficient for the induction of autophagy (Suppl. Fig. S4 and B ) and promoted increased cell viability, whereas >2.5 mM caffeine decreased cell viability (Fig. 6A ). In addition, a significant decrease in cell viability was noted in cells treated with >2.5 mM caffeine without MPP +. Also, mitochon-drial membrane potentials assessed by JC-1 were significantly preserved by 1 mM caffeine treatment compared to the control with MPP +, while those were lost by >5 mM caffeine treatment (Fig. 6B and Suppl. Fig. S5A ). These data suggest that caffeine-induced autophagy is not protective in these cell lines and leads to cell death.

Autophagy and apoptosis may act independently in parallel pathways or may influence one another.7 To confirm the relation-ship between these pathways in cells treated with caffeine, we examined caffeine effects on the cell cycle with a propidium iodide (PI) staining assay. Treatment with 2.5–10 mM caffeine increased the percentage of cells in the sub-G 1 peak, which is indicative of

Because caffeine induces autophagy dependently of mTOR inhibition, we hypothesized that combination treatment of caf-feine with rapamycin would not have additive effects on auto-phagy. However, caffeine and rapamycin showed an additive effect on the enhancement of LC3-I I /actin ratio compared to the single treatment of caffeine or rapamycin (Fig. 5A and B ). Several lines of evidences support the hypothesis that resistance to rapamycin results from a positive feedback loop from mTOR/S6K1 to Akt, resulting in enhancement of Akt phosphorylation at Ser 473.26-28 Recently, mutual suppression of the PI 3K/Akt/mTOR pathway by combination of rapamycin with perifosine, an Akt inhibitor, induces synergistic effects on autophagy-induced apoptosis as well as enhancement of autophagy, suggesting that dual inhibition of the PI3K/Akt/mTOR by rapamycin with caf-feine would be also a rational treatment for cancer.29

Several anti-cancer agents are known to inhibit the PI3K/Akt/mTOR/p70S6K pathway and simultaneously activate ERK1/2, resulting in induction of autophagy in tumor cell lines.30,31 The upregulation of this process has beneficial effects in neu-rodegenerative diseases, such as Parkinson and Huntington dis-eases, whereas an excess of autophagy can lead to cell death.32,33

Figure 1H–K. caffeine increases autophagic flux in various cell lines. (h and i) sh-sY5Y cells treated with various concentrations of caffeine for 24 or 48 hours were analyzed by immunoblotting with antibodies against p62 and actin. Densitometry analysis of p62 levels relative to actin (i) was performed using three independent experiments. (J and K) heLa cells stably expressing eGFP-Lc3 were treated with various concentrations of caffeine for 24 hours and analyzed using confocal microscopy. The percentage of eGFP-positive heLa cells with >5 eGFP-Lc3 vesicles was assessed (K) described previously in reference 43. error bars, s.D.; *p < 0.05; **p < 0.01.

Figure 1A–G (See opposite page). caffeine increases autophagic flux in various cell lines. (A) structural formula of caffeine. (B and c) sh-sY5Y cells treated with various concentrations of caffeine for 24 or 48 hours were analyzed by immunoblotting (B) with antibodies against Lc3 and actin. Densi-tometry analysis of Lc3-ii levels relative to actin (c) was performed using three independent experiments. (D and e) sh-sY5Y cells treated with 25 mM caffeine for 3–24 hours were analyzed by immunoblotting (D) with antibodies against Lc3 and actin. Densitometry analysis of Lc3-ii levels relative to actin (e) was performed using three independent experiments. (F) electron microscopic examination of sh-sY5Y cells treated with various concentra-tions of caffeine for 24 or 48 hours. Autophagic vacuoles accumulating in the cytoplasm are shown by arrows. (G) Morphometric analysis of auto-phagic vacuoles was performed with 30 different areas of the cytoplasm of control and caffeine-treated cells.

?2011L a n d e s B i o s c i e n c e.

D o n o t d i s t r i b u t e.

Figure 2. caffeine does not block autophagosome-lysosome fusion. (A–D) sh-sY5Y (A) or heLa (c) cells treated with 10 mM caffeine with or without

e64d (10 μg/ml) and pepstatin A (10 μg/ml) were analyzed by immunoblotting with antibodies against Lc3 and actin. Densitometry analysis of Lc3

levels relative to actin in sh-sY5Y (B) and heLa (D) cells was performed using three independent experiments. (e and F) heLa cells treated with various concentrations of caffeine for 48 hours were analyzed using confocal microscopy (e). Number of the autolysosomes and autophagosomes were auto-matically counted using imageJ “colocalization” Plugin and the ratios were calculated (F).

?2011 L a n d e s B i o s c i e n c e .

D o n o t d i s t r i b u t e .

manner (Suppl. Fig. S4C and D ). As expected, the level of

caffeine-induced cell death (positive to trypan blue staining) in Atg7-/- MEFs was less than that in Atg7+/+ MEFs (Fig. 7A ). The numbers of early apoptotic cells (annexin V positive, PI negative) were significantly increased in both a time-dependent and dose-dependent manner by caffeine treatment of Atg7+/+ MEFs com-pared to Atg7-/- MEFs (Fig. 7B–D ). Also, apoptotic or necrotic cells (annexin V positive) were significantly increased by caffeine treatment of Atg7+/+ MEFs compared to Atg7-/- MEFs (Suppl. Fig. S6). Together, these results indicate that caffeine-induced auto-phagy partly occurs upstream of apoptosis and is not a protective response to caffeine.

In various tumor cell lines, higher concentrations of caffeine alone induce p53-dependent G 1 phase arrest and under certain conditions apoptosis can also occur in a p53-independent man-ner.1 Furthermore, disruption at the G 2/M checkpoint by caffeine allows cells time to repair DNA damage by driving them through mitosis, eventually resulting in apoptosis.36,38,39 Consistent with these reports, the results of our study indicate that increased con-centrations of caffeine treatment cause a dose-dependent increase in apoptosis. More recently, autophagy, a process long known to

apoptosis (Fig. 6C ). To confirm whether caffeine-induced cell death is apoptotic, we examined the activity of caspase-3, a well-known inducer of apoptosis. Treatment with 10 mM caffeine markedly increased levels of cleaved caspase-3 and decreased full-length caspase-3 in PC12D cells (Fig. 6D ), consistent with previ-ous reports on the induction of apoptosis by caffeine.35-37

To test whether caffeine-induced apoptosis is dependent on autophagy, we determined whether the inhibition of autophagy by 3-methyladenine (3-MA) or Atg7 siRNA knockdown affects caffeine-induced cytotoxicity in PC12D cells. Treatment with 1 or 5 mM 3MA or Atg7 knockdown significantly decreased the percentage of cell death or cells with reduced mitochon-drial membrane potentials caused by caffeine treatment (5 or 10 mM) (Fig. 6E and F and Suppl. Fig. S6B ). As can be seen from the increased caffeine-induced apoptosis shown in Figure 6A and C , our data suggests that caffeine-induced autophagy is necessary for apoptotic cell death. To further confirm this, we compared autophagy-deficient mouse embryonic fibroblasts (MEFs), lacking the Atg7 gene (Atg7-/-), without LC3-II expres-sion (Suppl. Fig. S4E ), and matched wild-type (Atg7+/+) MEFs, in which autophagy is induced by caffeine in a dose-dependent

Figure 3. caffeine inhibits the Akt/mTOR/p70s6 signaling pathway and activates eRK1/2 signaling. (A and B) sh-sY5Y cells treated with or without 10 mM caffeine for 24 hours were analyzed for mTOR activity by immunoblotting for levels of phosphor- and total p70 ribosomal s6 protein, s6, 4e-BP1 (A), Akt (B) and actin. (c) sh-sY5Y cells treated with or without 10 mM caffeine for 0, 3, 6 or 9 hours were analyzed by immunoblotting for levels of phosphor- and total eRK1/2 and actin. (D) sh-sY5Y cells treated with 10 mM caffeine for various time periods were analyzed by immunoblotting for levels of phosphor- and total p70 ribosomal s6 protein, s6, 4e-BP1 and actin.

?2011 L a n d e s B i o s c i e n c e .

D o n o t d i s t r i b u t e .

treatment or Atg7 knockout, apoptosis is partially attenuated, suggesting that caffeine-induced autophagy occurs upstream of caffeine-induced apoptosis. It also indicates the involvement of other pathways in caffeine-induced apoptosis. These results pro-vide new insight into the effects of caffeine on cell death and survival and its use as a possible intervention strategy for the upregulation of apoptosis by a harnessing of its autophagic activ-ity in tumor treatment.

provide a survival advantage to cells undergoing nutrient depriva-tion and other stresses, has also been linked to the cell death pro-cess.7 The cross-talk between apoptosis and autophagy is complex and sometimes contradictory; however, it is critical to the overall fate of the cell. In this study, we have shown that autophagy is induced by higher concentrations of caffeine without starvation, mainly via the inhibition of PI3K/Akt/mTOR/p70S6K signaling. Likewise, when caffeine-induced autophagy is blocked by 3-MA Figure 4. caffeine-induced autophagy is dependent on Pi3K/Akt/mTOR pathway. (A) sh-sY5Y cells treated with 25 mM caffeine for 3 hours followed

by treatment with or without 200 nM insulin for 30 minutes were analyzed by immunoblotting. (B) Densitometry analysis of Lc3-ii levels relative to actin was performed using three independent experiments. (c) sh-sY5Y cells treated with 25 mM caffeine, 50 μM Akt1/2 inhibitors or 25 mM caffeine with 50 μM Akt1/2 inhibitors for 6 hours were analyzed by immunoblotting. (D) Densitometry analysis of Lc3-ii levels relative to actin was performed using three independent experiments. (e) sh-sY5Y cells were transfected for 24 hours with either a control plasmid DNA (pcDNA3.1) or a plasmid

encoding constitutively active Akt (myr-Akt), and then treated with h 2O or 10 mM caffeine for 6 hours. immunoblotting was performed using antibod-ies against Akt, p-Akt (ser 473) and actin. (F) sh-sY5Y cells treated with 25 mM caffeine with or without 20 μM U0126 for 6 hours were analyzed by immunoblotting using antibodies against actin, Lc3, p-eRK and eRK. (G) Densitometry analysis was performed using three independent experiments. error bars, sD; *p < 0.05; **p < 0.01; N.s., not significant.

?2011 L a n d e s B i o s c i e n c e .

D o n o t d i s t r i b u t e .

(Wako, 106-00131) according to the manufacturer’s proto-col. Detection of early apoptotic cells was determined using an

annexin V/propidium iodide (PI ) detection kit (I nvitrogen), according to the manufacturer’s protocol. Briefly, 0.5 x 106 Atg7+/+ or -/- MEFs were exposed to caffeine (0–25 mM) for 24 hours and washed twice. Then, they were incubated at room temperature with annexin V/Alexa488 and PI for 15 minutes. Annexin V +PI - cells, considered as early apoptotic cells, were enumerated using FACScan (BD Biosciences). Data were analyzed with CellQuest (BD Biosciences) and FlowJo softwares (Tree Star I nc.). Cells positive or negative for annexin V were regarded as apoptotic or non-apoptotic cells, respectively.

Cell cycle analysis. To examine apoptosis, 1.0 x 104 cells/well PC12D cells were seeded onto 96-well culture plate and incubated for 48 h in DMEM with NGF and treated with caffeine for 72 h. The cells were harvested and washed with PBS and fixed with ice-cold 70% ethanol at 4°C for 2 h. The cells were then stained with PI solution according to pre-viously reported protocol.41 DNA content was analyzed by flow cytometry using FACScan and CellQuest software (BD Biosciences).

Compounds. Compounds used included caffeine (Wako, 031-06792), E64d (Sigma, E8640), pepstatin A (Sigma, P5318), rapamycin (LC Laboratories, R5000), CCI-779 (Selleck Chemicals, S1044), MPP + (Sigma, M0896), bafilomycin A1 (Sigma, B1793), 3-methyladenine (Sigma, M9281), insulin (Sigma, I0516), U0126 (Sigma, U120), Akt1/2 inhibitors (Sigma, A6730), staurosporine (Cell Signaling Technology, 9953) and DMSO (Sigma, D2650).

Materials and Methods

Cell line. HeLa cells were maintained in DMEM (Sigma) sup-plemented with 10% fetal bovine serum (FBS) (Sigma) and 100 U/ml penicillin/streptomycin (Sigma) at 37°C and 5% CO 2. PC12D and SH-SY5Y cells were maintained in DMEM (Sigma) supplemented with 10% FBS (Sigma), 5% horse serum and 100 U/ml penicillin/streptomycin at 37°C and 5% CO 2. All experiments with PC12D were performed after differentia-tion with NGF treatment for 48 hours. Atg7+/+ and -/- MEFs were maintained in DMEM (Sigma) supplemented with 10% FBS, 100 U/ml penicillin/streptomycin, 1% sodium pyruvate (Gibco, 11360), 1% non-essential amino acid (NEAA) and 4.2 μl 2% beta-mercaptoethanol at 37°C.

To establish a HeLa GFP-LC3 stable cell line, proliferat-ing HeLa cells were transfected with a GFP-LC3 plasmid.14 Forty-eight hours post-transfection with Lipofectamine 2000 (Invitrogen), positive stable clones were selected by growing cells with G418 (400 μg/ml) for 2 weeks and maintained in DMEM (Sigma) supplemented with 10% FBS (Sigma), 100 U/ml peni-cillin/streptomycin and 200 μg/ml G418 at 37°C and 5% CO 2. All cellular experiments were performed with cells cultured in complete medium with FBS as explained above.

Cell viab ility assays. A trypan blue dye (Invitrogen, 15250-061) exclusion assay was used to examine cell viability and performed according to previously reported protocols.40,41 Changes of mito-chondrial m embrane p otentials w ere a ssessed a lso w ith t he l ipophilic cationic membrane potential-sensitive dye JC-1 (5,5',6,6'-tet-

rachloro-1,1',3,3'-tetraehylbenzimidazolylcarbocyanineiodide)

Figure 5. Rapamycin treatment with caffeine has an additive effect on enhancement of autophagy. (A) sh-sY5Y cells treated with various concentrations of rapamycin with or without 10 mM caffeine for 48 hours were analyzed by immunoblotting. (B) Densitometry analysis was performed using three independent experiments. error bars, sD; *p < 0.05; **p < 0.01; N.s., not significant.

Figure 6 (See next page). caffeine induces apoptosis by enhancement of autophagy. (A) After Pc12D cells were treated with 0, 1, 2.5, 5 or 10 mM caf-feine with DMsO or MPP + for 72 hours, cell viability was measured using trypan blue dye exclusion assay. Data are the means of triplicate experiments. (B) After cells were treated with 0, 1, 2.5, 5 or 10 mM caffeine with DMsO or MPP + for 48 hours, mitochondrial membrane potential was analyzed by Jc-1 using a flow cytometry. Data are the means of triplicate experiments. (c) After Pc12D cells were treated with 0, 1, 2.5, 5 or 10 mM caffeine with DMsO or MPP + for 72 hours, caffeine-induced sub G 1 area was analyzed by propidium iodide staining assay using a flow cytometry. Data are the means of triplicate experiments. (D) Pc12D cells were treated with h 2O or caffeine for 24 hours or staurosporine (positive control) for 3 hours and analyzed with immunoblotting for levels of caspase-3 and cleaved caspase-3. (e) After Pc12D cells were treated with 0, 1, 2.5, 5 or 10 mM caffeine with or without 1, 3 or 5 mM 3MA for 24 hours, cell viability was measured by trypan blue dye exclusion assay. (F) Pc12D cells were transfected with control siRNA or siR-NAs targeting Atg7. Forty eight hours later, they were treated with 0, 1, 2.5 or 10 mM caffeine for 24 hours and mitochondrial membrane potential was analyzed using Jc-1. The knockdown effects on Atg7 were confirmed by immunoblotting using antibodies against Atg7 and actin. Data are the means of triplicate experiments. error bars, s.D. Ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

?2011 L a n d e s B i o s c i e n c e .

D o n o t d i s t r i b u t e .

Western blotting. Cell pellets were lysed on ice in RIPA buf-fer for 20 minutes in the presence of protease inhibitor (Roche). Western blotting was performed according to a previously pub-lished report.42 The antibodies used were as follows: anti-p70 ribosomal protein (Cell Signaling Technology, 2708), anti-ribo-somal protein (Cell Signaling Technology, 2217), anti-4E-BP1

Plasmid DNAs. Myrystoylated Akt (21–151), a constitutively

active form of Akt, was purchased from Millipore.

siRNA knockdown experiments. PC12D cells were trans-fected with rat Atg7 siRNAs (I nvitrogen, 10620318-9) using Lipofectamine RNAiMAX (I nvitrogen, 13778-075) according to the manufacturer’s protocol.

?2011L a n d e s B i o s c i e n c e.

D o n o t d i s t r i b u t e.

Figure 7. cells without Atg7 expression are more resistant to caffeine-induced apoptosis. (A) After Atg7+/+ or -/- mouse embryonic fibroblasts (MeFs)

were treated with 0, 1, 2.5, 5, 10, 25 mM caffeine for 24 hours, the cell viability was measured by trypan blue dye exclusion assay. Data are the means

of triplicate experiments. (B–D) Fluorescence-activated cell-sorting analysis for annexin V/propidium iodide (Pi). Atg7+/+ or -/- MeFs were cultured with various concentrations of caffeine for 24 hours (B) or with 25 mM caffeine for various times (0, 8, 16 or 24 hours) (c and D). Annexin V/Pi staining was subsequently performed to assess early or late apoptosis and necrosis. 5 x 103 cells were analyzed by flow cytometry and the percentage of early apoptotic cells (annexin V-positive and Pi-negative cells, the lower right region in (D) was determined). Data are the means of triplicate experiments.

error bars, sD. Ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

?2011 L a n d e s B i o s c i e n c e .

D o n o t d i s t r i b u t e .

LAMP2 vesicles. Experiments were done in triplicate at least twice.

Quantification of cells with GFP-LC3 vesicles. HeLa cells stable expressing GFP-LC3 were treated with various concentra-tions of caffeine for 24 or 48 hours and then fixed as described above. Analyses in triplicate were done for counting the propor-tion of GFP-positive cells with GFP-LC3 vesicles as previously described in reference 43.Electron microscopy. SH-SY5Y cells treated with various concentrations of caffeine were prefixed in 2% glutaraldehyde in PBS at 4°C, treated with 1% OsO 4 for 3 hours at 4°C, dehy-drated in a graded series of ethanol and flat embedded in epon. Ultra-thin sections were doubly stained with uranyl acetate and observed using a JEOL JEM-2000EX electron microscopy at 80 kV.Statistical analysis. Densitometry analysis was performed using I mageJ 1.43 on immunoblots from three independent experiments. A t-test was performed with SYSTAT software (Hulinks).Acknowledgements We thank Dr. Takashi Ueno (Department of Biochemistry, Juntendo University) for critical comments and Drs. Masaaki Komatsu and Yu-Shin Sou for providing Atg7+/+ and -/- MEFs. We are very grateful for a grant from Hayashi Memorial Foundation for Female Natural Scientists (Y.S.), the Grant-in-Aid for Young Scientists (B) (S. Saiki and F. Sato), grants from the All Japan Coffee Association (S. Saiki), the Takeda Scientific Foundation (S. Saiki) and the Nagao Memorial Fund (S. Saiki).Note Supplementary materials can be found at:

https://www.sodocs.net/doc/449398986.html,/journals/autophagy/article/14074

(Cell Signaling Technology, 9452), anti-Akt (Cell Signaling Technology, 9272), anti-p44/42 MAP kinase (Cell Signaling Technology, 9102), anti-phospho-p70 ribosomal protein (Thr389) (Cell Signaling Technology, 9205), anti-phospho-S6 ribosomal protein (Ser235/236) (Cell Signaling Technology, 2211), anti-phospho-4E-BP1 (Thr37/46) (Cell Signaling Technology, 9459), anti-phospho-p44/p42 MAPK (Thy202/Tyr204) (Cell Signaling Technology, 9101), anti-Atg7 (Cell Signaling Technology, 2631), anti-phospho-Akt (Cell Signaling Technology, 4060), anti-actin (Millipore, clone C4), anti-LC3 (MBL, clone 4E12), anti-p62 (Progen Biotechnik, GP62-C) antibodies. Antibody signals were enhanced with chemifluores-cent methods from GE HealthCare.Immunofluorescent microscopy. Cells were embedded with 4% paraformaldehyde for 20 minutes. Following this, they were permeabilized with 0.1% Triton-X in 1x PBS. After incubation with 10% FBS and 1% bovine serum albumin in 1x PBS for 30 minutes, cells were immunostained with anti-LC3B (x500) (Sigma, L7543), anti-LAMP2 (x50) (Development Studies Hybridoma Bank, clone H4B4) overnight and incubated with

anti-rabbit IgG tagged with AlexaFluor 488 or anti-mouse IgG

tagged with AlexaFluor 546 for 1 hour. The cover slips were embedded with VectaShield, stained with DAPI and images were acquired on a Zeiss LSM510 META confocal microscope (63 x 1.4 NA) or a Leica TCS SP5 confocal microscope at room tem-perature using Zeiss LSM510 v.3.2 software or Leica LAS AF software. Adobe Photoshop 7.0 (Adobe Systems Inc.) was used for subsequent image processing. For colocalization assay in HeLa cells, an appropriate confocal image was taken with Leica LAS AF software. Then, these images were analyzed automatically

with the ImageJ “Colocalization” Plugin (Settings: Each thresh-old: 25, Ratio: 75%) followed by “Analyze particles” (Settings: threshold 25; Pixel: 1) between endogenous LC3 positive and References

1.

Bode AM, Dong Z. The enigmatic effects of caffeine in cell cycle and cancer. Cancer Lett 2007; 247:26-39.2. Jang MH, Shin MC, Kang IS, Baik HH, Cho YH, Chu

JP , et al. Caffeine induces apoptosis in human neuro-blastoma cell line SK-N-MC. J Korean Med Sci 2002; 17:674-8.

3. Gururajanna B, Al-Katib AA, Li YW, Aranha O,

Vaitkevicius VK, Sarkar FH. Molecular effects of taxol and caffeine on pancreatic cancer cells. Int J Mol Med 1999; 4:501-7.

4. Qi W , Qiao D, Martinez JD. Caffeine induces TP53-independent G(1)-phase arrest and apoptosis in human lung tumor cells in a dose-dependent manner. Radiat Res 2002; 157:166-74.

5. Mizushima N, Levine B, Cuervo AM, Klionsky DJ.

Autophagy fights disease through cellular self-digestion. Nature 2008; 451:1069-75.

6. Rubinsztein DC. The roles of intracellular protein-degradation pathways in neurodegeneration. Nature 2006; 443:780-6.

7. Eisenberg-Lerner A, Bialik S, Simon HU, Kimchi A.

Life and death partners: apoptosis, autophagy and the cross-talk between them. Cell Death Differ 2009; 16:966-75.

8. Espert L, Denizot M, Grimaldi M, Robert-Hebmann

V , Gay B, Varbanov M, et al. Autophagy is involved in T cell death after binding of HIV-1 envelope proteins to CXCR4. J Clin Invest 2006; 116:2161-72.9.

Foukas LC, Daniele N, Ktori C, Anderson KE, Jensen J, Shepherd PR. Direct effects of caffeine and theophyl-line on p110delta and other phosphoinositide 3-kinas-es. Differential effects on lipid kinase and protein kinase activities. J Biol Chem 2002; 277:37124-30.10. Kudchodkar SB, Yu Y, Maguire TG, Alwine JC.

Human cytomegalovirus infection alters the substrate specificities and rapamycin sensitivities of raptor- and rictor-containing complexes. Proc Natl Acad Sci USA 2006; 103:14182-7.

11. Winter G, Hazan R, Bakalinsky AT, Abeliovich H.

Caffeine induces macroautophagy and confers a cytoci-dal effect on food spoilage yeast in combination with benzoic acid. Autophagy 2008; 4:28-36.

12. Rubinsztein DC, Cuervo AM, Ravikumar B, Sarkar S,

Korolchuk V, Kaushik S, Klionsky DJ. In search of an “autophagomometer”. Autophagy 2009; 5:585-9.

13. Tanida I, Ueno T, Kominami E. LC3 and Autophagy.

Methods Mol Biol 2008; 445:77-88.

14. Kabeya Y, Mizushima N, Ueno T, Yamamoto A,

Kirisako T , Noda T , et al. LC3, a mammalian homo-logue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 2000; 19:5720-8.15. Yamamoto A, Tagawa Y, Yoshimori T, Moriyama

Y, Masaki R, Tashiro Y. Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepa-toma cell line, H-4-II-E cells. Cell Struct Funct 1998; 23:33-42.

16. Mizushima N, Yoshimori T, Levine B. Methods in

mammalian autophagy research. Cell 140:313-26.17. Hanahan D, Weinberg RA. The hallmarks of cancer.

Cell 2000; 100:57-70.

18. I kenoue T, Hong S, I noki K. Monitoring mamma-lian target of rapamycin (mTOR) activity. Methods Enzymol 2009; 452:165-80.

19. Sinn B, Tallen G, Schroeder G, Grassl B, Schulze J,

Budach V, Tinhofer I. Caffeine confers radiosensitiza-tion of PTEN-deficient malignant glioma cells by enhancing ionizing radiation-induced G 1 arrest and negatively regulating Akt phosphorylation. Mol Cancer Ther 9:480-8.

20. Sarkaria JN, Busby EC, Tibbetts RS, Roos P , Taya Y,

Karnitz LM, Abraham RT. I nhibition of ATM and ATR kinase activities by the radiosensitizing agent, caf-feine. Cancer Res 1999; 59:4375-82.

21. Inoki K, Li Y, Xu T, Guan KL. Rheb GTPase is a direct

target of TSC2 GAP activity and regulates mTOR signaling. Genes Dev 2003; 17:1829-34.

22. Inoki K, Li Y, Zhu T, Wu J, Guan KL. TSC2 is phos-phorylated and inhibited by Akt and suppresses mTOR signalling. Nat Cell Biol 2002; 4:648-57.

23. Garami A, Zwartkruis FJ, Nobukuni T, Joaquin M,

Roccio M, Stocker H, et al. Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhib-ited by TSC1 and 2. Mol Cell 2003; 11:1457-66.

24. Muise-Helmericks RC, Grimes HL, Bellacosa A,

Malstrom SE, Tsichlis PN, Rosen N. Cyclin D expres-sion is controlled post-transcriptionally via a phospha-tidylinositol-3-kinase/Akt-dependent pathway. J Biol Chem 1998; 273:29864-72.

?2011 L a n d e s B i o s c i e n c e .

D o n o t d i s t r i b u t e .

39. Takagi M, Shigeta T , Asada M, Iwata S, Nakazawa S,

Kanke Y, et al. DNA damage-associated cell cycle and cell death control is differentially modulated by caffeine in clones with p53 mutations. Leukemia 1999; 13:70-7.40. Ormerod MG, Collins MK, Rodriguez-Tarduchy G,

Robertson D. Apoptosis in interleukin-3-dependent haemopoietic cells. Quantification by two flow cyto-metric methods. J Immunol Methods 1992; 153:57-65.41. Kawatani M, Uchi M, Simizu S, Osada H, Imoto M.

T ransmembrane domain of Bcl-2 is required for inhibi-tion of ceramide synthesis, but not cytochrome c release in the pathway of inostamycin-induced apoptosis. Exp Cell Res 2003; 286:57-66.

42. Kawajiri S, Saiki S, Sato S, Sato F , Hatano T , Eguchi H,

Hattori N. PI NK1 is recruited to mitochondria with parkin and associates with LC3 in mitophagy. FEBS Lett 2010; 584:1073-9.

43. Sarkar S, Davies JE, Huang Z, Tunnacliffe A,

Rubinsztein DC. T rehalose, a novel mTOR-indepen-dent autophagy enhancer, accelerates the clearance of mutant huntingtin and alpha-synuclein. J Biol Chem 2007; 282:5641-52.

32. Rubinsztein DC, Gestwicki JE, Murphy LO, Klionsky

DJ. Potential therapeutic applications of autophagy. Nat Rev Drug Discov 2007; 6:304-12.

33. Ravikumar B, Vacher C, Berger Z, Davies JE, Luo S,

Oroz LG, et al. I nhibition of mTOR induces auto-phagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat Genet 2004; 36:585-95.

34. Kotake Y, Ohta S. MPP + analogs acting on mitochon-dria and inducing neuro-degeneration. Curr Med Chem 2003; 10:2507-16.

35. Hagan MP , Hopcia KL, Sylvester FC, Held KD.

Caffeine-induced apoptosis reveals a persistent lesion after treatment with bromodeoxyuridine and ultravio-let-B light. Radiat Res 1997; 147:674-9.

36. Efferth T, Fabry U, Glatte P , Osieka R. Expression

of apoptosis-related oncoproteins and modulation of apoptosis by caffeine in human leukemic cells. J Cancer Res Clin Oncol 1995; 121:648-56.

37. Shinomiya N, Takemura T, Iwamoto K, Rokutanda M.

Caffeine induces S-phase apoptosis in cis-diamminedi-chloroplatinum-treated cells, whereas cis-diamminedi-chloroplatinum induces a block in G 2/M. Cytometry 1997; 27:365-73.

38. Lau CC, Pardee AB. Mechanism by which caffeine

potentiates lethality of nitrogen mustard. Proc Natl Acad Sci USA 1982; 79:2942-6.

25. Degtyarev M, De Maziere A, Orr C, Lin J, Lee BB,

Tien JY, et al. Akt inhibition promotes autophagy and sensitizes PTEN-null tumors to lysosomotropic agents. J Cell Biol 2008; 183:101-16.

26. Wan X, Harkavy B, Shen N, Grohar P , Helman LJ.

Rapamycin induces feedback activation of Akt sig-naling through an GF-1R-dependent mechanism. Oncogene 2007; 26:1932-40.

27. Sun SY, Rosenberg LM, Wang X, Zhou Z, Yue P , Fu

H, Khuri FR. Activation of Akt and eI F4E survival pathways by rapamycin-mediated mammalian target of rapamycin inhibition. Cancer Res 2005; 65:7052-8.28. O’Reilly KE, Rojo F , She QB, Solit D, Mills GB, Smith

D, et al. mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res 2006; 66:1500-8.

29. Cirstea D, Hideshima T , Rodig S, Santo L, Pozzi S,

Vallet S, et al. Dual inhibition of akt/mammalian target of rapamycin pathway by nanoparticle albumin-bound-rapamycin and perifosine induces antitumor activity in multiple myeloma. Mol Cancer Ther 2010; 9:963-75.30. Aoki H, Takada Y, Kondo S, Sawaya R, Aggarwal

BB, Kondo Y. Evidence that curcumin suppresses the growth of malignant gliomas in vitro and in vivo through induction of autophagy: role of Akt and extra-cellular signal-regulated kinase signaling pathways. Mol Pharmacol 2007; 72:29-39.

31. Ellington AA, Berhow MA, Singletary KW. Inhibition

of Akt signaling and enhanced ERK1/2 activity are involved in induction of macroautophagy by triter-penoid B-group soyasaponins in colon cancer cells. Carcinogenesis 2006; 27:298-306.

自己翻译的罗氏tunel检测细胞凋亡试剂盒说明书

罗氏tunel检测细胞凋亡试剂盒说明书 注意:Label溶液含有甲次砷酸盐和二氯化钴,严禁吸入和食入。 反应悬浮物收集于密闭、不易碎、有明确标识的容器中,按有毒废物处理。 需要自己配置的其他物品: 除上表所列试剂外,还需准备以下溶液。下表列出每步所需物品概览:

产品概述: 特异性:TUNEL 反应优先标记凋亡产生的DNA 链断裂,从而辨别凋亡与坏死、以及由抑 制细胞生长的药物或放射线产生的primary DNA 链断裂 实验干扰:假阴性:在某些型式的凋亡细胞中DNA 链断裂可能缺失或不完全。空间位阻, 如细胞外元件可能阻止TdT 到达DNA 断裂处。两种情况均能产生假阴性。 假阳性:在坏死晚期,可能产生大量的DNA 片段 DNA 链断裂也可能在具有高增殖和代谢活动的细胞中出现。两种情况均能产生 假阳性。为确认细胞死亡的凋亡型式,应认真进行每种细胞的形态学检查 凋亡过程中产生的形态学改变尤其特征形式,因此,对于可以结果进行解释时, 细胞形态评估是一项重要的参数 样本:细胞离心涂片和细胞涂片 在chamber slides 上培养的黏附细胞 冰冻或福尔马林固定、石蜡包埋样本 分析时间:2-3小时,除外培养、固定和渗透 检测次数:一个试剂盒50T

步骤和所需材料: 1 流程图: 2 样品准备 黏附细胞、细胞涂片和细胞离心涂片 需准备的其他试剂:Washing buffer:磷酸盐缓冲液(PBS) Blocking buffer封闭溶液:甲醇稀释的3% H2O2 Fixation solution固定溶液:PBS配制的4%多聚甲醛,ph ,新鲜配制 Permeabilisation solution 渗透液:%Triton1)X-100溶于%柠檬酸钠溶液 中,新鲜配制 步骤:下表描述了细胞固定、内源性过氧化物酶封闭和细胞渗透过程。 组织部分 福尔马林-包埋组织 福尔马林包埋组织的预处理:可按4种不同的方式预处理。如用蛋白酶K,不含核酸酶,浓 度、孵育时间和温度应按组织类型优化 注意:只用罗氏应用科学的蛋白酶K,因其经检测不含核酸酶, 核酸酶可导致假阳性。 另外3中替代方法在下表中描述(step 2) 需准备的其他试剂:二甲苯和乙醇(浓度:95%,90%,80%,70%,溶于双蒸水中)

细胞凋亡实验步骤及注意事项

细胞凋亡实验步骤及注意事项 一、实验目的 1、掌屋凋亡细胞的形态特征 2、学会用荧光探针对细胞进行双标记来检测正常活细胞、凋亡细胞与坏死 细胞的方法 二、实验原理 细胞死亡根据其性质、起源及生物学意义区分为凋亡与坏死两种不同类型。凋亡普遍存在于生命界,在生物个体与生存中起着非常重要的作用。它就是细胞在一 定生理条件下一系列顺序发生事件的组合,就是细胞遵循一定规律自己结束生命 的自主控制过程。细胞凋亡具有可鉴别的形态学与生物化学特征。 在形态上可见凋亡细胞与周围细胞脱离接触,细胞变园,细胞膜向内皱缩、胞浆浓缩、内质网扩张、细胞核固缩破裂呈团块状或新月状分布、内质网与细胞膜进一步融合将细胞分成多个完整包裹的凋亡小体,凋亡小体最后被吞噬细胞吞噬消化。在凋亡过程中细胞内容物并不释放到细胞外,不会影响其它细胞,因而不引起炎症反应。 在生物化学上,多数细胞凋亡的过程中,内源性核酸内切酶活化,活性增加。核DNA 随机地在核小体的连接部位被酶切断,降解为180-200bp或它的整倍数的各种片断。如果对核DNA进行琼脂糖电泳,可显示以180-200bp为基数的DNA ladder(梯状带纹)的特征。 相比之下,坏死就是细胞处于剧烈损伤条件下发生的细胞死亡。细胞在坏死早期 即丧失质膜完整性,各种细胞器膨胀,进而质膜崩解释放出其中的内容物,引起炎症反应,坏死过程中细胞核DNA虽也降解,但由于存在各种长度不等的DNA片断,不能形成梯状带纹,而呈弥散状。 一些温与的损伤刺激及一些抗肿瘤药物可诱导细胞凋亡,通常这些因素在诱导凋亡的同时,也可产生细胞坏死,这取决于损伤的剧烈程度与细胞本身对刺激的敏感 程度。 三尖杉酯碱(HT)就是我国自行研制的一种对急性粒细胞白血病,急性单核白血病等有良好疗效的抗肿瘤药物。研究表明HT在0、02~5μg/ml范围内作用2小时,即可诱导HL-60细胞凋亡,并表现出典型的凋亡特征。本实验用1μg/ml HT在体外诱导培养的HL-60细胞发生凋亡,同时也有少数细胞发生坏死。用 Hoechst33342与碘化丙啶(propidium iodide,PI)对细胞进行双重染色,可以区别凋亡、坏死及正常细胞。 细胞膜就是一选择性的生物膜,一般的生物染料如PI等不能穿过质膜。当细胞坏死时,质膜不完整,PI就进入细胞内部,它可嵌入到DNA或RNA中,使坏死细胞着

细胞凋亡试验常用的方法

细胞凋亡试验常用的方法(MTT法、荧光法、DNA琼脂糖凝胶电泳法与流式细胞仪检测法) (一)药物对肿瘤细胞的抑制效应的MTT法: 用培养基将肿瘤细胞调整至2 X108个/L,在96孔板中每孔加入100ul细胞悬液于37℃、5% CO2下培养过夜。 次日每孔加入不同浓度的药物100mg/L作为试验组,设加完全培养基不加药物的阴性对照,并用功能明确的药物为阳性对照和0.5%的乙醇溶剂对照,每组均设4-6个复孔(平行孔)、37℃、5% CO2继续培养。 培养至12h、24h、48h、实验终止前4-6h加入10ulMTT(5g/L),培养4-6h后,阴性对照孔中已形成明显的蓝紫色颗粒结晶时加100ul/孔SDS-HCl终止反应,于37℃存放过夜。 用酶标仪在A570波长下测吸光度值,按下式计算抑制率 抑制率(%)=(1-试验组平均吸光度值/阴性对照组平均吸光度值)x 100%。 (二)荧光法: 选用上述最佳浓度作用于肿瘤细胞,培养细胞48h后,收货细胞用PBS洗2-3次后用0.4%多聚甲醛室温下固定30min。 弃去固定液,并用PBS洗2次后,用1%Triton X-100作用4min加入适量的0.5mg/L DAPI 荧光染色60min,用PBS冲洗3次,取10ul滴片,干燥后于荧光显微镜下检测断裂的颗粒和片状荧光。 (三)DNA琼脂糖凝胶电泳法: 1、DNA提取: 用大方瓶培养肿瘤细胞,每瓶10ml,细胞浓度为3 x 108个/ml,每隔药物浓度、作用时间均设2瓶,共分3个时间段,4个药物浓度。共培养26瓶细胞。 分别于细胞中加入不同浓度的药物,于37℃、5% CO2中分别培养12h、24h、48h,收货细胞,用PBS洗2-3次。 于-20℃将细胞冷却处理10min后将细胞收集至离心管中,加1ml细胞裂解液,再加蛋白酶K,轻轻振摇使悬液混匀,成黏糊状,50℃过夜。 冷却后加入等体积的饱和酚溶液,混合后10000r/min离心10min,吸出上层水相,移至另一离心管中,再加入等体积饱和酚溶液重复抽提一次,直到无蛋白为止。 吸上清加入氯仿/异戊醇(24:1)按上述方法再抽提一次。 吸取水相层加入1/10体积的3mol/L的醋酸钠溶液,混匀。 再加入2.5倍体积冷无水乙醇,混合置-20℃处理30min后,10000r/min离心10min,沉淀部分为提供的DNA,弃去无水乙醇后用70%乙醇漂洗2次,将离心管倒扣在吸水纸上,吸干乙醇。 加入200ulTE缓冲液融解DNA,再加入25ul的RNA酶,置37℃作用30min,置4℃冰箱保存。 2、琼脂糖凝胶电泳: TBE缓冲液配制1.8%琼脂糖凝胶。在微波炉内煮沸至琼脂糖融解,待冷却至60℃时,加入溴化乙锭,使其终浓度为0.5mg/ml,混匀后灌胶。 待凝胶固定后放入含TBE电泳液的电泳槽内,使TBE电泳液盖过凝胶。 取10-15ul提取的各组DNA样品液与上样缓冲液按4:1比例混匀后点样。 60V电泳1h,用紫外透射仪观察梯形条带。

细胞生物学实验细胞凋亡观察

实验目的: 1.了解凋亡细胞的形态学特征,加深对于细胞凋亡现象及本质的理解。 2.了解并掌握细胞凋亡检测的方法和基本原理。 实验原理: 细胞凋亡时,出现一系列形态学变化,包括凋亡细胞的染色质浓缩、边缘化,核膜裂解、染色质分割成块状,染色质的DNA出现缺口甚至断裂,出现DNA碎片,并逐渐形成凋亡小体等,经相应的染色后可以在普通光学显微镜和荧光显微镜下观察到这些变化。从而把凋亡的细胞和正常的细胞区分开来。

细胞凋亡是指细胞对环境的生理、病理性刺激信号、环境条件的变化或缓和性损伤产生的应答有序变化的死亡过程。细胞凋亡是一个主动过程,涉及一系列基因的激活、表达以及调控等的作用,它并不是病理条件下的自体损伤,而是为更好地适应生存环境的一种死亡过程。 1.细胞凋亡与细胞程序性死亡: 细胞程序性死亡的概念是指一个多细胞生物体中某些细胞的死亡是个体发育中一个预定的,并受到严格程序控制的正常组成部分。例如蝌蚪变成青蛙,其变态过程中尾部的消失伴随大量细胞死亡,高等哺乳类动物指间蹼的消失、颚融合、视网膜发育以及免疫系统的正常发育都必须有细胞死亡的参与。这些形形色色的在机体发育过程中出现的细胞死亡有一个共同特征:即散在的、逐个地从正常组织中死亡和消失,机体没有炎症反应,而且这种死亡对整个机体的发育是有利和必须的。因此认为动物发育过程中存在的细胞程序性死亡是一个发育学概念,而细胞凋亡则是一个形态学的概念,但是一般认为这两个概念可以交互使用,具有同等意义。2.细胞凋亡与坏死的区别: 虽然凋亡与坏死的最终结果极为相似,但它们的过程与表现却有很大差别。坏死是细胞受到强烈理化或生物因素作用引起细胞无序变化的死亡过程。表现为细胞胀大、胞膜破裂、细胞内容物外溢、核变化较慢、DNA降解不充分、有局部严重的炎症反应。坏死是一个被动的过程,其细胞及组织的变化与凋亡有明显的不同。

一步法TUNEL细胞凋亡检测试剂盒说明书

一步法TUNEL细胞凋亡检测试剂盒说明书 货号:T2190 规格:20次 保存:-20oC保存,荧光标记液需避光保存。 产品简介: 细胞在发生凋亡时,会激活一些DNA内切酶,这些内切酶会切断核小体间的基因组DNA。细胞凋亡时抽提DNA进行电泳检测,可以发现180-200bp的DNA ladder。基因组DNA断裂时,暴露的3’-OH可以在末端脱氧核苷酸转移酶(Terminal Deoxynucleotidyl Transferase,TdT)的催化下加上绿色荧光探针荧光素(FITC)标记的dUTP(fluorescein-dUTP),从而可以通过荧光显微镜或流式细胞仪进行检测,这就是TUNEL(TdT-mediated dUTP Nick-End Labeling)法检测细胞凋亡的原理。 一步法TUNEL细胞凋亡检测试剂盒(One Step TUNEL Apoptosis Assay Kit)为您提供了一种高灵敏度又快速简便的细胞凋亡检测方法。对于经过固定和洗涤的细胞或组织,只要经过一步染色反应,洗涤后就可以通过荧光显微镜或流式细胞仪检测到呈现绿色荧光的凋亡细胞。 TUNEL法特异性检测细胞凋亡时产生的DNA断裂,但不会检测出射线等诱导的DNA断裂(和细胞凋亡时的断裂方式不同)。这样一方面可以把凋亡和坏死区分开,另一方面也不会把射线等诱导发生DNA断裂的非凋亡细胞判断为凋亡细胞。极少数细胞凋亡时没有DNA断裂,此时不适用TUNEL法检测。在个别类型的坏死细胞中也发现TUNEL检测呈阳性。在需要严格判断细胞凋亡的情况下,最好同时检测多个凋亡指标。产品内容: 1.TdT酶100μl 2.荧光标记液900μl 3.TdT酶稀释液(选用)500μl

2018_2019学年高中生物每日一题细胞凋亡和细胞坏死的区别含解析新人教版必修1

细胞凋亡和细胞坏死的区别 高考频度:★★★☆☆难易程度:★★☆☆☆ 脑缺氧、心缺血、急性胰腺炎、动脉粥样硬化等疾病都是由细胞坏死引起的。近日,厦门大 学生命科学学院韩家淮教授课题组的一项研究表明,存在于人体内的一种名为RIP3的蛋白 激酶,能够将细胞凋亡转换成细胞坏死,通过调控这种酶的合成,就可以调控细胞的死亡方式。下列有关叙述错误的是 A.从以上分析可知细胞坏死过程中存在基因的选择性表达 B.—些细胞的坏死对人体也有益处,比如被病原体感染的细胞在免疫系统的作用下死亡 C.抑制RIP3的活性,能在一定程度上对急性胰腺炎起治疗、防御的作用 D.在人体的癌细胞中,也可能存在控制RIP3合成的基因 【参考答案】B 【试藍翼祈】由细运碍亡特銮弟鈿迸环芒寸虽该追越白基医逵淫土爰注'A正聽:由越丹熄思繆不出譎縫琢死痔身徳有莖,B 樂唳;抑割该薛的洁席.则毅別条锻坏死,C正碍:艳籍踰胞前全寵惟,号令鑰無占的基因鑫是一縊的尸D正菇’ ■ ”推荐 -------------- ” 1有关细胞凋亡和细胞坏死的叙述正确的是 A.细胞凋亡的速率会因其功能不同而不同 B.被病毒侵染的细胞的清除属于细胞坏死 C.细胞凋亡和细胞坏死都有利于个体的生长发育 D.细胞凋亡和细胞坏死都受环境影响较大,机体难以控制 2.下列关于细胞凋亡和细胞坏死的叙述中,错误的是 A.细胞凋亡是一种自然的生理过程 B.细胞坏死是一种病理性变化

C.被病原体感染的细胞的清除是通过细胞坏死完成的 D.蝌蚪尾的消失,是由基因决定的细胞自动结束生命的过程 3?下列关于细胞凋亡和细胞坏死的叙述中,错误的一项是 A.细胞凋亡是主动的,细胞坏死是被动的 B.细胞凋亡是生理性的,细胞坏死是病理性的 C.细胞凋亡是基因调控的,细胞坏死是外界因素引起的 D.细胞凋亡是急性的,细胞坏死是慢性的 4?细胞凋亡也称为细胞编程性死亡,其大致过程如图所示。下列有关叙述错误的是 A.细胞皱缩、染色质固缩表明细胞处于衰老状态 B.图示过程只发生在胚胎发育过程中 C.吞噬细胞吞噬凋亡小体与细胞膜的流动性密切相关 D.细胞凋亡是由遗传物质控制的,与细胞坏死有明显区别 5?香烟中含有大量的有害物质,如尼古丁等会造成吸烟者肺部细胞的死亡。这种细胞的死亡过程属于 A.生理性死亡 B.正常衰亡 C.细胞坏死 D.细胞凋亡 1.【答案】A 【解析】功能不同的细胞凋亡速率不同,如神经细胞可能一生都不凋亡,口腔上皮细胞 则在短时间内发生凋亡,A正确;被病毒侵染的细胞的清除属于细胞凋亡,B错误;细胞凋亡是指由基因控制的细胞自动结束生命的过程,有利于个体的生长发育;细胞坏死是 1E常细 胞 细胞皱缩细胞励SL吞噬细胞 核染色质分解战多个包袅、呑噬 凋亡小体凋亡小体

淋巴细胞分离和细胞凋亡诱导及形态学观察

细胞生物学实验报告 淋巴细胞分离和细胞凋亡诱导及形态学观察lymphocyte isolation and morphological observation of apoptosis induction 2012年6月2日

淋巴细胞分离和细胞凋亡诱导及形态学观察 lymphocyte isolation and morphological observation of apoptosis induction 摘要:目的了解细胞凋亡的原理,掌握离体诱导细胞凋亡的方法,及用普通光学显微镜和荧光显微镜观察凋亡细胞的形态学变化,并从观察结果初步推断和识别凋亡细胞具体阶段。方法实验所用Hoechst 33342/PI双染检测细胞凋亡、Giemsa染色。结果实验结束后得到了染色的结果以及照片。结论所提取的淋巴细胞发生凋亡。 Abstract:Objective Understand the principles of cell apoptosis, master the methods of cell apoptosis induced by in vitro, and by ordinary optical microscopy and fluorescence microscopy observation of morphological changes of apoptotic cells and preliminary inferred from observations and identifying apoptotic cells specific stages. Method Experimental method used in,Hoechst 33342/PI double dye test and the Giemsa stain. Results Be dyed after the end of the experiment results and photos. Conclusions The extraction of lymphocyte apoptosis. 关键词:细胞凋亡、Giemsa染色、Hoechst 33342/PI双染。 Keyword:cell apoptosis、Giemsa stain、Hoechst 33342/PI double staining 1.实验原理 1.1 关于淋巴细胞的分离 外周血中淋巴细胞比重约为1.070,而红细胞和粒细胞的比重较大,为1.902左右。因此,利用相对密度为1.077±0.002的淋巴细胞分离液(称为分层液)离心,可使一定比重的细胞按相应密度梯度分布(使比重中较大的红细胞和粒细胞沉于管底,淋巴细胞浮于分层液与血浆的界面上),从而将淋巴细胞分离出来。 1.2 关于细胞凋亡 细胞凋亡又称编程性死亡或程序性死亡。细胞凋亡是多细胞生物体在发育过程中或在某些环境因子的作用下发生的受基因调控的主动的死亡方式。细胞凋亡对于多细胞生物个体的正常发育、保持自稳平衡、抵御外界各种因素的干扰及多种病理过程具有极其重要的意义,起着非常重要的作用。细胞凋亡是多种生理、病理因子参与的由凋亡相关基因启动的细胞程序性死亡过程,其中由氧应激造成大量活性氧(reactive oxygen species, ROS)的产生以及继发性细胞损伤过程在细胞凋亡中起着重要作用,如电离辐射或紫外线照射产生大量H2O2、OH-等。 细胞凋亡的诱导因子包括三大类: 1.物理因子:包括射线、较温和的温度刺激(如热激或冷激)等; 2.化学因子:包括活性氧基团和分子、重金属离子等; 3.生物因子:肿瘤坏死因子、生物毒素、抗肿瘤药物、DNA和蛋白质合成的抑制剂等。 细胞凋亡的主要特征包括: 1.染色质凝集、质膜出芽、核裂解及凋亡小体的形成。 2.DNA特异性降解成200bp或其整数倍片断,通过凝胶电泳形成梯状条带,称为DNA Ladder。 3.由于DNA特异性降解而产生大量3’-OH末端,可被末端脱氧核糖核苷酸移换酶介导的dUTP 缺口末端原位标记法(TUNEL)标记,从而产生亮绿色荧光等。

凯基TUNEL细胞凋亡原位检测试剂盒

凯基TUNEL细胞凋亡原位检测试剂盒(通用)(BIOTIN标记POD法,适用于细胞、组织样本) 使用说明书 一、TUNEL制品说明 凯基TUNEL细胞凋亡检测试剂盒是用来检测细胞在凋亡过程中细胞核DNA 的断裂情况,其原理是生物素(biotin)标记的dUTP在脱氧核糖核苷酸末端转移酶(TdT Enzyme)的作用下,可以连接到凋亡细胞中断裂的DNA的3‘-OH末端,并可与连接了的辣根过氧化酶的链霉亲和素(Streptavidin-HRP)特异结合,在辣根过氧化酶底物二氨基联苯胺(DAB)的存在下,产生很强的颜色反应(呈深棕色),特异准确地定位正在凋亡的细胞,因而在普通显微镜下即可观察和计数凋亡细胞;由于正常的或正在增殖的细胞几乎没有DNA的断裂,因而没有3'-OH 形成,很少能够被染色。 本试剂盒适用于组织样本(石蜡包埋、冰冻和超薄切片)和细胞样本(细胞涂片)的凋亡原位检测。 本试剂盒特点 ●操作简便:使用Ready-to-Use型试剂,并配有Proteinase K 和DAB。 ●高灵敏度:可以单一检出初期的凋亡细胞。 ●高特异性:能特异性染色凋亡细胞。 ●快速操作:整体操作约需3小时。 ●用途广泛:可应用于组织切片、细胞样本等。 ●方便观察:使用光学显微镜观察实验结果。 ●高正确性:有阳性对照片的制备方法,可以确认试剂盒的有效性

使用注意事项 1.使用前请认真阅读本说明书,提前准备好相关试剂。 2.因本试剂盒中组分均为微量,使用前请离心集液。 3.为避免试验误差、降低试剂的损耗,建议使用精密度高的进口微量移液枪及枪头。 4. TdT 酶反应液最好在使用前根椐样本数量集中配制,再分别滴加于各样本片上,避免每个样本单独配制而产生的试剂损耗。 5. 为防止样本脱落,请使用硅烷(Silane)处理的载玻片或采用多聚赖氨酸铺片。 6. 固定好的样本可以在-20℃的70%乙醇中放置30分钟或至过夜,以改善细胞的渗透性。 7. 使用PBS清洗细胞样本时,不要直接加在细胞样本上,以防止细胞样本的脱落。 8. 进行PBS清洗时,以5分钟清洗3次为标准。 9. DAB为固体粉末,使用前加入PBS配制成20×DAB(10 mg/ml)后,按说明书显色使用。 二、TUNEL试剂盒组分 试剂盒以外自备仪器和试剂

细胞坏死凋亡和细胞程序性死亡

细胞坏死necrosis、细胞凋亡apoptosis、 细胞程序性死亡programmed cell death(PCD) 死亡是生命的普遍现象,但细胞死亡并非与机体死亡同步,在正常的组织中,经常发生“正常”的细胞死亡,它是维持组织机能和形态所必需的;细胞死亡的方式通常有3种:即①细胞坏死(necrosis)。②细胞凋亡(apoptosis)。③细胞程序性死亡(programmed cell death,PCD)。 Ⅰ.细胞坏死 细胞坏死是细胞受到化学因素(如强酸、强碱、有毒物质)、物理因素(如热、辐射)和生物因素(如病原体)等环境因素的伤害,引起细胞死亡的现象。坏死细胞的形态改变主要是由下列2种病理过程引起的,即酶性消化和蛋白变性;参与此过程的酶,如来源于死亡细胞本身的溶酶体,则称为细胞自溶(autolysis);若来源于浸润坏死组织内白细胞溶酶体,则为异溶(heterolysis)。 细胞坏死初期,胞质内线粒体和内质网肿胀、崩解,结构脂滴游离、空泡化,蛋白质颗粒增多,核发生固缩或断裂。随着胞质内蛋白变性、凝固或碎裂,以及嗜碱性核蛋白的降解,细胞质呈现强嗜酸 性,故坏死组织或细胞在苏木精 /伊红染色切片中,胞质呈均一 的深伊红色,原有的微细结构消 失。在含水量高的细胞,可因胞 质内水泡不断增大,并发生溶 解,导致细胞结构完全消失,最 后细胞膜和细胞器破裂,DNA 降解,细胞内容物流出,引起周 围组织炎症反应(图1)。 图1细胞坏死与凋亡的形态区别

Ⅱ.细胞凋亡 细胞凋亡(cell apoptosis)是借用古希腊语,表示细胞象秋天的树叶一样凋落的死亡方式,1972年Kerr最先提出这一概念,他发现结扎大鼠肝的左、中叶门静脉后,其周围细胞发生缺血性坏死,但由肝动脉供应区的实质细胞仍存活,只是范围逐渐缩小,其间一些细胞不断转变成细胞质小块,不伴有炎症,后在正常鼠肝中也偶然见到这一现象。 凋亡是一个形态学概念,是指为维持内环境稳定,由基因控制的细胞自主的有序的死亡现象,不是一件被动的过程,而是主动过程,是为更好地适应生存环境而主动争取的一种死亡过程,涉及一系列基因的激活、表达以及调控等的作用。 凋亡细胞的主要特征见表1,共有:①染色质聚集、分块、位于核膜上,胞质凝缩,最后核断裂,细胞通过出芽的方式形成许多凋亡小体(图1、2);②凋亡小体内有结构完整的细胞器,还有凝缩的染色体,可被邻近细胞吞噬消化,因始终有膜封闭,没有内溶物释放,故不会引起炎症;③凋亡细胞中仍需要合成一些蛋白质,但是在坏死细胞中ATP和蛋白质合成受阻或终止;④核酸内切酶活化,导致染色质DNA在核小体连接部位断裂,形成约200bp整数倍的核酸片段,凝胶电泳图谱呈梯状;⑤凋亡通常是生理性变化,而细胞坏死是病理性变化。 表1细胞凋亡和细胞坏死的区别 区别点细胞凋亡细胞坏死 起因生理或病理性病理性变化或剧烈损伤 范围单个散在细胞大片组织或成群细胞 细胞膜保持完整,一直到形成凋亡小体破损 染色质凝聚在核膜下呈半月状呈絮状 细胞器无明显变化肿胀、内质网崩解 细胞体积固缩变小肿胀变大 凋亡小体有,被邻近细胞或巨噬细胞吞噬无,细胞自溶,残余碎片被 巨噬细胞吞噬 基因组DNA 有控降解,电泳图谱呈梯状随机降解,电泳图谱呈涂抹 状 蛋白质合成有无 调节过程受基因调控被动进行 炎症反应无,不释放细胞内容物有,释放内容物。

常用细胞凋亡检测方法(图)

常用细胞凋亡检测方法(图) 转载请注明来自丁香园 发布日期:2012-02-16 13:41 文章来源:丁香通 关键词:丁香园生物专题义翘神州细胞培养点击次数:951 一、细胞凋亡的形态学检测 1、光学显微镜和倒置显微镜 ①未染色细胞:凋亡细胞的体积变小、变形,细胞膜完整但出现发泡现象,细胞凋亡晚期可见凋亡小体。贴壁细胞出现皱缩、变圆、脱落。 ②染色细胞:常用姬姆萨染色、瑞氏染色等。凋亡细胞的染色质浓缩、边缘化,核膜裂解、染色质分割成块状和凋亡小体等典型的凋亡形态。 2、荧光显微镜和共聚焦激光扫描显微镜 一般以细胞核染色质的形态学改变为指标来评判细胞凋亡的进展情况。常用的DNA 特异性染料有:HO 33342 (Hoechst 33342),HO 33258 (Hoechst 33258), DAPI。三种种染料与DNA的结合是非嵌入式的,主要结合在DNA的A-T碱基区。紫外光激发时发射明亮的蓝色荧光。Hoechst是与DNA特异结合的活性染料,储存液用蒸馏水配成1mg/ml的浓度,使用时用PBS稀释,终浓度为10 ug/ml。DAPI为半通透性,用于常规固定细胞的染色。储存液用蒸馏水配成1mg/ml的浓度,使用终浓度一般为10 ug/ml。结果评判:细胞凋亡过程中细胞核染色质的形态学改变分为三期:Ⅰ期的细胞核呈波纹状(rippled)或呈折缝样(creased),部分染色质出现浓缩状态;Ⅱa期细胞核的染色质高度凝聚、边缘化;Ⅱb期的细胞核裂解为碎块,产生凋亡小体(图1)。 3、透射电子显微镜观察 结果评判:凋亡细胞体积变小,细胞质浓缩。凋亡Ⅰ期(pro-apoptosis nuclei)的细胞核内染色质高度盘绕,出现许多称为气穴现象(cavitations)的空泡结构(图2);Ⅱa期细胞核的染色质高度凝聚、边缘化;细胞凋亡的晚期,细胞核裂解为碎块,产生凋亡小体。 二、磷脂酰丝氨酸外翻分析(Annexin V法) 磷脂酰丝氨酸(Phosphatidylserine, PS)正常位于细胞膜的内侧,但在细胞凋亡的早期,PS可从细胞膜的内侧翻转到细胞膜的表面,暴露在细胞外环境中(图3)。Annexin-V是一种分子量为35~36KD的Ca2+依赖性磷脂结合蛋白,能与PS高亲和力特异性结合。将Annexin-V进行荧光素(FITC、PE)或biotin标记,以标记了的Annexin-V作为荧光探针,利用流式细胞仪或荧光显微镜可检测细胞凋亡的发生。 碘化丙啶(propidine iodide, PI)是一种核酸染料,它不能透过完整的细胞膜,但在凋亡中晚期的细胞和死细胞,PI能够透过细胞膜而使细核红染。因此将Annexin-V 与PI匹配使用,就可以将凋亡早晚期的细胞以及死细胞区分开来。 方法

细胞凋亡检测方法

细胞凋亡检测方法 一、细胞凋亡的形态学检测 1 光学显微镜和倒置显微镜 (1)未染色细胞:凋亡细胞的体积变小、变形,全面皱缩,细胞膜完整但出现发泡现象,细胞凋亡晚期可见凋亡小体,凋亡小体为数个圆形小体围绕在细胞周围。贴壁细胞出现皱缩、变圆、脱落。 (2)染色细胞: 姬姆萨(Giemsa)染色、瑞氏染色等:正常细胞核色泽均一;凋亡细胞染色质浓缩、边缘化,核膜裂解、染色质分割成块状和凋亡小体等典型的凋亡形态;坏死细胞染色浅或没染上颜色。 苏木素-伊红(HE)染色:细胞核固缩碎裂、呈蓝黑色、胞浆呈淡红色(凋亡细胞),正常细胞核呈均匀淡蓝色或蓝色,坏死细胞核呈很淡的蓝色或蓝色消失。 2 荧光显微镜和共聚焦激光扫描显微镜 一般以细胞核染色质的形态学改变为指标来评判细胞凋亡的进展情况。 常用的DNA特异性染料有:Hoechst 33342,Hoechst 33258,DAPI。三种染料与DNA 的结合是非嵌入式的,主要结合在DNA的A-T碱基区。紫外光激发时发射明亮的蓝色荧光。 Hoechst是与DNA特异结合的活性染料,能进入正常细胞膜而对细胞没有太大细胞毒作用。Hoechst 33342在凋亡细胞中的荧光强度要比正常细胞中要高。 DAPI为半通透性,用于常规固定细胞的染色。 PI和Hoechst33342双标:PI、Hoechst33342均可与细胞核DNA(或RNA)结合。但PI不能通过正常细胞膜,Hoechst则为膜通透性荧光染料,故细胞在处于坏死或晚期调

亡时细胞膜被破坏,这时可为PI着红色。正常细胞和中早期调亡细胞均可被Hoechst着色,但是正常细胞核的Hoechst着色的形态呈圆形,淡兰色,内有较深的兰色颗粒;而调亡细胞的核由于浓集而呈亮兰色,或核呈分叶,碎片状,边集。故PI着色为坏死细胞;亮兰色,或核呈分叶状,边集的Hoechst着色的为调亡细胞。 凋亡细胞体积变小,细胞质浓缩。细胞凋亡过程中细胞核染色质的形态学改变分为三期:Ⅰ期的细胞核呈波纹状(rippled)或呈折缝样(creased),部分染色质出现浓缩状态;Ⅱa期细胞核的染色质高度凝聚、边缘化;Ⅱb期的细胞核裂解为碎块,产生凋亡小体(图1)。 3 透射电子显微镜观察 凋亡细胞体积变小,细胞质浓缩。凋亡Ⅰ期(pro-apoptosis nuclei)的细胞核内染色质高度盘绕,出现许多称为气穴现象(cavitations)的空泡结构(图2);Ⅱa期细胞核的染色质高度凝聚、边缘化;细胞凋亡的晚期,细胞核裂解为碎块,产生凋亡小体。 二、磷脂酰丝氨酸外翻分析(Annexin V法) 磷脂酰丝氨酸(Phosphatidylserine, PS)正常位于细胞膜内侧,但在细胞凋亡早期,PS可从细胞膜内侧翻转到细胞膜表面,暴露在细胞外环境中。磷脂酰丝氨酸的转位发生在凋亡早期阶段,先于细胞核的改变、DNA断裂、细胞膜起泡。体内的吞噬细胞可通过识别

一步法TUNEL细胞凋亡检测试剂盒红色TRITC标记荧光检

一步法 TUNEL 细胞凋亡检测试剂盒(红色 TRITC 标记荧光检测法,通用型) 说明书修订日期:2018.07.31 Catalog No.:KGA7061 / KGA7062 / KGA7063 Storage:-20℃ for 12 months,避光 For Research Use Only(科研专用) 一、TUNEL 检测原理 凯基一步法 TUNEL 细胞凋亡检测试剂盒(TRITC红色荧光标记,通用型)提供一种高灵敏度又快速简便的细胞凋亡检测方法,可检测细胞在凋亡过程中细胞核 DNA 的断裂情况,其原理是红色荧光素(Tetramethylrhodamine,TRITC)标记的 dUTP 在脱氧核糖核苷酸末端转移酶(TdT Enzyme)的作用下,可以连接到凋亡细胞中断裂 DNA 的3′-OH 末端,可用荧光显微镜检测。由于正常的或正在增殖的细胞几乎没有 DNA 的断裂,因而没有3′-OH 形成,很少能够被标记。 本试剂盒适用于组织样本(石蜡包埋、冰冻和超薄切片)和细胞样本(细胞涂片或爬片)的凋亡原位检测。 对于经过固定和洗涤的细胞或组织,只要经过一步染色反应,洗涤后就可以通过荧光显微镜检测到凋亡细胞。 二、TUNEL 试剂盒组分 运输及保存条件: 2-8℃低温运输,收到后-20℃避光保存,保质期一年。 实验前准备: 实验前按说明书准备好相应试剂与器具,多聚甲醛、二甲苯、乙醇、1×PBS pH 7.4、H2O2、Triton X-100、甲醇、多聚赖氨酸铺载玻片(细胞样本)、盖玻片、染色缸、温盒、量筒等。 三、操作步骤 各个样本请用免疫组化笔做好标记,另外加 2 张样本切片分别用于阳性片和阴性片制备并做好标记。 在每步反应或浸洗的间隙时,配制下一步即用的工作液。

TUNEL法检测细胞凋亡

细胞在发生凋亡时,会激活一些DNA内切酶,这些内切酶会切断核小体间的基因组DNA。基因组DNA 断裂时,暴露的3'-0H 可以在末端脱氧核苷酸转移酶(Terminal Deox yn ucleotidyl Tran sferase,TdT)的催化下加上荧光素(FITC)标记的dUTP(fluorescein-dUTP),从而可以通过荧光显微镜或流式细胞仪进行检测,这就是TUNEL 法检测细胞凋亡的原理。 TUNEL法特异性检测细胞凋亡时产生的DNA断裂,但不会检测出射线等诱导的DNA断裂(和细胞凋亡时的断裂方式不同)。这样一方面可以把凋亡和坏死区分开,另一方面也不会把 射线等诱导发生DNA断裂的非凋亡细胞判断为凋亡细胞。 针对问题2(TUNEL法的实验原理是什么?): 基本原理:对不同组织切片先增加细胞膜通透性,然后让rTDT和bio标记的dUTP进入细 胞内,在rTDT的辅助下dUTP与核断裂的DNA 3 -0H结合,再用HRP标记的链霉亲和素与dUTP 上的biot in 结合(每个链霉亲和素至少可以再结合3个biot in 分子),最后用DAB 过氧化氢与SP上的辣根过氧化物酶HRP发生氧化、环化反应,形成苯乙肼聚合物而呈现棕褐色,最终通过计数每张切片上不同视野中TUNEL阳性细胞的比例来判断细胞凋亡发生情 况。■ 1. TUNEL工作原理:简单说就是一一TUNEL细胞凋亡检测试剂盒是用来检测细胞在凋亡过程中细胞核DNA的断裂情况。 其原理是;生物素(biot in )标记的dUTP在脱氧核糖核苷酸末端转移酶(TdT En zyme)的 作用下,可以连接到凋亡细胞中断裂的DNA的3' - 0H末端,并可与连接了的辣根过氧化酶的 链霉亲和素(Streptavidin-HRP )特异结合,在辣根过氧化酶底物二氨基联苯胺(DAB的存在下,产生很强的颜色反应(呈深棕色),特异准确地定位正在凋亡的细胞,因而在普通 显微镜下即可观察和计数凋亡细胞;由于正常的或正在增殖的细胞几乎没有DNA的断裂,因而没有3'-0H形成,很少能够被染色。 针对问题3 (TUNEL实验中几个关键步骤是什么?): 1. 充分脱蜡和水化。脱蜡可以先60度20min,再用二甲苯两次5~10min ;而水化用梯度乙 醇从高浓度到低浓度浸洗,这些以便后面的结合反应充分、均匀; 2. 把握好细胞通透的时间。一般根据切片的厚薄,选择蛋白酶k的孵育时间,常用10~30min, 几um切片用短时间;几十um切片用长时间,通过摸索达到既不脱片,有能够使后面的酶和 抗体进入胞内。 3. 适当延长TUNEL反应液的时间。一般是37度1h,你也可以根据你的凋亡损伤程度,选择更长的时间,可长至2h,但要结合你最终的背景着色。 4. DAB显色条件的选择。一般DAB反应10分钟左右,结合镜下控制背景颜色,最长不超过 30min;我不喜欢用promega公司提供的DAB液(桃红色),不利于辨认棕褐色。 5. PBS的充分清洗。我个人认为,在TUNEL反应后和酶标反应后的清洗应十分严格,可增加 次数达5次,因为这些清洗直接决定最后切片的非特异性着色。 6. 此外,内源性POD的封闭也十分关键。对于肝脏、肾脏等血细胞含量多的组织,我的经 验是适当延长封闭时间和升高过氧化氢的浓度,可以达到很好的封闭效果,且不影响最终的 特异性染色。 针对问题5.细胞通透的原理、通透剂的浓度、孵育时间及其配制方法? 1. 蛋白酶K是消化膜蛋白,从而起打孔作用,增加

第17讲 细胞凋亡、细胞坏死和细胞癌变

第十七讲细胞凋亡、细胞坏死和细胞癌变 常见考法: 1、以选择题的形式考查细胞生命历程个过程的实质、特点。 2、以选择题的形式考查细胞凋亡和细胞坏死的区别。 1.1下列有关人体细胞生命历程的叙述,不正确的是( ) A. 细胞分化形成不同功能的细胞,但这些细胞也有相同的蛋白质 B. 衰老的细胞内多种酶活性降低,但有的基因还在表达 C. 癌细胞不能进行正常地分化,机体清除癌细胞与细胞凋亡有关 D. 细胞的分裂、分化、衰老和坏死对生物体均有积极的意义 1.2下列关于细胞分化、衰老、凋亡和癌变的叙述,正确的是( ) ①个体发育过程中细胞的衰老对生物体都是有害的 ②正常细胞癌变后在体外培养可无限增殖 ③由造血干细胞分化成红细胞的过程是不可逆的 ④癌细胞容易在体内转移,与其细胞膜上糖蛋白等物质减少有关 ⑤人胚胎发育过程中尾的消失是细胞坏死的结果 ⑥原癌基因和抑癌基因的变异是细胞癌变的内因 ⑦低温引起的细胞冻伤和死亡属于细胞凋亡. A. 1项 B. 2项 C. 3项 D. 4项 任务2、个体衰老和细胞衰老有什么关系?细胞衰老的特征和原因是什么?? 关系:单细胞生物体的衰老或死亡就是个体的衰老或死亡。但多细胞生物体总是在不断更新,总有一部分细胞处于衰老或走向死亡。总体来说,个体的衰老的过程也是细胞普遍衰老的过程。细胞衰老特征:一大(细胞核体积增大)一小(细胞体积变小)一多(色素增多)三低(水分减少;酶活性降低;膜通透性改变,运输功能降低)

细胞衰老原因:自由基学说和端粒学说 2.1下列有关衰老细胞特征的叙述,错误是( ) A、膜通透性改变,物质运输功能下降 B、酶活性降低,呼吸减弱,代谢减慢 C、染色质收缩,遗传信息表达受影响 D、细胞体积增大,水分增加,色素积累 任务3、细胞凋亡的含义、实例、原因、发生时期、意义? 细胞凋亡含义(课本P123) 实例:胎儿手的发育,蝌蚪尾巴的消失 原因:由遗传机制决定的程序性调控 时期:个体发育全过程 意义:保证多细胞生物体完成正常发育;维持内部环境的稳定;抵御外界各种因素的干扰。 3.1细胞凋亡大致过程如图所示.下列有关叙述不正确的是 ( ) A. 图示该过程只发生在胚胎时期 B. 与凋亡相关的基因是机体固有的 C. 吞噬细胞吞噬凋亡小体与水解酶有关 D. 细胞凋亡过程与基因的选择性表达有关 3.2下列有关衰老细胞特征和细胞凋亡的叙述,错误是( ) A: 细胞凋亡是由细胞内的遗传物质决定的 B 细胞的功能不同,凋亡的速率也不同 C: 细胞凋亡对生物体维持内部环境的稳定起关键作用 D: 在人体神经系统的发育过程中,神经细胞发生凋亡的数量很少 任务4、细胞癌变的主要特征?原癌基因和抑癌基因的功能是什么?细胞癌变的外因和内因是什么?癌症都是不治之症吗?是遗传病吗?癌变是可遗传的变异吗?如何防癌? 主要特征:①“不死”:无限增殖;②“变态”:形态结构改变;③“扩散”:糖蛋白减少,粘着性降低,易分散和转移 原癌基因和抑癌基因的功能(课本P126) 癌变外因:致癌因子;内因:原癌基因和抑癌基因发生基因突变 癌症是不治之症,不是遗传病,癌变是可遗传的变异。 防癌:远离致癌因子,健康的饮食和生活习惯,良好心态 4.1下列关于原癌基因和抑癌基因的说法,不正确的是( ) A. 两者和细胞周期的调控有关 B. 只有在癌细胞中才存在这两种基因 C. 原癌基因和抑癌基因突变过程中存在累积效应 D. 原癌基因的脱氧核苷酸序列改变可导致遗传信息的变化 4.2、2011年4月15日~21日是第17届全国肿瘤防治宣传周,主题为“科学抗癌,关爱生命”。下列相关叙述不正确的是( ) A、放射性治疗可以引发癌细胞凋亡

实验14-细胞凋亡的诱导和检测

实验14 细胞凋亡的诱导和检测 20世纪60年代人们注意到细胞存在着两种不同形式的死亡方式:凋亡(apoptosis)和坏死(necrosis)。细胞坏死指病理情况下细胞的意外死亡,坏死过程细胞膜通透性增高,细胞肿胀,核碎裂,继而溶酶体、细胞膜破坏,细胞容物溢出,细胞坏死常引起炎症反应。 细胞凋亡apoptosis一词来源于古希腊语,意思是花瓣或树叶凋落,意味着生命走到了尽头,细胞到了一定时期会像树叶那样自然死亡。凋亡是细胞在一定生理或病理条件下遵守自身程序的主动死亡过程。凋亡时细胞皱缩,表面微绒毛消失,染色质凝集并呈新月形或块状靠近核膜边缘,继而核裂解,由细胞膜包裹着核碎片或其他细胞器形成小球状凋亡小体凸出于细胞表面,最后凋亡小体脱落被吞噬细胞或邻周细胞吞噬。凋亡过程中溶酶体及细胞膜保持完整,不引起炎症反应。细胞凋亡时的生化变化特征是核酸切酶被激活,染色体DNA被降解,断裂为50~300 kb长的DNA片段,再进一步断裂成180~200bp整倍数的寡核苷酸片断,在琼脂糖凝胶电泳上呈现“梯状”电泳图谱(DNA Ladder)。细胞凋亡在个体正常发育、紫稳态维持、免疫耐受形成、肿瘤监控和抵御各种外界因素干扰等方面都起着关键性的作用。 1.细胞凋亡的检测方法 凋亡细胞具有一些列不同于坏死细胞的形态特征和生化特征,据此可以鉴别细胞的死亡形式。细胞凋亡的机制十分复杂,一般采用多种方法综合加以判断,同时不同类型细胞的凋亡分析方法有所不同,方法选择依赖于具体的研究体系和研究目的(表?)。

形态学观察方法:利用各种染色法可观察到凋亡细胞的各种形态学特征: (1)DAPI时常用的一种与DNA结合的荧光染料。借助于DAPI染色,可以观察细胞核的形态变化。 (2)Giemsa染色法可以观察到染色质固缩、趋边、凋亡小体形成等形态。 (3)吖啶橙(AO)染色,荧光显微镜观察,活细胞核呈黄绿色荧光,胞质呈红色荧光。凋亡细胞核染色质呈黄绿色浓聚在核膜侧,可见细胞膜呈泡状膨出及凋亡小体。 (4)吖啶橙(A())/溴化乙啶(EB)复染可以更可靠地确定凋亡细胞的变化,AO只进入活细胞,正常细胞及处于凋亡早期的细胞核呈现绿色;EB只进入死细胞,将死细胞及凋亡晚期的细胞的核染成橙红色。 (5)台盼蓝染色对反映细胞膜的完整性,区别坏死细胞有一定的帮助,如果细胞膜不完整、破裂,台盼蓝染料进入细胞,细胞变蓝,即为坏死。如果细胞膜完整,细胞不为台盼蓝染色,则为正常细胞或凋亡细胞。使用透射电镜观察,可见凋亡细胞表面微绒毛消失,核染色质固缩、边集,常呈新月形,核膜皱褶,胞质紧实,细胞器集中,胞膜起泡或出“芽”及凋亡小体和凋亡小体被临近巨噬细胞吞噬现象。 (6)木精-伊红(HE)染色是经典的显示细胞核、细胞质的染色方法,染色结果清晰。发生凋亡的细胞经HE染色后,其细胞大小的变化及特征性细胞核的变化:染色质凝集、呈新月形或块状靠近核膜边缘,晚期核裂解、细胞膜包裹着核碎片“出芽”凸出于细胞表面形成凋亡小体等均可明显显示出来。 DNA凝胶电泳:细胞发生凋亡或坏死,其细胞DNA均发生断裂,细胞小分子 质量DNA片段增加,高分子DNA减少,胞质出现DNA片段。但凋亡细胞DNA断裂点均有规律的发生在核小体之间,出现180~200 bp DNA片段,而坏死细胞的DNA断裂点为无特征的杂乱片段,利用此特征可以确定群体细胞的死亡,并可与坏死细胞区别。

细胞凋亡的几种检测方法

细胞凋亡的几种检测方法 1、形态学观察方法 (1)HE(苏木精—伊红染色法)染色、光镜观察:凋亡细胞呈圆形,胞核深染,胞质浓缩,染色质成团块状,细胞表面有“出芽”现象。 (2)丫啶橙(AO)染色,荧光显微镜观察:活细胞核呈黄绿色荧光,胞质呈红色荧光。凋亡细胞核染色质呈黄绿色浓聚在核膜内侧,可见细胞膜呈泡状膨出及凋亡小体。 (3)台盼蓝染色:如果细胞膜不完整、破裂,台盼蓝染料进入细胞,细胞变蓝,即为坏死。如果细胞膜完整,细胞不为台盼蓝染色,则为正常细胞或凋亡细胞。此方法对反映细胞膜的完整性,区别坏死细胞有一定的帮助。

(4)透射电镜观察:可见凋亡细胞表面微绒毛消失,核染色质固缩、边集,常呈新月形,核膜皱褶,胞质紧实,细胞器集中,胞膜起泡或出“芽”及凋亡小体和凋亡小体被临近巨噬细胞吞噬现象。 2、DNA凝胶电泳 细胞发生凋亡或坏死,其细胞DNA均发生断裂,细胞内小分子量DNA片断增加,高分子DNA减少,胞质内出现DNA片断。但凋亡细胞DNA断裂点均有规律的发生在核小体之间,出现180-200bpDNA片断,而坏死细胞的DNA断裂点为无特征的杂乱片断,利用此特征可以确定群体细胞的死亡,并可与坏死细胞区别。正常活细胞DNA 电泳出现阶梯状(LADDER)条带;坏死细胞DNA电泳类似血抹片时的连续性条带 3、酶联免疫吸附法(ELISA)核小体测定

凋亡细胞的DNA断裂使细胞质内出现核小体。核小体由组蛋白及其伴随的DNA片断组成,可由ELISA法检测。 检测步骤 1、将凋亡细胞裂解后高速离心,其上清液中含有核小体; 2、在微定量板上吸附组蛋白体’ 3、加上清夜使抗组蛋白抗体与核小体上的组蛋白结合‘ 4、加辣过氧化物酶标记的抗DNA抗体使之与核小体上的DNA结合’ 4、加酶的底物,测光吸收制。 用途 该法敏感性高,可检测5*100/ml个凋亡细胞。可用于人、大鼠、小鼠的凋亡检测。该法不需要特殊仪器,

相关主题