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AIM: To investigate the effect of ischemic preconditioning (IP) on myocardial Bcl-2 expression and mitochondrial structure during heart valve replacement surgery under cardiopulmonary bypass. METHODS: Fifty-four patients were prospectively randomized to receive or not ischemic preconditioning (IP) before cold cardioplegic arrest. Ischemic preconditioning in the IP patients (n=22) was induced by a single 2-min ischemia followed by 3-min reperfusion just before aortic clamping and cold crystalloid cardioplegia for myocardial protection. The control group (n=32) received no ischemic preconditioning before cold cardioplegic arrest. The levels of ejection fraction (EF), fractional shortening(FS) and stroke volume (SV) in both groups were measured and compared. troponin T (c-TnT) level, Bcl-2 protein expression and microscopic changes of myocardial mitochondrial structure were recorded for each group before and after surgery. RESULTS: The level of EF, FS and SV in IP group was higher than those in control group (P<0.05). No significant difference in preoperative c-TnT levels between two groups was observed. The level of c-TnT in IP group was lower than that in control group and with a declining trend over time of 6 h, 24 h, 48 h, 72 h and 5 d after surgery, respectively. The preoperative positive unit of Bcl-2 expression between two groups showed no statistical difference (P> 0.05). Postoperatively, the positive unit of Bcl-2 expression in IP group was 19.85±5.88, significantly increased as compared to the preoperative value (P<0.05). In control group, the positive unit of Bcl-2 expression was 14.17±3.39, showed no statistically significant difference to the preoperative value (P>0.05). Postoperative Bcl-2 expression between two groups showed a significant difference (P<0.05). In the control group, microscopic observation revealed swollen mitochondrion, with a hardly visible or disrupted membrane for some mitochondrion;mitochondrial crista were obviously dissolved and loose with a large number of vacuoles formation. However in IP group, myocardial mitochondrion appeared with intact membrane, concentrated mitochondrial cristae with high electron density and no vacuoles formation was observed. CONCLUSION: IP may up-regulate the expression of myocardial anti-apoptotic protein Bcl-2 to protect the mitochondrion, thus protecting cardiocytes and cardiac functions.
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Objective: To investigate the effect of cinobufacini on apoptosis of U937 cells and its possible mechanism. Methods: Cell viability was measured by MTT assay. The morphological changes were observed by Wrightś staining and immunofluorescence staining. DNA fragmentation was visualized by agarose gel electrophoresis. Apoptotic rate was evaluated by teminal deoxynucleotidyl transferase (TdT) labeling in situ. Expression of bel-2 protein was analyzed by flow cytometry. The levels of Fas and Fas-1 mRNA were measured by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Results: Compared with the control group, treatment with cinobufacini at 0.225 to 1.8 μg/ mL for 1-3 d significantly inhibited growth of U937 cells in a time and dose dependent manner. The IC50 value was 1.36 μg/ mL at 24 h. The typical morphological changes of apoptosis and typical apoptotic DNA ladder were observed after incubation with cinobufacini at 0.9 μg/mL for 1 d. The apoptotic rate evaluated by TdT labeling in situ were 4.8%, 13.57%, and 24.33% after exposure to cinobufacini at 0.225, 0.45, and 0.9 μg/ mL for 1 d, respectively. The expression levels of bcl-2 protein and Fas-1 mRNA significantly decreased and the expression of Fas mRNA significantly increased in apoptotic cells. Conclusions: Cinobufacini inhibits growth and inducs apoptosis of U937 cells via inhibition of bcl-2 and Fas-1 genes and activation of Fas gene.
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Objective To observe the changes in Bcl-2 protein expression in rat kidney tissue after severe burn with delayed fluid resuscitation in areas of different altitude,and explore the relationship between its expression and cell apoptosis.Methods The experiments were performed at two altitudes(1 517m and 3 848m).A 30% TBSA Ⅲ? scald model was reproduced with 240 male Wistar rats(120 rats for each altitude).Rats were randomly assigned into delayed fluid resuscitation group(DFR,n=50),immediate fluid resuscitation group(IFR,n=60) and normal control group(NC,n=10).Renal tissue samples were harvested at 1,6,12,24,72 and 168 hours after scald,respectively.The cell apoptosis was detected by tissue chips technic and terminal uridin nick end labeling(TUNEL).The expression of Bcl-2 was detected by immunohistochemistry and image analysis.Results In higher altitude,cellular edema,granular degeneration,necrosis and disintegration of renal tubular epithelial cells;dilation and engorgement of renal glomerular capillaries,degeneration and necrosis of endothelial cells,and congestion,edema and inflammatory cell infiltration in renal interstitium were found.The pathological changes were more serious in DFR group than that in IFR group,and they were worse in the rats at 3 848m altitude than that in those rats at 1 517m altitude.The levels of cell apoptosis and Bcl-2 protein expression were higher in DFR group than that in IFR group,and in the rats at 3 848m altitude than that in those rats at 1 517m altitude(P
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Objective:In order to study the correlation between The expression of CD44v6 protein and Bcl-2 protein in thyroid cancer were examined.Methods:The expression of CD44v6 and Bcl-2 in 50 thyroid cancers,20 adjacent noncancerous portion,45 adenoma and 10 normal thyroid tissue were respecitively investigated by catalyzed signal amplification immunohistochemical technique. Results:The positive rate of CD44v6 and Bcl-2 in thyroid cancer was 64.0% and 46.0%, which was significantly higher than that in adenoma or adjacent noncancerous ( P
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AIM:To evaluate the antitumor effect of caffeic acid Ge on U14 tumor bearing mice.METHODS:The tumor inhibitory ratios of caffeic acid Ge on the growth of U14 in mice was observed.Apoptosis morphological transformation of U14 cells induced by caffeic acid Ge was detected by electronic scan microscope and MG-P staining.Alteration of cell cycle was analyzed by flow cytometry.Apoptosis-related protein levels of Bax and Bcl-2 were determined by immunity histochemistry technology.MTT assay was applied to study the antitumor activities of caffeic acid Ge in U14 cell lines in vitro.RESULTS:Tumor inhibitory rates in caffeic-acid Ge groups were 38.50%,47.17% and 64.02%(from low dose to high dose)(P
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AIM: To investigate the apoptosis and Bcl-2/Bax expression in the early follicles of women at reproductive age. METHODS: 12 ovarian specimens were collected from reproductive women (aged 23-38 years) undergoing gynaecological operation. Histopathological examination of these specimens was performed to confirm its' morphological normalities. Using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay and immunohistochemistry method, cell apoptosis and Bcl-2/Bax expression were examined in the early follicles including mainly primordial, intermediary and primary follicles. RESULTS: 18.75% of the oocytes were found TUNEL positive in the early follicles, but no granulosa cells in these follicles were found TUNEL positive. Bax expression was detected in 76.07% of the oocytes in the early follicles, but Bcl-2 expression was negative in these oocytes. In addition, Bcl-2/Bax expression were not present in the granulosa cells in early follicles. CONCLUSION: The oocyte apoptosis occurs in the early follicles of reproductive woman, and pro-apoptotic protein Bax may play a role in regulating this process. It suggests that Bax mediated oocyte apoptosis may be the molecular mechanism of the early follicle atresia in the ovaries of reproductive woman. [
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AIM: To probe into the role of 1,4,5-trisphosphate inositol(IP3) and bcl-2 gene expression in inhibiting hepatocellular carcinoma of nude mice by genistein.METHODS: Animals with hepatocellular carcinoma were treated with genistein 1 mg?kg-1?d-1(ip) for 3 weeks.The volume and weight of tumaor were measured.IP3,bcl-2 mRNA,Bcl-2 protein were assayed by IP3- Birtrak assay,RT-PCR,Western blotting,respectively.RESULTS: The tumor volume and weight of animals treated with genistein were lower than those in control(42.7mm3?27.8mm3 vs 52.3mm3?26.5mm3,42.7mg?27.8 mg vs 91.3mg?31.4 mg).IP3 content was lower than that in control [(13.4?1.4)nmol/g protein vs(35.3?6.6)nmol/g protein].bcl-2 mRNA expression was lower in group treated with genistein than that in control(RI which was the gray degree multiply area of bcl-2 / the gray degree multiply area of ?-actin 0.48?0.02 vs 0.56?0.15).Bcl-2 protein expression was lower in group treated with genistein than that in control(RI 1.69?0.52 vs 1.37?0.48).CONCLUSION: Genistein inhibits growth of transplanted hepatocellular carcinoma in nude mouse liver by reducing IP3 production and down-regulating bcl-2 gene expression.
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BACKGROUND: There has been a growing realization that a variety of anticancer drugs can induce apoptotic cell death. In the present study, an attempt was made to investigate the responsiveness of gastric cancer cells to various anticancer drugs and to identify which apoptosis-related proteins could be correlated to chemosensitivity. METHODS: Nine human Korean gastric cancer cell lines (SNU-1, -5, -16, -484, -601, -620, -638, -668, and -719) were analyzed. The cytotoxicity of each cell line to camptothecin, cisplatin, mitomycin C, vincristine, 5-FU, epirubicin, and doxorubicin was determined by using a MTT (dimethylthiazole- diphenyltetrazolium-bromide) assay. Apoptosis-related proteins (p53, p21, Bcl-2, Bcl-x, and Bax) were detected using a Western blot assay. RESULTS: Of the nine gastric cancer cell lines, SNU-1 was resistant while SNU-5 was sensitive to anticancer drugs. Mutated p53 was detected in all the cell lines. The highest expression of Bcl-2 was observed in SNU-1 while less or no expression of Bcl-2 was observed in SNU-5, -484, and -601. Bcl-xL was less expressed in SNU-5 than in the other cell lines. CONCLUSIONS: Chemosensitivity in gastric cancer cell lines was correlated mainly with the level of Bcl-2 and partly with that of Bcl-xL. There was no correlation between the chemosensitivity and other apoptosis-related proteins, such as p21, p53, Bax, and Bcl-xS in the studied gastric cancer cell lines.
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Humans , Blotting, Western , Camptothecin , Cell Death , Cell Line , Cisplatin , Doxorubicin , Epirubicin , Fluorouracil , Mitomycin , Stomach Neoplasms , VincristineABSTRACT
Objective: To investigate the expression of Bcl-2, the apoptosis inhibitor, and Bax, the apoptosis promoter in hepatocellular carcinomas (HCCs). Methods: 35 formalin-fixed and paraffin-embedded samples of histologically confirmed HCC, were examined using immunohis to chemical staining for Bcl-2 and Bax protein. Results : 7 of 35 HCCs were positive for Bcl-2 protein and 13 of 35 positive for Bax protein. Immunoreactivity for Bcl-2 protein was present in 24 of 35 HCC cases and Bax protein in 25 of 35 cases. Furthermore, all positive cells were cytoplasmic stained. 6 of 7 Bcl-2 protein positive HCCs were (histological grade Ⅱ ), Bax protein expression had little association with histological grading. Co-expression of Bcl-2 and Bax protein was only present in 4 cases. At any rate, a significant high Bcl-2 and Bax protein expression was detected in small HCCs. Also, Bcl-2 protein expression tended to decrease with clinical stage. Conclusion: Bcl-2 and Bax immunohistochemical expression in HCCs seems to be associated with histological grading. Also, Bcl-2 protein seems to be relative to tumors with good prognosis. It supports the hypothesis that Bcl-2 expression has prognostic value.
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AIM:To study the senescence of human umbilical vein endothelial cells(HUVECs) and Bcl-2,Bax gene expression associated with apoptosis induced by angiotensinⅡ(AngⅡ).METHODS:HUVECs were cultured in vitro and the cell viability was observed by methyl thiazolyl tetrazolium(MTT).HUVECs were intervened by AngⅡ and valsartan(AngⅡ type 1 receptor blocking) and divided into 3 groups:the control group,AngⅡ group(stimulated with AngⅡ10-6mol/L for 48 h),valsartan group(valsartan was added to cells 1 h before 10-6mol/L AngⅡ treatment).?-gal staining aod cell cycle analysis were used to identify the cell aging status.Morphologic changes and percentage of apoptosis were assayed with Hoechst33258 under fluorescent microscope.The expressions of Bcl-2 and Bax,and the apoptosis-associated genes were detected by immunocytochemical staining,RT-PCR and Western blotting.RESULTS:The cell viability by AngⅡ-induced cells was(81.9%?4.1)%,the positive cell number of ?-gal staining was significantly higher in AngⅡ-induced cells(80.10%?6.81)% than that in the control cells.The cell cycle was at G0-G1(91.36%?6.45)%,the apoptotic cells significantly increased(31.84?2.86)% under fluorescent microscope.In valsartan group,Bcl-2 mRNA and protein expression increased markedly(P
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AIM:To observe the protective effect of Nano-Se on myocardium of experimental diabetes mice.METHODS:Sixty healthy male KM mice were chosen,ten of which were selected randomly as the normal control group.After fasted for 24 h,the rest 50 mice were injected with streptozotocin(STZ,50 mg/kg)intraperitoneally for 5 d.At 7th d,the blood-sugar was measured from vena caudalis,40 mice,of which blood-sugar exceeded 16.65mmol/L,were selected and randomized into 4 groups:the positive control group,low dose(25 ?g/kg)Nano-Se group,mid dose(50 ?g/kg)Nano-Se group,high dose(50 ?g/kg)Nano-Se group.All mice were given intragastric administration of 0.2 mL normal saline and corresponding dose of Nano-Se.The body weights were measured every week,and the dose of which was adjusted according to the change of the body weights.8 weeks later,the mice were killed and cardiac muscle of the left ventricle was taken.The myocardium was prepared to 10% homogenate for measuring SOD,GSH-Px activity and MDA content.The myocardial cell apoptosis was measured by TUNEL.The expressions of Bc1-2 and Bax proteins were determined by immunohistochemistry.RESULTS:Compared to normal group,the SOD and GSH-Px activities in positive control group decreased,MDA level increased,the rate of myocardial cell apoptosis increased significantly,Bc1-2 protein expression deceased and Bax protein expression increased.Compared to positive control group,the SOD and GSH-Px activities in low and mid dose Nano-Se groups expression increased,MDA level decreased,myocardial cell apoptosis rate decreased,Bc1-2 protein expression increased and Bax protein expression decreased.Moreover,the SOD and GSH-Px activities in high dose Nano-Se group decreased obviously compared to those in mid dose Nano-Se group.MDA level and myocardial cell apoptosis rate increased,Bc1-2 protein expression decreased and Bax protein expression increased,no significant difference in SOD,GSH-Px activity,MDA level and myocardial cell apoptosis rate was observed compared with positive control group.CONCLUSION:The damage of cardiac muscle is alleviated when a certain dose of Nano-Se is supplied to diabetes mice.The protective mechanism may be related to antioxidation,blood-sugar adjustment and the increase of Bc1-2 expressing.
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AIM: To investigate the role of mitochondrial ceramidase in mitochondrial functions, especially in the regulation of apoptosis. METHODS: pCDNA3.1/His-MtCDase plasmid, containing mitochondrial ceramidase cDNA sequence, was transfected into K562 cells by liposome, and G418 was used to screen the positive clones. A stable transfected K562 cell line was established and defined as ‘K562TC’. The differences between K562 and K562TC cells in serum withdrawal resistance and Bcl-2 protein expression were evaluated by annexin V/PI test, flow cytometry and Western blotting, respectively. RESULTS: K562TC cells with elevated Bcl-2 protein expression level identified by FCM or Western blotting showed stronger resistance to apoptosis induced by serum withdrawal than their parental cells. Inhibition of mitochondrial ceramidase expression in K562TC cells by its specific antisense oligodeoxynucleotide was correlated with a decrease in Bcl-2 protein level. N, N'-dimethylsphingosine (DMS), a sphingosine kinase inhibitor, depleted intracellular sphingosine-1-phosphate (SPP) production, also abrogated Bcl-2 protein expression in K562TC cells, while exogenous sphingosine-1-phosphate up-regulated Bcl-2 protein level in K562 cells. CONCLUSION: Mitochondrial ceramidase overexpression in K562 cells leads to markedly elevated level of Bcl-2 protein and results in more resistance to serum withdrawal. This effect is initiated not by sphingosine, the direct metabolite of mitochondrial ceramidase, but via sphingosine-1-phosphate, its phosphorylated form, indicating that mitochondrial ceramidase, through its sphingoid metabolite sphingosine-1-phosphate, up-regulates Bcl-2 protein expression in K562 cells.
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AIM: To investigate the influence of bone marrow stromal cells (BMSCs) on the anoxia cardiomyocyte apoptosis. METHODS: Using the anaerobic culture apparatus, the apoptosis of the cardiomyocytes, the BMSCs alone and co-cultured with each other were detected by morphological observation, PI staining flowcytometry, electrophoretic gel mobility analysis of DNA fragmentation. Western blotting was used to detect Bax and Bcl-2 protein expression. RESULTS: Compared with control, the BMSCs were unsensitive to anoxic cultured while the anoxic cardiomyocytes were prone to apoptosis. Apoptosis of cardiomyocytes was increased significantly, detected by PI staining and agarose gel elestrophoresis showed “DNA ladder”. However, when anoxia cardiomyocytes co-cultured with BMSCs, apoptosis cells were decreased, “DNA ladder” disappeared and the expression of protein Bax was also decreased. CONCLUSION: Bone marrow stromal cells prevent the anoxia cardiomyocytes from apoptosis, probably by suppressing the expression of Bax protein.
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AIM:To compare the effect of 1,2-propanediol (PROH) and ethylene glycol (EG) on apoptosis and expressions of P53,Bcl-2 of follicles in human ovarian tissue,in order to offer experimental foundation for selecting the best cryoprotectant. METHODS:Biopsies of ovarian tissue obtained from 12 women were cryopreserved,and ovarian tissue slice from each woman was divided into three groups:fresh control group,ethylene glycol group and 1,2-propanediol group. The slow-freezing /rapid-thawing protocol was used to freeze and thaw the slice of ovarian cortex. Apoptosis of follicles in fresh and frozen-thawed ovarian cortical tissue was detected by TUNEL experiment,and expressions of P53 and Bcl-2 were detected by immuno-histochemistry. RESULTS:The percentages of apoptosis follicles were 14.58%,23.08%,30.43% in fresh control group,PROH group and EG group,respectively,and the percentage of apoptosis follicles in EG group was higher than that in fresh control group (P0.05). The percentages of P53 positive follicles were 13.48%,25.00% and 33.93%,respectively. There was significant difference between fresh control and EG group (P0.05). CONCLUSION:The results of this study indicate that 1.5 mol/L PROH is more suitable for cryopreservation of human ovarian tissue rather than 1.5 mol/L EG in slow-freezing /rapid-thawing protocol. The protocol of slow-freezing/ rapid-thawing may preserve oocytes well,but it is not ideal for cryopreservation of granulosa cells. Cryopreservation may influence on apoptosis of follicles in human ovarian tissue.
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AIM:To investigate the apoptotic pathway of MCF-7 breast cancer induced by the grub extract in vitro.METHODS:MTT assay was used to determine the effect of the grub extract on proliferation of MCF-7 human breast cancer cell line and cell toxicity.Morphological changes of the apoptosis in cancer cells were observed by HE staining through invert microscope,light microscope,AO/EB double fluorescent staining under fluorescent microscope.FCM was used to assay the change of apoptotic rate.The expression of Bcl-2,Fas,caspase-9,caspase-3 in apoptotic pathway was detected with immunocytochemical method before and after exposure to the grub extract,and the effect of that on apoptotic pathway was explored.RESULTS:(1)The MTT test showed that the growth of MCF-7 human breast cancer cell line was significantly inhibited by the grub extract in dose and time dependent manners.The inhibitory rate in exposure group was significantly different from that in control group(P
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AIM: To investigate the inhibitory effect of arsenic trioxide on human glioma cell line U251 growth, change of gene expression and intracellular calcium content. METHODS: MTT method was used to observe the growth inhibition effect. Cell cycle, positive rate of proliferation cell nuclear antigen (PCNA), apoptosis associated protein Fas and Bcl-2, and intracellular calcium ion (IECa~ 2+ ) levels were measured by flow cytometry in U251 cells treated with different doses of As_2O_3. Apoptosis was detected with annexin V-FITC+PI dual parameter. RESULTS: As_2O_3 inhibited the growth of U251 cells dramatically. There were obvious dosage-effect and time-effect correlations (P0.05). The cell cycle was arrested in G_2M phase. Apoptosis occurred in U251 cells treated with As_2O_3 by annexin V-FITC+PI dual parameter detection. CONCLUSION: As_2O_3 inhibits the growth of U251 cells in vitro dramatically and induces apoptosis. The mechanism is probably associated with the improvement of Fas expression and IECa~ 2+ levels, decrease in PCNA protein expression and cell cycle arrest.
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AIM: To observe the effect of ginkgolide B (GB) on glutamate-induced apoptosis in the cultured neurons of rat retina. METHODS: Neurons of rat retina were cultured and apoptosis was induced by glutamate. The neurons were cultured with different concentration of GB and the survival rate was monitored by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The apoptosis in the cultured neurons and the expression of Bcl-2 and Bax were observed by flow cytometry. RESULTS: After exposed to glutamate, the survival rate and the number of Bcl-2 positive cells obviously decreased. At the same time, the number of Bax positive cells obviously increased, and the number of the apoptotic cells also obviously increased. Such phenomena were relieved by the treatment of ginkgolide B, with raise of survival rate and the expression of Bcl-2. Meanwhile, the expression of Bax and the apoptosis of neurocytes obviously decreased. CONCLUSIONS: Ginkgolide B protects retinal neurons from the virulence induced by glutamate. Such effects of GB might be brought about by increasing the expression of Bcl-2 while decreasing Bax, resulting in increasing the ratio of Bcl-2 to Bax and so reducing the apoptosis in the cultured neurons of rat retina.
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AIM: The cytoplasm Ca 2+, expression of Bcl-2 and Bax and germ cell apoptosis of testes in adult male rats with varicocele were observed to investigate the relationship between apoptosis and infertility. METHODS: Thirty-five healthy male Wistar rats were divided into two groups randomly: varicocele group (VG, n=20) and sham operation group (SOG, n=15). Bilateral testes were removed after ten weeks. One part of it was homogenized, the other part was performed for tissue sectioning. The atomic absorption method was used to detect the cytoplasm Ca 2+. The germ cell apoptosis was detected by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) technique. The expression of Bcl-2/Bax was detected by immunohistochemical method. RESULTS: The cytoplasm Ca 2+ in testes of rats with varicocele was decreased obviously. The apoptosis indexes of germ cells and Bax expression in VG were significantly increased as compared with those in the SOG, while Bcl-2 expression was obviously decreased. CONCLUSION: The results showed that apoptosis was increased in testes of VG rats, suggesting that male infertility with varicocele may be caused by some apoptosis agents such as high temperature, testes toxin and oxygen free radical. The cytoplasm Ca 2+ and change of expression of Bcl-2/Bax may contribute to germ cell apoptosis and cause male infertility.
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AIM: To observe the effect of crocetin on the apoptosis and the changes of its related regulating proteins caspase-3 and Bcl-2 expression induced by hydrogen peroxide (H_2O_2) in cultured cardiomyocytes. METHODS: Changes of cellular morphology were detected under microscope. Apoptosis rates of the cells were analyzed by PI staining with flow cytometry. Expressions of caspase-3 and Bcl-2 proteins in the cells were determined by immunofluorescence with flow cytometry. RESULTS: In the concentrations used, more severe morphological changes with higher apoptosis rate of the cultured myocardial cells were seen in each H_2O_2 group than that in control group. When treated with 1?10 -4 mol?L -1 H_2O_2, the caspase-3 was increased and Bcl-2 protein decreased remarkably in the cells. But each dosage of crocetin, especially the highest one (5?10 -5 mol?L -1, P
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AIM: To study the effects of M. vaccae on eosinophil apoptosis and Bcl-2 protein expression in lung tissues of asthmatic guinea pigs. METHODS: 30 guinea pigs were divided into normal saline (NS) group, asthma group and M. vaccae treatment group at random, every group included 10 guinea pigs. Guinea pigs in M. vaccae treatment group were injected intramuscularly with 22 5 ?g M. vaccae 10 days before OVA immunization. TdT-mediated dUTP nick end labeling (TUNEL) technique was used to investigate the apoptosis of eosinophils and immunohistochemistry method was used to study the expression of Bcl-2 protein in lung tissues. RESULTS: The apoptosis index (AI) of eosinophils in lung tissues in M. vaccae treatment group was significant higher than that in asthma group (23 78?5 42)% vs (4 56?0 68)%, P