线粒体与细胞凋亡

细胞凋亡是一种由基因控制的细胞自杀性死亡过程,是机体维持自身稳定的一种基本生理机制。机体通过细胞凋亡清除损伤、衰老与突变的细胞,维持生理平衡。通过对细胞凋亡的研究发现,线粒体在调节细胞凋亡中发挥着关键作用。尤其是被称为细胞凋亡主开关的线粒体通透性改变孔(MitochondrionPermeabilitytransitionPore,MPTP),它是细胞色素C、Smac、AIF等凋亡诱导因子的主要来源。本文以MPTP的开放、细胞色素C、AIF在细胞凋亡中的作用以及Bcl-2家族对细胞凋亡的调控做一综述,以便广大读者能系统地了解线粒体在细胞凋亡中的作用。     

Effect of cyclophosphamide on proliferation and apoptosis of rat mesangial cells in vitro

AIM:To investigate the effect of cyclophosphamide(CTX) on proliferation and apoptosis of mesangial cells(GMC) of rat in vitro. METHODS:GMC proliferat ion was detected by MTT method,GMC apoptosis was examined by inverted microscopy for phase-contract and fluoroscopy and flow cytometry analysis.The levels of Fas and Bcl-2 were also detected by immunohistology. RESULTS:The proliferation of GMC were inhibited by CTX, methylprednisolone(MP), low molecular weight heparin(LMWH). Apoptosis of GMC was induced by CTX, the apoptosis rate of GMC was 8.2%, and the Fas level was increased. CONCLUSION:CTX could inhibit proliferation and induce apoptosis of GMC possibly by enhancing the Fas level.     

Induction of apoptosis with Salvia miltiorrhiza monomer IH764-3 via downregulating focal adhesion kinase in H2O2-stimulated hepatic stellate cells

AIM: To investigate the effect of IH764-3 on the apoptosis of H₂O₂-stimulated hepatic stellate cells(HSCs) and the alteration of focal adhesion kinase (FAK). METHODS: The proliferation of HSCs was examined by direct cell count and the apoptosis was determined by Annexin-V/PI labeling, while the morphological change was observed by light microscopy and transmission electron microscopy. In addition, FAK mRNA was detected by RT-PCR. RESULTS: H₂O₂ promoted the proliferation of HSCs. IH764-3 induced the apoptosis of HSCs in a dose-dependent manner. The HSC apoptotic rates of different groups were 6. 35%,9. 28%,15. 10%,19. 69%,respectively, after treated with different concentrations of IH764-3 for 48 h while H₂O₂ group showed 2. 30%. In 30 mg/L group, the apoptosis rates were 6. 73%、10. 34%、15. 10% for the indicated time periods(12 h, 24 h, 48 h). In the presence of IH764-3, FAK mRNA decreased. The FAK mRNA reduction began at 2 h after adding IH764-3. CONCLUSION: IH764-3 induced the apoptosis of HSCs. Down-regulating the expression of FAK mRNA may be one of its mechanisms.     

Effect of cocaine on caspase-3 in myocardiac cells of male rats in different age

AIM: To study the effect of cocaine on caspase-3 in myocardiac cells of male rats in different age. METHODS: Three-week-old(n=16), six-week-old(n=16) and twelve-week-old (n=16) male Sprague-Dawley rats were all divided into control groups and experiment groups randomly, each group had eight animals, experiment groups were given cocaine hydrochloride (15 mg·kg^-1 body weight) subcutaneously daily for four weeks. The ratio of heart weight to body weight (HW/BW, mg/g) were measured. DNA fragmentation of myocardia cells was determined by gel electrophoresis, and caspase-3 activity in myocardia cells was tested by flow cytometry (FCM). RESULTS: In three experiment groups, the DNA isolated from myocardial cells displayed clear ladder pattern. The HW/BW and the caspase-3 activity were increased significantly than those of control groups (P<0.01). CONCLUSIONS: Cocaine induced apoptosis in rat myocardial cells. The increase in caspase-3 activity may be the one of important pathways related to the induction of apoptosis.     

Influence of ischemia-reperfusion on apoptosis of sinoatrial node cells in rabbits in vivo

AIM:To study influence of ischemia-reperfusion(IR) on apoptosis and expression of apoptosis-related genes Fas-L, Bax and Bcl-2 of sinoatrial node(SAN) cells in rabbits in vivo. METHODS:Ninety healthy adult rabbits were divided randomly into control group, ischemia groups (I_{10 min}, I_{30 min}, I_{60 min} and I_{120 min}) and IR groups (I_{10 min}R_4h, I_{30 min}R_4h, I_{60 min}R_4hand I_{120 min}R_4h). IR injury model of SAN was established by occluding and loosening the start section of right coronary artery. The apoptosis of SAN cells was detected by TUNEL staining. The expression of Fas-L, Bax and Bcl-2 of SAN cells was detected by immunohistochemistry. RESULTS:①No obvious apoptosis of SAN cells was observed in control group, I_{10 min} and I_{30 min} groups. Apoptosis of different degrees in SAN cells were found in 68.3%(41/60) rabbits in I_{60 min}, I_{120 min} and 4 subgroups of IR. ②The highest expression of Fas-L and Bax was observed in I_{120 min} group and that of Bcl-2 was in I_{60 min} group. ③The highest expression of Fas-L and Bax was observed in I_{60 min}R_4h group. The peak level of Bcl-2 was observed in I_{30 min}R_4h group. ④The expression of Fas-L and Bax was significant higher in IR group than that in ischemic group at the same time point. CONCLUSION:Ischemia and IR induced apoptosis of SAN cells in rabbit in vivo. Fas-L、Bax、Bcl-2 may participate in the regulation of apoptosis and the injury during IR aggravates the apoptosis of SAN cells.     

Effect of Yigu capsule drug-containing serum on apoptosis and tartrate resistant acid phosphatase secretion in rat osteoclasts

AIM:The effects of Yigu capsule on tartrate resistant acid phosphatase (TRAP) secretion and apoptosis in rat osteoclasts were investigated in order to further explore its mechanism of preventing and treating osteoporosis.METHODS:(1) Twenty-month-old Sprague-daweley rats were randomly divided into two groups(Yigu capsule group and saline group), and the drug-containing serum and control serum were prepared. (2) The newborn Sprague-daweley rat osteoclasts were cultured with different concentrations of Yigu capsule drug-containing serum. At different time point, TRAP activity was measured and the survival osteoclast was counted under reverse microscope.The percentage of osteoclast apoptosis was observed under fluorescence microscope after acridine orange staining.RESULTS:TRAP activity was lower and the percentage of osteoclast apoptosis was higher in drug-containing serum group than in control group at 24, 48 and 72 h(P<0.01), respectively, and the survival osteoclasts were less in drug-containing serum group than in control groups at 24, 48 and 72 h(P<0.01).CONCLUSIONS:These data suggest that Yigu capsule drug-containing serum induces apoptosis and inhibits TRAP activity in osteoclasts, which may be one of the mechanisms of Yigu capsule preventing and treating osteoporosis.     

Preparation of gfp-bcl-XL-contained recombinant adenovirus vector by the homologous recombination in bacteria

AIM: To prepare gfp-bcl-X_L-contained recombinant adenovirus(rAd-gfp-bcl-X_L).METHODS: Bcl-X_L gene was amplified from pEGFP-C₃-bcl-X_L, subcloned into shuttle plasmid and formed transfer plasmid of pAdTrack-CMV-bcl-X_L. Then pAdTrack-CMV-bcl-X_L was linealinzed with PmeI and co-transformed into BJ5183 bacteria with adenovirus genomic plasmid of pAdEasy-1. The identified recombinant adenovirus plasmid was digested with PacI and transfected into 293 cells to package recombinant adenovirus particles. The target gene was detected by PCR.RESULTS: There were about 35% positive recombinant bacterial clones after the co-transformation of pAdTrack-CMV-bcl-X_L and pAdEasy-1 into BJ5183. Recombinant adenovirus particle were produced and further amplified after the transfection of pAdEasy-1-gfp-bcl-X_L into 293 cells. PCR test indicated that the recombinant Ad contained bcl-X_L gene. The titer of the purified rAd-gfp-bcl-X_L was 6.5×10¹² PFU/L. CONCLUSIONS: The homologous recombination in bacteria is a convenient and high efficient method to prepare rAd-gfp-bcl-X_L. This affords a good gene transfer vector for the gene therapy in human’s diseases.     

Expression of alpha-smooth muscle actin in scar tissue and its relationship with apoptosis

AIM: To examine the expression of alpha-smooth muscle actin in scar tissue, and observe the phenomenon of apoptosis and its involvement in the process of pathological scarring and the presence of myofibroblasts or absence of cell in the dermis. To investigate the potential role of reparative cell apoptosis in hyperplastic scar formation. METHODS: The samples of scar were obtained from post-burn patients undergoing plastic operation in our burn unit recently, and the samples of control came from skin donor site of the same patient correspondingly. TUNEL assays were performed to evaluate the number of apoptotic cells in scar versus normal skin. In situ hybridization and immunohistochemistry staining technique were employed to determine the expression of different dermis cells markers in scar tissue and normal skin. RESULTS: There existed evident difference in apoptotic cells in the dermis between scars tissue and normal human skin. The expression positive cells were much more in hyperplastic scars than that in normal human skin; the apoptotic cells of proliferative stage were slight more than that of mature stage. However, in proliferative stage, the number of apoptotic cells was higher for the combination of hyperplastic scar than normally healed flat scars. But in mature stage, no obviously difference was detected between hyperplastic scar and normally healed flat scar. The monoclonal anti-α smooth muscle actin (ASMA) expression was significantly stronger in proliferative stage than that of mature stage. CONCLUSIONS: With reconstitution of dermal tissue, myofibroblasts containing alpha-SM actin disappear under normal wound healing, probably as a result of apoptosis. The myofibroblast play a critical role in wound closure and in the pathologic sequelae of healing.     

Effects of riboflavin and ascorbic acid on apoptosis and proliferative inhibition of mouse thymocytes induced by deoxynivalenol in vivo

AIM: To explore the effects of riboflavin and ascorbic acid on the apoptosis induced by deoxynivalenol(DON) in mouse thymocytes. METHODS: The effects of riboflavin and ascorbic acid on the apoptosis and proliferation inhibition of thymocytes induced by DON in KM mice were studied with animal experiment, DNA agarose gel electrophoresis and flow cytometric DNA content analysis. RESULTS: Apoptosis rate of thymocytes in DON (4 mg/kg) treated group was13.73%±15.3% The percentages of apoptosis in riboflavin (1.25 mg/kg-10mg/kg) and ascorbic acid (25 mg/kg-100mg/kg) pretreated thymocytes groups were significantly lower than that in DON group (P <0.05). The result of DNA agarose gel electrophoresis showed that the characteristic ladder pattern of apoptosis was found in DON-treated thymocytes, but not in control and riboflavin pretreatment and ascorbic acid pretreatment groups The significant differences in proliferation index were not found among DON-treated thymocytes and riboflavin and ascorbic acid-pretreated thymocytes CONCLUSION: Pretreatment with riboflavin and ascorbic acid inhibit apoptosis of mouse thymocytes induced by DON in certain extent and have no effect on proliferation inhibition by DON.     

IL-12 induces T lymphocytes apoptosis and influences the expression and signal transduction of Fas/FasL

AIM: IL-12 acts upon Tlymphocytes and activates its receptor complexes of β1/β2,and so IL-12 can regulate TH1/TH2 balance. Our study is aimed at IL-12-inducing apoptosis of Tcells and expression and signal transduction of Fas/FasLduring Tcell apoptosis. METHODS: The apoptosis of Tcells was detected by Annexin Vstaining cytometry and the expression of Fas/FasLunder different inhibitors were detected by semi-quantitative PCR. RESULTS: IL-12 induced the human leukemic Tcell line(TIB-152) and the human lymphoma Tcell line(HTB-176) and the normal human Tcells to undergo apoptosis. The FasLexpression at 6 hours after treatment with IL-12 increased apparently, and reached the max at 24 hours, and FasLexpression induced by IL-12 was inhibited by PKCinhibitor. But IL-12 did not influence Fas expression. CONCLUSIONS: IL-12 can induce Tcells to undergo apoptosis which is characterized by early membrane changes, the inducing effect is correlated with the concentration of IL-12 and the maturation of Tcells. FasLparticipates in the progression of Tcell apoptosis as a apoptosis mediator, and the effect of IL-12 on FasLexpression may be related with PKCpathway.