大鼠心肌缺血再灌注时心肌细胞凋亡caspase-3活性变化规律及药物对其影响

目的　探讨大鼠心肌缺血再灌注 (Ischemiareperfusion ,IR)不同时相的心肌细胞凋亡、caspase 3活性变化规律及caspase 3抑制剂Ac DEVD CHO的影响。方法　Wistar大鼠 12 2只 ,设立IR组 ,IR +Ac DEVD CHO组和假手术对照组并分设缺血 3 0min后再灌注 1、3、6、12、2 4h 5个时相点 ;以缺口末端标记法 (TUNEL)标记凋亡细胞 ,用荧光分析法检测caspase 3活性 ,行TTC染色测定心肌梗死范围。结果　心肌细胞凋亡与caspase 3活性随心肌再灌注不同时相而变化 ,心肌细胞凋亡指数 (Apoptosisindex ,AI)与caspase 3活性于再灌注 12h最高 [AI :( 3 4 83± 9 3 5 ) % ;caspase 3活性 :( 1 3 4±0 2 ) ] ,其后基本维持在平台状态 ;心肌梗死范围随IR时间逐渐增加 ,至 2 4h仍未见下降趋势 ,三者间呈显著正相关 (P <0 0 5 )。IR +Ac DEVD CHO组上述指标虽也明显增高 ,但比IR组明显减小 (P <0 0 5 )。结论　Caspase 3激活及心肌细胞凋亡参与了心肌缺血再灌注损伤过程 ,Ac DEVD CHO减轻心肌缺血再灌注损伤可能部分与其抑制心肌细胞凋亡有关。     

Caspase-dependent and -independent DNA fragmentation in Sertoli and germ cells from men with primary testicular failure: relationship with histological diagnosis

BACKGROUND: Germ cell elimination and sperm DNA fragmentationin men with primary testiculopathies involve apoptosis-relatedprocesses whose mechanisms are poorly understood. This studyexamines the participation of typical (caspase-dependent) andatypical (caspase-independent) pathways in these processes.METHODS: Caspase activity and DNA fragmentation were evaluatedin Sertoli and germ cells from 63 men with non-obstructive azoospermiaand with different histological diagnoses who were undergoingtesticular biopsy for an assisted reproduction attempt. In eightof these men, phosphatidylserine externalization was also examined.RESULTS: The percentage of Sertoli cells showing caspase activityand DNA fragmentation was low and uniform in all diagnoses.In germ cells that remained tightly associated with Sertolicells despite vigorous mechanical treatment, the incidence ofboth caspase activity and DNA fragmentation was high, particularlyin men with maturation arrest. In Sertoli cell-free germ cells,high incidence of DNA fragmentation contrasted with low incidenceof caspase activity and phosphatidylserine externalization.CONCLUSIONS: In men with primary testicular failure, apoptosisof Sertoli cells is insignificant. Some germ cells undergo caspase-dependentapoptosis, show phosphatidylserine externalization and are tightlyassociated with Sertoli cells. Other germ cells show caspase-independentDNA fragmentation, do not externalize phosphatidylserine andlack a tight association with Sertoli cells.     

凋亡抑制蛋白XIAP和促凋亡因子Smac在胰腺癌化疗抵抗中的作用

目的探讨x-相关凋亡抑制蛋白（XIAP）和促凋亡因子Smac在胰腺癌细胞化疗抵抗中的作用，以及转染胞浆表达型Smac基因靶向下凋XIAP对化疗药物诱导的胰腺癌细胞凋亡的影响。方法应用流式细胞术检测顺铂、5-FU介导的Panc-1、BXPC-3的凋亡率及胞浆染色分析细胞XIAP表达变化，Western blot分析XIAP、Smac、Caspase-3表达水平；构建pEGFP-NI／Smac真核表达载体并转染胰腺癌Panc-1细胞，流式细胞术检测转染Smac基因前后Panc-1细胞的凋亡敏感性。结果与BXPC-3细胞相比，Panc-1对顺铂或5-FU介导的凋亡具有较强抵抗性，Western blot分析显示Panc-1细胞高表达XIAP，在化疗药物作用下化疗敏感细胞BXPC-3胞浆内XIAP水平下降明显多于Panc-1细胞，而且凋亡的BXPC-3细胞释放入胞浆内的成熟Smac蛋白水平明显高于Panc-1细胞。转染胞浆表达型Smac基因至化疗抵抗Panc-1细胞，可明显下调其XIAP表达水平，促进效应Caspase-3分子活化，显著提高顺铂、5-FU诱导的细胞凋亡率。结论胰腺癌细胞XIAP的表达水平下调与其化疗敏感性有关，XIAP是克服化疗抵抗的重要靶分子，而上调Smac活性蛋白的胞浆表达作为一种有效调节信号，通过拮抗XIAP的凋亡抑制作用协同化疗药物促进胰腺癌细胞凋亡。     

Effect of IL-5, glucocorticoid, and Fas ligation on Bcl-2 homologue expression and caspase activation in circulating human eosinophils

IL-5 is a potent eosinophil viability-enhancing factor that has been strongly implicated in the pathogenesis of IgE-mediated inflammation in vivo. Recently published data have suggested that IL-5 (and related cytokines) may act by altering the expression of the anti-apoptotic regulator Bcl-2 or its homologues, but this is controversial. The behaviour of the recently described pro-apoptotic cysteine proteases (caspases) in eosinophils after IL-5 treatment has not been explored. We examined the effect of IL-5 on the expression of four major Bcl-2 homologues, as well as on the expression/activation of key members of the caspase cell death cascade in cultured circulating human eosinophils. The effect of relevant inducers of eosinophil apoptosis (glucocorticoid and Fas ligation) on these regulatory proteins was also examined. We observed baseline expression of the anti-apoptotic Mcl-1 and pro-apoptotic Bax proteins in immunoblots of eosinophil lysates, but not Bcl-x, Bcl-2. IL-5 treatment had the effect of maintaining this basal level of expression over time without altering the balance of Bcl-2 homologues. The (upstream) caspase 8 and (downstream) caspase 3 proenzymes were detected in eosinophils at baseline, and were processed during spontaneous and stimulated eosinophil death. IL-5 completely blocked caspase processing in spontaneous and dexamethasone-induced cell death, and significantly slowed processing during Fas ligation. Our data do not support the theory that IL-5 acts by altering the balance of anti-apoptotic and pro-apoptotic Bcl-2 homologues, but suggest that it may act by regulating activation of the caspase cell death cascade.     

Melanosis of the appendix: common in the paediatric age group

AIMS: The objective of this study was to assess apoptotic activity in gestational trophoblastic disease (GTD) and its prognostic value in hydatidiform mole (HM). METHODS AND RESULTS: Expression of the specific caspase cleavage site within cytokeratin 18 was assessed immunohistochemically using the monoclonal antibody M30 CytoDeath in 12 spontaneous abortions, 22 partial and 57 complete HM, eight choriocarcinoma (CCA) and 28 normal placentas. The M30 immunoreactivity occurred predominantly in the syncytiotrophoblasts. A significantly higher M30 index in HM and CCA was found when compared with normal placentas and spontaneous abortions (P < 0.001). The M30 index of those HM which spontaneously regressed was significantly higher than those HM which developed persistent disease requiring chemotherapy (P < 0.001). The M30 index correlated with another apoptotic index previously detected by TdT-mediated dUTP nick-end labelling (TUNEL) (P = 0.007) and the proliferation index assessed by the Ki67 antigen (P = 0.034). CONCLUSIONS: We conclude that apoptosis is important in the pathogenesis of GTD. Assessment of apoptotic activity in HM by the M30 index may be considered as an alternative prognostic indicator for predicting the clinical behaviour.     

Immunohistochemical analysis of cleaved caspase-3 detects high level of apoptosis frequently in diffuse large B-cell lymphomas of the central nervous system

The purpose of the present paper was to examine the level of apoptosis and the relationships among apoptosis, apoptosis-associated proteins, and proliferating potential in lymphoma tissues to clarify the characteristics of apoptosis in diffuse large B-cell lymphomas (DLBCL) of the central nervous system (CNS). The formalin-fixed, paraffin-embedded tissues of CNS and non-CNS DLBCL (20 cases each) were studied by terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) and immunohistochemistry, using antibodies against single-stranded DNA (ssDNA), cleaved caspase-3, bcl-2, bax, p53, Fas and Ki-67. The cleaved caspase-3 immunohistochemistry detected apoptosis of the lymphoma cells most sensitively compared to TUNEL and ssDNA immunohistochemistry. High expression (grade + + or + + +) of cleaved caspase-3 was found more frequently in CNS DLBCL (11 cases, 55%) than non-CNS DLBCL (three cases, 15%; P = 0.009). Bax-positivity of lymphoma cells was increased in six cases of CNS DLBCL, which also showed high positivity of cleaved caspase-3. There was no significant correlation between the cleaved caspase-3-positivity and the Ki-67 positivity. The present study indicates that the number of apoptotic cells and expression level of cleaved caspase-3 were significantly higher in CNS DLBCL than non-CNS DLBCL, and that the correlation of bax and cleaved caspase-3 expression was often present in CNS DLBCL.     

Caspase inhibitor Z-VAD-FMK potentiates heat shock-induced apoptosis and HSP70 synthesis in macrophages

Caspase inhibitor Z-VAD-FMK potentiated heat shock-induced apoptosis in macrophages. Z-VAD-FMK did not activate HSP70 synthesis, but significantly increased the intensity of this process during heat shock. It cannot be excluded that caspases abolish HSP70 accumulation under these conditions. The HSP70 synthesis inhibitor quercetin potentiated DNA fragmentation in macrophages cocultured with Z-VAD-FMK after heat shock. HSP70 play an important role in the protection of macrophages from caspase-independent apoptosis.Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 138, No. 9, pp. 261–263, September, 2004     

长春瑞滨诱导人肺癌Calu-3细胞凋亡及机制

目的　观察长春瑞滨 (VRB)诱导人肺癌Calu 3细胞凋亡时Bcl 2和半胱天冬酶 3的表达有无变化。方法　以不同浓度的VRB( 2 0 ,40和 60 μmol·L-1)作用于体外培养的人肺癌Calu 3细胞 2 4h后 ,TUNEL法和吖啶橙染色法观察肺癌细胞凋亡形态学特征 ;流式细胞仪检测肺癌细胞凋亡率和肺癌细胞Bcl 2蛋白表达水平 ;以半胱天冬酶 3荧光分析检测试剂盒测定肺癌细胞半胱天冬酶 3活性。结果　VRB( 2 0 ,40和60 μmol·L-1)处理细胞 2 4h ,TUNEL法及吖啶橙染色均观察到典型的凋亡细胞形态学特征。流式细胞仪检测VRB处理的肺癌细胞凋亡率分别为 ( 3 .1±0 .6) % ,( 7.8± 1 .2 ) %和( 1 9.6± 4.3 ) % ,较对照组 (凋亡率为 0 )显著增高且呈剂量依赖性 (P <0 .0 1 ) ;Bcl 2蛋白阳性表达细胞率分别为 ( 3 7.6±6.9) % ,( 2 5 .4±6.2 ) %和( 8.4±2 .5 ) % ,较对照组 ( 4 8.3±7.1 ) %显著降低且呈剂量依赖性 (P <0 .0 5 ) ;肺癌细胞半胱天冬酶 3活性分别为 ( 3 3 2± 1 6) ,( 4 1 7± 1 1 )和 ( 63 1± 2 7)μmol·L-1·h-1,较对照组 ( 1 95±1 2 ) μmol·L-1·h-1显著增高且呈剂量依赖性(P <0 .0 1 )。结论　VRB可以诱导肺癌细胞凋亡 ,抑制Bcl 2表达及增强半胱天冬酶 3活性     

去甲斑蝥素诱导人黑色素瘤A375-S2细胞凋亡

目的:研究去甲斑蝥素(NCTD)诱导人黑色素瘤A375-S2细胞凋亡的机制.方法:采用MTT法、形态学观察、DNA凝胶电泳及Western blot检测法.结果:去甲斑蝥素可诱导A375-S2细胞发生凋亡.半胱氨酸天冬氨酸酶(caspase)-3,-9抑制剂可以部分的抑制NCTD诱导的细胞死亡.细胞凋亡时caspase-3,8,-9酶活力升高,caspase-3底物-caspase-3激活的DNA酶抑制物(ICAD)蛋白表达下降,同时Bcl-2/Bax、Bcl-xL/Bax蛋白表达比率明显降低.结论:去甲斑蝥素通过激活caspase和Bcl-2家族诱导A375-S2细胞凋亡.