Caspase-9与Apaf-1在锰中毒大鼠生精细胞表达及XIAP与Smac的调控作用

目的研究氯化锰导致的大鼠生精细胞caspase-9及Apaf-1表达中XIAP和Smac的调节机制,探讨锰导致的雄性不育机制。方法雄性SD大鼠随机分为对照组,低剂量(15 mg/kg Mn Cl2)和高剂量(30 mg/kg Mn Cl2)组,腹腔注射MnCl24周和6周,免疫组织化学检测生精细胞caspase-9、Apaf-1、XIAP和Smac表达。结果各组生精细胞caspase-9、Apaf-1和Smac表达均显著升高,XIAP表达降低;同时间的高剂量组与低剂量组比较,同剂量的6周组与4周组比较,生精细胞caspase-9、Apaf-1和Smac表达均显著升高,XIAP表达降低;各组caspase-9与Apaf-1表达正相关,XIAP与Smac表达成负相关。结论锰可促进生精细胞caspase-9、Apaf-1和Smac表达,抑制XIAP表达,导致细胞凋亡,产生雄性生殖毒性效应。     

染锰大鼠睾丸Caspase-9,Smac,Apaf-1和XIAP的表达

目的研究氯化锰导致的大鼠生精细胞caspase-9及Apaf-1表达中XIAP和Smac的调节机制,探讨锰导致的雄性不育机制。方法雄性SD大鼠随机分为对照组,氯化锰低剂量(15mg/kg MnCl_2)和氯化锰高剂量(30mg/kg MnCl_2)组,腹腔注射MnCl_24周和6周,免疫组织化学SABC法和Western blot检测生精细胞caspase-9、Apaf-1、XIAP和Smac表达。结果氯化锰组生精细胞caspase-9和Smac蛋白表达水平显著高于对照组,随时间和剂量增加而升高;而Apaf-1和XIAP蛋白表达水平则降低,并有时间和剂量依赖性。结论 caspase-9,Apaf-1,XIAP和Smac可能参与锰引起雄性生殖毒性的发病机制。     

染锰大鼠生精细胞凋亡中caspase-3 mRNA、凋亡蛋白酶活化因子-1及多聚ADP核糖聚合酶的表达变化

目的:研究染锰诱导的大鼠生精细胞凋亡过程中,天冬氨酸特异性半胱氨酸酶-3(caspase-3)mRNA和凋亡蛋白酶活化因子-1(Apaf-1)、多聚ADP核糖聚合酶(PARP)的表达及其关系,探讨它们在生精细胞凋亡过程中的作用。方法:雄性SD大鼠,设立空白对照组、低剂量(15 mg/kg MnCl_2)和高剂量(30 mg/kg MnCl_2)组。实验组分别染锰4周和6周,空白对照组给予等容生理盐水,给药途径均为腹腔注射,TUNEL法检测生精细胞凋亡,原位杂交法和免疫组织化学法检测生精细胞caspase-3 mRNA、Apaf-1和PARP的表达。结果:与空白对照组比较,各染锰组生精细胞凋亡指数(AI)和caspase-3mRNA阳性细胞率均显著升高,Apaf-1和PARP阳性细胞率均显著降低。染锰剂量相同,6周与4周组比较,以及染锰时间相同,高剂量组与低剂量组比较,生精细胞AI和caspase-3 mRNA阳性细胞率均显著升高,Apaf-1和PARP阳性细胞率均显著降低。各组大鼠生精细胞AI与caspase-3 mRNA阳性细胞率呈正相关,与Apaf-1和PARP阳性细胞率呈负相关。结论:锰可影响大鼠生精细胞caspase-3 mRNA表达,促进Apaf-1和PARP分解,导致生精细胞凋亡,产生生殖毒性效应。     

锰诱导大鼠生精细胞凋亡过程中半胱氨酸酶-3mRNA与增殖细胞核抗原表达变化

目的:研究氯化锰对生精细胞凋亡过程半胱氨酸酶3(caspase-3)mRNA和增殖细胞核抗原(PCNA)表达的影响,探讨两者在生精细胞凋亡过程中的作用。方法:雄性SD大鼠随机分为空白对照组、低剂量(15 mg/kg MnCl_2)和高剂量(30mg/kg MnCl_2)组。MnCl_2组分别染锰4周和6周,空白对照组给予等容生理盐水,给药途径均为腹腔注射,TUNEL法检测睾丸生精细胞凋亡,原位杂交和免疫组织化学(SABC)检测生精细胞caspase-3 mRNA和PCNA和表达。结果:与空白对照组比较,各染锰组生精细胞凋亡指数(AI)均升高,caspase-3 mRNA阳性细胞率均显著升高,染锰4周:PCNA阳性细胞率显著降低,染锰6周反而升高。染锰剂量相同,6周与4周组比较,生精细胞AI与caspase-3 mRNA阳性细胞率、PCNA阳性细胞率均显著升高。染锰时间相同,高剂量组与低剂量组比较,生精细胞AI与caspase-3 mRNA阳性细胞率均显著升高,PCNA阳性细胞率显著降低。各组大鼠生精细胞AI与caspase-3 mRNA阳性细胞率呈正相关,与PCNA阳性细胞率呈负相关,而caspase-3 mRNA阳性细胞率与PCNA阳性细胞率呈负相关。结论:锰可诱导大鼠生精细胞caspase-3mRNA表达,促使生精细胞凋亡并且抑制细胞增殖,产生生殖毒性效应。     

Caspase-3mRNA与PARP在染锰大鼠生精细胞中表达变化

目的研究氯化锰对生精细胞凋亡过程半胱氨酸酶3(caspase-3)mRNA和多聚ADP核糖聚合酶(PARP)表达的影响,探讨氯化锰的生殖毒性效应及可能机制。方法雄性SD大鼠48只随机分为空白对照组,低剂量(15 mg/kg MnCl_2)和高剂量(30 mg/kg MnCl_2)组,8只/组。MnCl_2组分别染锰4周和6周,空白对照组给予等容生理盐水,给药途径均为腹腔注射,TUNEL法检测睾丸生精细胞凋亡,原位杂交法和免疫组织化学(SABC)法检测生精细胞caspase-3mRNA和PARP和表达。结果随染锰时间延长和剂量增加,各染锰组生精细胞凋亡指数(AI)及caspase-3 mRNA表达水平显著升高,PARP表达水平显著降低,凋亡指数AI分别与caspase-3 mRNA和PARP表达呈正相关和负相关。结论锰可诱导大鼠生精细胞caspase-3 mRNA表达,促进PARP分解,导致生精细胞凋亡,产生生殖毒性效应。     

染锰大鼠生精细胞半胱氨酸蛋白酶3的表达及锌的保护作用

目的:研究氯化锰对生精细胞半胱氨酸蛋白酶3(caspase-3)表达的影响,探讨锰诱发大鼠生精细胞caspase-3表达的机制及锌的拮抗作用.方法:雄性SD大鼠随机分为空白对照组,ZnCl_2对照组,15mg/kg和30mg/kg MnCl_2组;15mg/kg和30mg/kg MnCl_2+ZnCl_2组.免疫组织化学法检测睾丸caspase-3表达.结果:各染锰组,染锰补锌组及染锰剂量相同的6周组,染锰时间相同的30mg/kg MnCl_2组caspase-3阳性细胞率均显著升高,染锰补锌组显著降低.结论:染锰15mg/kg 4周可诱发大鼠生精细胞caspase-3表达,且随染锰时间和剂量的增加而增加,两者存在一定时间-效应和剂量-效应关系;摄入含锌100mg/kg的食物可拮抗锰诱发的生精细胞caspase-3表达.     

锰诱导大鼠生精细胞凋亡过程中生殖激素和乳酸脱氢酶及PARP的作用

目的研究锰对大鼠体内生殖激素和乳酸脱氢酶(LDH)和多聚ADP核糖聚合酶(PARP)表达的影响,探讨它们在生精细胞凋亡过程中的作用。方法雄性SD大鼠48只,随机分为空白对照组,低剂量组和高剂量组,腹腔注射Mn Cl24周和6周,TUNEL法检测生精细胞凋亡,放射免疫测定血清睾酮(T)、卵泡刺激素(FSH)、黄体生成素(LH)含量,化学比色测定血清和睾丸LDH含量。免疫组织化学法检测生精细胞PARP表达。结果各染锰组生精细胞凋亡指数、血清FSH、LH及LDH含量升高,血清T、睾丸LDH含量及PARP表达降低。结论锰可使大鼠T和睾丸LDH降低,FSH、LH和血清LDH活性升高,抑制大鼠生精细胞PARP表达,导致生精细胞凋亡。     

兔抗凋亡蛋白酶活化因子－1多克隆抗体的制备、纯化与鉴定

1997年,Zou等[1]首次从HeLa细胞胞质内纯化和克隆出凋亡蛋白酶活化因子-1(apoptotie protease activating factor-1,Apaf-1)的cDNA,该蛋白质的相对分子质量(Mr)为130.近来研究发现在细胞色素C和dATP存在的情况下,Apaf-1可聚集成凋亡体(apoptosome)[2],凋亡体负责将半胱天冬酶-9酶原蛋白(pmcaspase-9)裂解成半胱天冬酶-9(easpase-9),以及维持caspase-9的酶活性,进一步激活caspase-3而诱导细胞凋亡[2],因此Apaf-1可被认为是凋亡体的真正核心.     

沉默Apaf-1 基因对氧糖剥夺/ 复氧复糖PC12 细胞线粒体凋亡通路的影响

目的:探讨RNA干扰沉默Apaf-1基因对氧糖剥夺/复氧复糖PC12细胞线粒体凋亡通路的影响。方法:PC12细胞随机分为3组:正常组(Control)、模型组(Model)、Apaf-1基因沉默组(Apaf-1-siRNA)。正常组于CO2培养箱内正常培养,其余2组给予氧糖剥夺2 h、复氧复糖24 h处理,Apaf-1-siRNA组于造模前将化学合成的siRNA通过脂质体转染于PC12细胞靶向沉默Apaf-1基因。用荧光标记的siRNA检测Apaf-1转染效率,Western blot检测转染后PC12细胞Apaf-1蛋白表达,CCK-8检测细胞存活率,TUNEL染色检测细胞凋亡指数,流式细胞术检测细胞凋亡率,免疫荧光染色检测Bax/Bcl-2比值,Western blot检测线粒体凋亡通路关键蛋白Apaf-1、caspase-9、caspase-3表达。结果:Apaf-1-siRNA可有效沉默PC12细胞Apaf-1蛋白表达(P<0.05)。与Control组相比,Model组细胞存活率明显降低(P<0.05),细胞凋亡指数和凋亡率显著升高(P<0.05),Bax/Bcl-2比值升高(P<0.05),线粒体凋亡通路关键蛋白Apaf-1、caspase-9、caspase-3表达显著升高(P<0.05);与Model组相比,Apaf-1-siRNA组细胞存活率显著升高(P<0.05),细胞凋亡指数和凋亡率显著降低(P<0.05),Bax/Bcl-2比值降低(P<0.05),Apaf-1、caspase-9、caspase-3蛋白表达均明显降低(P<0.05)。结论:靶向沉默Apaf-1基因可有效降低氧糖剥夺/复氧复糖PC12细胞线粒体凋亡通路关键蛋白Apaf-1、caspase-9、caspase-3表达,抑制细胞凋亡,提高细胞存活率。     

Nodosin通过Apaf-1和caspase-3诱导HepG2细胞凋亡

目的:研究传统中药提取物nodosin对人肝细胞癌Hep G2细胞凋亡的影响,并探讨其作用机制。方法:将nodosin设置为1. 25μmol/L、2. 5μmol/L、5μmol/L、10μmol/L和20μmol/L不同浓度组,作用于Hep G2细胞24 h后,用Hoechst 33258染色和电镜观察不同浓度的药物对细胞形态学的影响,用流式细胞术检测细胞凋亡率,用RT-qPCR检测凋亡蛋白酶激活因子1 (Apaf-1) mRNA的表达,用Western blot检测caspase-3及其前体和活化体的蛋白水平。结果:形态学结果显示,随着用药剂量的增加,细胞皱缩和细胞核偏移越明显,凋亡小体在5μmol/L、10μmol/L和20μmol/L剂量组明显增多。Apaf-1 mRNA的表达增加,caspase-3及其前体的表达和cleaved caspase-3的蛋白水平随着用药剂量的增加逐渐增加(P 0. 01)。结论:Nodosin能够诱导Hep G2细胞凋亡。该作用可能是通过增加Apaf-1 mRNA的表达继之激活caspase-3实现的。     

hTERT干扰对HepG2肝癌细胞Smac表达的影响

目的检测RNAi靶向抑制hTERT基因表达促HepG2肝癌细胞凋亡与Smac的关系,探讨hTERT干扰促进细胞凋亡的可能机制。方法收集hTERT基因RNAi真核表达载体稳定转染第20代的HepG2肝癌转染细胞、对照细胞（转染空载体质粒）及未转染细胞,通过Westernblot检测胞内XIAP和caspase-3,以及线粒体和胞浆Smac的表达;采用共聚焦显微镜观察线粒体膜电位变化。结果与未转染细胞和对照细胞相比,转染细胞内XIAP表达明显减少（P〈0．05）,caspase-3表达明显增加（P〈0．05）,线粒体Smac表达明显减少（P〈0．05）,而胞浆Smac表达明显增加（P〈0．05）;转染细胞线粒体膜电位明显降低。结论RNAi靶向抑制hTERT基因表达可能通过降低HepG2肝癌细胞线粒体膜电位,引起Smac从线粒体释放入胞浆,进而抑制XIAP表达,促进caspase-3表达增加诱导细胞凋亡。     

Expression of apoptosis-related proteins and its clinical implication in surgically resected gastric carcinoma

Apoptosis, via caspase cascade, is involved in tumorigenesis and progression, and thus, altered apoptosis-related protein expressions have clinical and prognostic significance. Moreover, the apoptosis pathway is highlighted due to the recent introduction of apoptosis-targeted therapy for several genes such as the X-linked inhibitor of apoptosis protein (XIAP). XIAP is the most potent direct inhibitor of caspase, and XIAP-associated factor 1 (XAF1) and secondary mitochondrial activator of caspase/direct IAP-binding protein with low PI (Smac/DIABLO) are negative regulators of XIAP. In this study, we evaluated the expression of these proteins and investigated their clinical and prognostic significance in gastric carcinomas. Immunohistochemical analysis by using the tissue array method was performed for XIAP, survivin, Bcl-2, XAF1, Smac/DIABLO, and cleaved caspase-3 proteins in 1,162 surgically resected gastric carcinoma cases. XIAP expression was related to the advanced stage. The expression of XIAP showed negative relationship with XAF1 and Smac/DIABLO expressions. In addition, XIAP expression was associated with a poor prognosis and was also proved to be an independent prognostic factor. Cleaved caspase-3 expression was related to the early stage. In addition, cleaved caspase-3 expression was associated with a favorable prognosis and was also proved to be an independent prognostic factor. The expression of XIAP showed an inverse relationship with cleaved caspase-3. In addition, the expression of XAF1 and Smac/DIABLO had a positive relationship with cleaved caspase-3. These findings are consistent with their known functions, and they may help to identify individuals best suited for apoptosis-targeted therapy as a baseline data in gastric carcinoma.     

红景天苷对急性心肌缺血大鼠心肌细胞凋亡的作用及机制研究

目的：观察红景天苷对急性心肌缺血大鼠心肌细胞凋亡的影响,探讨其可能的分子机制。方法：制备Sprague Dawley大鼠急性心肌缺血模型,随机分为红景天苷高、低剂量组(40 mg/kg、10 mg/kg)、急性心肌缺血组和对照组。原位末端转移酶标记染色法检测心肌组织凋亡情况,免疫印迹法检测心肌组织Bcl-2、Bax、Cytochrome c (Cyt-c)、cleaved caspase-3以及cleaved caspase-9蛋白的表达水平。结果：原位末端转移酶标记染色法显示红景天苷浓度依赖性抑制急性心肌缺血所引起心肌细胞的凋亡;免疫印迹法结果显示,与急性心肌缺血组相比,红景天苷干预后心肌组织中Bcl-2的蛋白表达显著增加,Bax的蛋白表达显著减少,胞浆(cyto)中Cyt-c的蛋白表达水平显著下降,cleaved caspase-3和cleaved caspase-9表达均显著降低。结论：红景天苷具有抗心肌细胞凋亡的作用,其作用机制可能是通过抑制线粒体通路减少细胞的凋亡。本实验为红景天苷可作为临床上治疗缺血性心脏病提供实验依据。     

TRAIL-induced apoptosis requires Bax-dependent mitochondrial release of Smac/DIABLO.

Recent reports suggest that a cross-talk exists between apoptosis pathways mediated by mitochondria and cell death receptors. In the present study, we report that mitochondrial events are required for apoptosis induced by the cell death ligand TRAIL (TNF-related apoptosis-inducing ligand) in human cancer cells. We show that the Bax null cancer cells are resistant to TRAIL-induced apoptosis. Bax deficiency has no effect on TRAIL-induced caspase-8 activation and subsequent cleavage of Bid; however, it results in an incomplete caspase-3 processing because of inhibition by XIAP. Release of Smac/DIABLO from mitochondria through the TRAIL-caspase-8-tBid-Bax cascade is required to remove the inhibitory effect of XIAP and allow apoptosis to proceed. Inhibition of caspase-9 activity has no effect on TRAIL-induced caspase-3 activation and cell death, whereas expression of the active form of Smac/DIABLO in the cytosol is sufficient to reconstitute TRAIL sensitivity in Bax-deficient cells. Our results show for the first time that Bax-dependent release of Smac/DIABLO, not cytochrome c, from mitochondria mediates the contribution of the mitochondrial pathway to death receptor-mediated apoptosis.     

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。     

谷胱甘肽对染锰大鼠生精细胞凋亡与增殖的影响

目的:研究谷胱甘肽(GSH)对染锰大鼠生精细胞凋亡与增殖的作用及机制。方法:雄性SD大鼠48只随机分为6组,各组给锰途径为腹腔注射。采用TUNEL法检测生精细胞凋亡指数(AI);免疫组织化学法检测生精细胞增殖细胞核抗原(PCNA)表达。结果:与空白对照组比较,GSH对照组和15mg/kg GSH组生精细胞AI与生精细胞PCNA增殖指数(PI)均无显著性差异;与各自对应的单纯染锰组比较,15mg/kg GSH组和30mg/kg GSH组生精细胞AI均显著降低而PI均显著升高;30mg/kg组与15mg/kg组比较,生精细胞AI显著升高而PI显著降低;各组大鼠生精细胞AI和PI呈负相关。结论:15mg/kg和30mg/kg氯化锰可诱发大鼠睾丸生精细胞凋亡,两者存在剂量-效应关系;各组大鼠睾丸生精细胞AI和PI呈显著负相关;GSH对锰诱发大鼠生精细胞凋亡具有拮抗作用;GSH可能拮抗锰对大鼠生精细胞增殖的抑制作用。     

Role of inhibitor of apoptosis protein in gentamicin-induced cochlear hair cell damage

Apoptotic cell death is considered to play a key role in gentamicin-induced cochlear hair cell loss. Inhibitor of apoptosis proteins (IAPs) are important regulators of apoptosis that can prevent activation of effector caspases. This study was designed to investigate the possible involvement of X-linked inhibitor of apoptosis protein (XIAP) in hair cell death due to gentamicin. Basal turn organ of Corti explants from postnatal day (p) p3 or p4 rats were maintained in tissue culture and were exposed to 35 muM gentamicin for up to 48 h. Effects of specific XIAP inhibitors on gentamicin-induced hair cell loss and caspase-3 activation were examined. XIAP inhibitors increased gentamicin-induced hair cell loss but an inactive analog had no effect. Caspase-3 activation was primarily observed at 36 or 48 h in gentamicin-treated hair cells, whereas caspase-3 activation peaked at 24-36 h when explants were treated with gentamicin and an XIAP inhibitor. The inhibitors alone had no effect on hair cells. Finally, a caspase-3 inhibitor decreased caspase-3 activation and hair cell loss induced by gentamicin and an XIAP inhibitor, but caspase-8 and -9 inhibitors did not. The results indicate that XIAP normally acts to decrease caspase-3 activation and hair cell loss during gentamicin ototoxicity, as part of a protective response to potentially damaging stimuli.