N-乙酰-L-半胱氨酸和过氧化氢酶抑制晶状体上皮细胞凋亡及对caspase-3酶活性的影响

目的　观察抗氧化剂N 乙酰 L 半胱氨酸 (NAC)和过氧化氢酶 (catalase ,CAT)对晶状体上皮细胞凋亡的抑制作用及对凋亡关键蛋白酶caspase 3酶活性的影响。方法　Sprayue Dawley(SD)大鼠 84只分成 4组 ,每组 2 1只。取晶状体于MEM培养液中培养 ,过氧化氢 (H2 O2 )组加入终浓度为 2mmol/L的H2 O2 ,对照组不加H2 O2 ,H2 O2 +NAC组及H2 O2 +CAT组除加入 2mmol/LH2 O2 外再分别加入 10 0 μmol/LNAC和 9× 10 5U/LCAT。培养 2 4h后观察晶状体透明度改变 ,透射电镜下观察细胞超微结构改变 ,流式细胞AnnexinV PI双染色法检测细胞凋亡率 ,并用Westernblot法分析caspase 3相对分子量为 2 0 0 0 0的活性亚单位的表达。结果　 2mmol/LH2 O2 作用 2 4h后可引起晶状体明显混浊 ,透射电镜下晶状体上皮细胞出现典型的凋亡形态学改变 ,细胞凋亡率上升为 (31 2± 3 3) % ,并伴随caspase 3酶活性增高 ;分别加入抗氧化剂NAC和CAT后 ,晶状体混浊度明显减轻 ,细胞凋亡率下降为(2 0 9± 3 2 ) %和 (15 0± 2 4 ) % (P <0 0 1) ,caspase 3酶活性均降低。结论NAC和CAT可有效抑制氧化损伤引起的晶状体混浊及上皮细胞凋亡 ,其抑制作用可能与降低caspase 3酶活性有关。     

Caspase抑制剂联合Cocktail蛋白酶抑制剂对胰岛细胞凋亡的保护

背景：目前关于联合应用Caspase抑制剂及Cocktail蛋白酶抑制剂在胰岛细胞分离纯化过程中对胰岛细胞的影响的研究还较少有报道。 目的：比较Caspase抑制剂及Cocktail蛋白酶抑制剂对胰岛细胞在分离纯化及培养过程中的保护作用。 方法：取新生猪,体外分离、纯化和培养猪胰岛,培养24 h后分为3组：①空白对照组。②消化时加抑制剂组仅在消化过程中加入Caspase抑制剂及Cocktail蛋白酶抑制剂。③消化及培养时加抑制剂组在消化及培养的过程中均加入Caspase抑制剂及Cocktail蛋白酶抑制剂。AO-EB染色定性观察细胞形态及凋亡情况,流式细胞术定量检测细胞活力及凋亡。 结果与结论：空白对照组β细胞所占百分比为66.91%,消化时加抑制剂组为84.58%,消化及培养时加抑制剂组为87.15%;空白对照组活细胞、凋亡细胞及死亡细胞的比例分别为56.52%、16.15%、21.25%,消化时加抑制剂组分别为62.27%、14.66%、14.47%,消化及培养时加抑制剂组分比为73.09%、6.83%、10.28%。结果表明,在消化以及体外培养的过程中均加入Caspase抑制剂及Cocktail蛋白酶抑制剂可明显减少细胞的损失,并且可以增加胰岛β细胞的百分比。中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

硫酸镁对胎儿生长受限孕鼠胎盘组织半胱氨酸天冬氨酸蛋白酶3表达的影响

目的探讨硫酸镁对胎儿生长受限(FGR)孕鼠胎盘组织半胱氨酸天冬氨酸蛋白酶3(caspase-3)表达的影响,及硫酸镁治疗FGR的机理。方法烟熏法构建FGR模型。实验对象分为对照组(10只)、治疗组(18只)、FGR组(10只)。治疗组中低剂量(硫酸镁300 mg／kg)治疗10只、高剂量(硫酸镁600 mg／kg)治疗8只,皮下注射给药。络合指示剂方法测孕鼠血清Mg2+(血镁)浓度。物理测量胎鼠的各项生理指标。链霉菌抗生物素蛋白-过氧化物酶连接(SP)法及RT-PCR法检测胎盘组织caspase-3的表达情况。结果(1)FGR组孕鼠血镁浓度为(0．55±0．03)mmol／L,高、低剂量治疗组孕鼠血镁浓度分别为(0．72±0．13)、(0．61±0．03)mmol／L,高、低剂量治疗组分别与FGR组比较,差异均有统计学意义(P<0．01)。(2)FGR组孕鼠胎盘重量为(0．63±0．05)g,其胎鼠体重为(2．95±0．46)g,高剂量治疗组分别为(0．80±0．16)、(3．58±0．10)g,两组分别比较,差异均有统计学意义(P<0．05、P<0．01)。(3)FGR组胎盘组织caspase-3 mRNA表达量为0．626±0．036,其蛋白表达量为199．5±4．7,高剂量治疗组分别为0．361±0．030、183．0±3．3,差异均有统计学意义(P< 0．05),低剂量治疗组caspase-3 mRNA表达量为0．525±0．029,与高剂量治疗组比较,差异也有统计学意义(P<0．05)。(4)孕鼠血镁浓度与胎鼠重量、胎盘组织caspase-3 mRNA及蛋白表达量有显著相关性(r=0．899,P=0．038;r=-0．747,P=0．033;r=-0．915,P=0．001)。结论硫酸镁能降低胎盘组织caspase-3 mRNA及蛋白表达,硫酸镁可能通过抑制胎盘caspase-3的表达,减少胎盘滋养细胞、血管内皮细胞等功能细胞的凋亡,从而改善FGR胎鼠的低体重现象。     

Role of lysosomal cathepsins in naphthazarin- and Fas-induced apoptosis in oral squamous cell carcinoma cells

Conclusion. Intracellular cysteine cathepsins are pro-apoptotic factors involved in activation of caspases in two oral squamous cell carcinoma (SCC) cell lines. Objective. To study the possible involvement of lysosomal cathepsins in oral SCC cell apoptosis. Material and methods. Apoptosis was induced in the two human oral SCC cell lines UT-SCC-20A and UT-SCC-24A using naphthazarin or anti-Fas antibodies, and was studied by analysis of caspase activity and nuclear morphology. Involvement of lysosomal cathepsins was investigated using the cysteine cathepsin inhibitor z-FA-FMK and the cathepsin D inhibitor pepstatin A. The amounts of cellular and soluble Fas death receptor were determined by ELISA. Results. Release of cathepsins from the lysosomes to the cytosol was observed early in apoptosis. Cysteine cathepsins were found to be involved in activation of caspases in response to treatment with naphthazarin or anti-Fas antibodies, but inhibition of cysteine cathepsin activity was not sufficient to prevent cell death. Moreover, inhibition of cysteine cathepsin activity resulted in increased expression of the Fas death receptor, suggesting involvement of extracellular cysteine cathepsins in death receptor shedding.     

Roles of the Bcl-2/Bax Ratio,Caspase-8 and 9 in Resistance of Breast Cancer Cells to Paclitaxel

The goal of this study was to establish paclitaxel resistant MCF-7 cells, as in vitro model, to identify themolecular mechanisms leading to acquired chemoresistance in breast cancer cells. Resistant cells were developedby stepwise increasing exposure to paclitaxel. Gene expression levels of Bax and Bcl-2 along with protein levelsof caspase-8 and caspase-9 were evaluated in two resistant cell lines (MCF -7/Pac64 and MCF -7/Pac5 nM).Morphological modifications in paclitaxel resistance cells were examined by light microscopy and fluorescenceactivated cell sorting (FACS). As an important indicator of resistance to chemotheraputic agents, the Bcl-2/Baxratio showed a significant increase in both MCF-7/Pac5nM and MCF-7/Pac 64nM cells (p<0.001), while caspase-9levels were decreased (p<0.001) and caspase-8 was increased (p<0.001). FACS analysis demonstrated that MCF-7/Pac64 cells were smaller than MCF-7 cells with no difference in their granularity. Our results support theidea that paclitaxel induces apoptosis in a mitochondrial-dependent manner. Identifying breast cancer patientswith a higher Bcl-2/Bax ratio and caspase 9 level and then inhibiting the activity of these proteins may improvethe efficacy of chemotheraputic agents.     

硫酸链霉素引起的大鼠体外培养耳蜗毛细胞凋亡

目的 探讨在离体培养条件下硫酸链霉素是否通过半胱氨酸天冬氨酸蛋白酶(caspase)的激活而导致大鼠耳蜗毛细胞凋亡.方法 将出生后3～4 d的大鼠耳蜗基底膜取出进行离体培养,培养过夜后用0.1 mmol/L硫酸链霉素培养液继续培养24 h.终止培养前1 h应用碳氧荧光素标记的肽荧光甲基酮(carboxyfluorescein labeled peptide fluoromethyl ketone,FAM-peptide-FMK)标记的caspase-6,caspase-8,或caspase-9指示剂作用1 h,然后应用异硫氰酸四甲基罗丹明(tetramethyl rhodamine iso-thiocyanate,TRITC)标记的鬼笔环肽(phalloidin)标记毛细胞的纤毛和表皮板,再应用Topro-3 DNA探针对细胞核进行染色,最后在共聚焦显微镜下观察.结果 1 mmol/L硫酸链霉素引起的耳蜗毛细胞缺失在底回约为80%,在顶回约为20%.经链霉素作用后,耳蜗毛细胞的细胞核发生浓缩和碎裂,同时可见cmpase-6,caspase-8和caspase-9阳性表达.结论 硫酸链霉素可以引起离体培养的大鼠耳蜗毛细胞凋亡,上游和下游的caspase激活可能是其主要途径.     

Inhibition of apoptosis by ascorbic and dehydroascorbic acids in Xenopus egg extracts

Purpose  The viability of mammalian eggs after ovulation is reported to be improved by the presence of ascorbic acid in the culture medium. However, the pro-survival mechanisms of ascorbic acid are poorly understood. The molecular pathways of apoptosis are evolutionarily conserved among animal species, and Xenopus eggs are technically and ethically more suitable for biochemical analyses than mammalian eggs. We used Xenopus egg cytoplasmic extracts to examine the direct intracellular effects of ascorbic acid. Methods  Incubation of egg extracts for more than 4 h induces the spontaneous release of cytochrome c from mitochondria. This event triggers the activation of caspases, cleavage of substrate proteins, and execution of apoptosis. Multiple signal transduction pathways including proteolysis and protein phosphorylation are also involved in this process. We examined whether any of these events might be inhibited by the addition of ascorbic acid. Results  Ascorbic acid showed no effect against cytochrome c release, but prevented caspase activation and substrate cleavage. Ascorbic acid also blocked the proteolysis of apoptosis inhibitor proteins and the dephosphorylation of p42 MAP kinase. However, dehydroascorbic acid (oxidized form of ascorbic acid) and acetate (unrelated acid) were equally effective, indicating that these effects were primarily due to their acidity. In addition, dehydroascorbic acid inhibited caspase activities directly in vitro. Conclusions  The anti-apoptotic effect of ascorbic acid in Xenopus egg extracts is mainly due to cytoplasmic acidification rather than its intracellular antioxidant activity. Instead, oxidative conversion of ascorbic acid into dehydroascorbic acid may inhibit apoptosis through the inhibition of caspases.     

依赖天冬氨酸特异性半胱氨酸蛋白酶-3的肝细胞凋亡在肝硬化大鼠肝缺血再灌注损伤中的作用

目的:探讨肝硬化大鼠肝缺血/再灌注(I/R)损伤是否与肝细胞凋亡相关及天冬氨酸特异性半胱氨酸蛋白酶-3(caspase-3)活性变化与肝细胞凋亡的关系。方法:Pringle法复制肝I/R模型,将肝硬化大鼠随机分为2组:A组:单纯肝门阻断;B组:血流阻断+抑制剂:N-苯甲基氧化碳酰-缬氨酸-丙氨酸-天冬氨酸-氟化丙酮(ZVAD-fmk)15mg/kg;取无肝硬化大鼠,作单纯肝门阻断为C组。各组肝门阻断时间均为30min,再灌注72h。比较3组的血清天冬氨酸转氨酶(AST)、肝组织的caspase-3活性和肝细胞凋亡数。结果:A组大鼠肝组织caspase-3活性、肝细胞凋亡数在再灌注后6h达高峰,分别为(18.1±1.8)μmolAMC·h^-1·g^-1(tissue)和20.9%±4.9%,与I/R前的(6.6±2.0)μmolAMC·h^-1·g^-1(tissue)和0.5%±0.3%相比,P＜0.01。肝细胞凋亡数、caspase-3的活性随灌注时间的延长而减低,两者随时间的变化一致。3组中A组肝损伤最严重,表现为再灌注后6h血清AST最高,与B、C组比较有显著差异,大鼠7d生存率只为62.5%。进一步研究表明,再灌注后6h,A组的肝组织caspase-3活性、肝细胞凋亡数亦明显比B、C组高。结论:肝细胞凋亡是肝硬化大鼠肝I/R损伤的主要病理改变。肝细胞凋亡的发生可能主要依赖于肝组织caspase-3活性的改变,抑制caspase-3能明显减轻肝I/R损伤。肝硬化肝脏比无硬化肝脏对缺血损伤敏感性高的病理机制与依赖caspase-3的肝细胞凋亡密切相关。     

大鼠坐骨神经损伤后脊髓组织中Caspase 3、Bcl-xl表达变化的实验研究

目的：探讨大鼠坐骨神经损伤后脊髓组织中Caspase3、Bcl-xl的表达变化以及与脊髓神经细胞凋亡的关系。为进一步研究坐标神经损伤后的神经再生提供依据。方法：成年Wistar大鼠96只,随机分为对照组（12只）和实验组（84只）,实验组大鼠切除右侧长约6mm的坐骨神经,分为伤后3d和1,2,3,4,5,6周组,采用Caspase3、Bcl-xl免疫组化检测大鼠脊髓组织Caspase3、Bcl-xl表达变化,测定Caspase3的活性;采用流式细胞仪AnnexinⅤ/PI双标检测和原位末端脱氧核苷酸转移酶介导的生物素脱氧尿嘧啶核苷酸缺口末端标记法（TUNEL）检测脊髓细胞凋亡情况。结果Caspase3、Bcl-xl免疫组化阳性反应主要位于灰质神经元细胞,高表达区在前角。伤后1周实验组伤侧脊髓组织中Caspase3表达轻度升高,2－4周显著升高,5周后下降,伤后1－3周脊髓Bcl-xl表达减弱,4周后明显升高,高水平持续到伤后6周;伤后2－4周在鼠伤侧脊髓有较多TNEL阳性标记细胞;伤后3d伤侧Caspase3活性荧光值开始升高,1－3周明显升高,4周逐渐下降;流式细胞仪检测,伤后1周伤侧脊髓凋亡细胞开始增加,2,3周为高峰,4周伤侧凋亡细胞逐渐减少。实验组末伤侧与对照组比较,各项检查差异无显著性意义。结论：Caspase3的活化是细胞凋亡的早期生化。与细胞凋亡的形态学表现相互关系;Bcl-xl对减轻大鼠坐骨神经损伤后脊髓组织细胞凋亡有重要作用,及早识别细胞凋亡的早期特征,对促进神经再生具有重要意义。     

Ethanolic Neem (Azadirachta indica) Leaf Extract Induces Apoptosis in the Hamster Buccal Pouch Carcinogenesis Model by Modulation of Bcl-2, Bim,Caspase 8 and Caspase 3

Induction of apoptosis is one of the most active strategies in cancer chemoprevention and the ability of medicinal ‍plants in this regard has attracted major research interest. The present study was designed to investigate the apoptosis ‍inducing capacity of an ethanolic neem leaf extract (ENLE) during 7,12-dimethylbenz[a]anthracene (DMBA)-induced ‍hamster buccal pouch carcinogenesis using the apoptosis-associated proteins Bcl-2, Bim, caspase 8 and caspase 3 as ‍markers. Topical application of DMBA to the hamster cheek pouch for 14 weeks resulted in well developed squamous ‍cell carcinomas associated with increased expression of Bcl-2 and decreased expression of Bim, caspase 8 and caspase ‍3. Administration of ENLE inhibited DMBA-induced hamster buccal pouch (HBP) carcinogenesis, as revealed by ‍the absence of neoplasms, with induction of Bim and caspases 8 and 3 and inhibition of Bcl-2 expression. Our results ‍suggest that the chemopreventive effects of ENLE may be mediated by induction of apoptosis.