Caspase-3在血管内皮细胞凋亡中的作用研究进展

半胱氨酸天冬氨酸蛋白水解酶(Caspase)家族在细胞凋亡中起着核心的控制作用,家族中的Cas-pase-3是凋亡过程中的关键执行子,它在整个凋亡过程中可被多次激活,并激活下游的细胞死亡蛋白酶。在目前所有能够检测的细胞凋亡过程中的染色质凝结和DNA裂解方面,Caspase-3是必不可少的;在某些细胞凋亡末期的细胞裂解及凋亡小体形成过程中,Caspase-3也是不可或缺的,而且有可能在凋亡早期细胞活力开始降低时,Caspase-3就起到激活作用。研究发现,血管内皮细胞凋亡的主要信号传导通路为线粒体通路和死亡受体通路,两条通路均需激活Caspase-3才能引起凋亡,均表现为经典的染色质凝结和DNA裂解。在血管瘤的增生与消退病理周期转变中,Caspase-3也发挥着重要作用。本文对Caspase-3在血管内皮细胞凋亡中的作用研究进展做一综述。     

hTERT启动子调控caspase 3基因的特异性表达及其抑癌作用

目的：探讨端粒酶催化亚基(KFERT)启动子调控caspase3(半胱天冬酶-3)基因在端粒酶阳性肿瘤细胞中表达的特异性及在体内外的抑癌作用。方法：构建hTERT启动子调控的caspase3基因的真核表达载体，通过RTPCR、瞬时转染、MTT法、流式细胞术和动物实验方法，检测hTERT启动子调控caspase3基因表达对肿瘤细胞生长的影响。结果：hTERT启动子调控caspase3基因在端粒酶阳性肿瘤细胞HepG2中有明显表达，而在正常人胚肺成纤维细胞(human embryonic lung fibroblast，HEL)中未见表达；caspase3表达能对端粒酶阳性肿瘤细胞HepG2、HeLa、Glc和A549细胞的增殖产生明显抑制作用，抑制率为10．5％-37．9％；同时也可以诱导HepG2、HeLa、Glc和A549细胞发生凋亡，凋亡率为15．39％-35．19％；而对端粒酶阴性的正常细胞HEL没有明显的影响。动物实验结果显示，hTERT启动子调控的caspase3基因转染对HepC2细胞的体内增殖具有抑制作用。结论：hTERT启动子调控的caspase3基因表达载体是一种有应用潜力的肿瘤基因治疗载体。     

凋亡相关蛋白Caspase研究进展

细胞凋亡，又称细胞程序性死亡，是指细胞在一定的生理或病理条件下，遵循自身的程序，自己结束其生命的过程。细胞凋亡是一个主动的、信号依赖的过程，可以由许多因素所诱导，如放射线照射、毒素、药物、缺血缺氧、病毒感染等。和细胞增殖一样．细胞凋亡也是受基因调控的精确过程．其途径主要有两条．一条是通过细胞膜上的死亡受体激活cas．pase，另一条是通过胞质内的线粒体途径释放细胞凋亡因子激活caspase。     

脑创伤后细胞凋亡及其基因调控

脑创伤后的继发性损伤因素可诱发脑细胞凋亡的发生,凋亡是脑创伤后细胞死亡的重要原因之一,该凋亡过程受bcl-2、半胱氨酸门冬氨酸特异性蛋白酶(caspase)等凋亡相关基因家族的调控。     

辛基酚对MDA-MB-231细胞中bcl2和caspase3表达的影响

目的:观察辛基酚(OP)对乳腺癌MDA-MB-231细胞凋亡的影响。方法:以流式细胞分析、免疫细胞化学和RT-PCR等方法观察OP对MDA-MB-231细胞凋亡、bcl2、bax和caspase3表达的影响。结果:4μmol/L、8μmol/L-OP作用MDA-MB-231细胞48 h,其凋亡率分别为26.93±6.76、42.48±8.21(对照4.05±1.14);bcl2表达量荧光值分别为78.05±9.47、42.74±6.49(对照103.12±11.26)。OP降低细胞中bcl2 mRNA表达,增加caspase3 mRNA及其蛋白的表达。结论:OP能诱导MDA-MB-231细胞凋亡,其机制可能是通过上调细胞中caspase3的表达,下调bcl2的表达来诱导细胞凋亡。     

Bcl—2基因家庭与细胞凋亡

目前已知在多细胞生物体中细胞有两种不同的死亡形式 :坏死 (Ｎｅｃｒｏｓｉｓ)和凋亡 (Ａｐｏｐｔｏｓｉｓ)。细胞凋亡是指细胞在一定生理或病理条件下 ,由基因调控的主动而有序的自我消亡过程 ,在形态学、生化和分子水平上与细胞坏死有明显区别。目前公认的凋亡形态学改变为 :细胞体积缩小、胞浆浓缩、核固缩、染色质密度增高呈半月形并凝集于核膜周边、核仁裂解及凋亡小体的形态。细胞凋亡的生化特征是细胞核ＤＮＡ被核酸酶降解成 180～ 2 0 0ｂｐ左右的ＤＮＡ片段 ,电泳呈特征性梯状带 (ＤＮＡＬａｄｄｅｒ)。细胞凋亡与增殖的动态平衡…     

粒-巨噬细胞集落刺激因子通过蛋白激酶Cα途径挽救辐射导致的白血病细胞TF-1的凋亡

蛋白激酶C(PKC)作为细胞内的传递介质参与了一系列细胞活动过程。PKC能激活与抗辐射有关的蛋白,这意味着辐射介导的细胞反应中存在着PKC信号通路。细胞凋亡对PKC激活或抑制的不同反应是不同异构体的表达所致。为了证实PKC异构体在凋亡中的作用,对辐射介导细胞凋亡在用与不用粒-巨噬细胞集落刺激因子(GM-CSF)时,PKC异构体在细胞内的分布和量的变化进行了研究。方法:TF-1细胞用含有20%的胎牛血清常规培养。蛋白定量用Pierce试剂盒。细胞裂解,分离,抽提蛋白。免疫组化分析:蛋白样品变性后经电泳转移到硝酸纤维膜上,与兔抗鼠PKC抗体…     

腺病毒介导p53基因转染增加人胃癌细胞的放射敏感性

目的评价腺病毒介导p53基因(Adp53)转染对人胃癌细胞的凋亡效应和放射增敏作用。方法以重组腺病毒介导p53基因感染4种不同p53状况的人胃癌细胞，用免疫组织化学法和Western blot法检测P53蛋白在胃癌细胞中的表达；用细胞集落形成法检测细胞存活率；用TUNEL法检测细胞凋亡。胃癌细胞感染Adp53后照射4Gy，用流式细胞仪检测细胞周期分布和凋亡；胃癌细胞种植肿瘤内注射Adp53后照射6Gy，以肿瘤相对体积增长曲线观察肿瘤抑制情况。结果1：100效靶比(MOI)Adp53产生细胞高转染率，以及p53基因在4种胃癌细胞中均高表达，并产生G2/M期阻滞、凋亡增加和细胞增殖抑制。如果以凋亡评价放射效应，Adp53转染对4Gy照射4种细胞的凋亡率比值为：W细胞3．0，M细胞3．6，neo细胞2．2，823细胞2．5。体内实验结果显示，Adp53对w细胞肿瘤6Gy照射的抑瘤率比值为1．41，而对M细胞肿瘤为1．91。结论腺病毒介导p53基因转染产生细胞凋亡并提高人胃癌细胞的放射敏感性，这种作用不依赖于细胞内在的p53状况。     

Caspases在消炎痛所致胃黏膜细胞凋亡中的作用

目的　研究大鼠在体情况下消炎痛(IND) 所致胃黏膜细胞凋亡过程中caspase 3、8基因及蛋白表达的变化,以探讨其黏膜损伤机制。方法　SD大鼠以不同剂量IND(30、60、90、120mg/kg)灌胃后3h处死,另设对照组。采用TUNEL标记技术检测黏膜细胞凋亡;分别采用原位分子杂交和RT PCR检测caspase 3基因表达的变化,应用免疫组化方法同步检测caspase 3、8蛋白表达的变化。结果　TUNEL标记显示对照组大鼠胃黏膜仅见少量凋亡细胞,IND组凋亡细胞数明显增加,计算机图像分析显示阳性细胞平均像素点在IND 30、60、90、120mg/kg组分别为对照组的6 3、8 0、12 6和17 1倍(P<0 01);原位分子杂交显示,caspase 3 mRNA在对照组胃黏膜呈弱阳性表达,IND 30~90mg/kg组呈中等至强阳性,120mg/kg组呈强阳性表达,与对照组相比均有显著差异(P<0 01), caspase 3 mRNA表达变化同细胞凋亡之间呈明显正相关(r=0 9642,P<0 01);RT PCR显示对照组胃黏膜中caspase 3 mR NA表达量极少,IND各剂量组的caspase 3/βactin吸光度比值较对照组明显升高(P<0 01);免疫组化染色显示对照组胃黏膜caspase 3、8蛋白均呈弱阳性表达,caspase 3表达水平强于caspase 8(P<0 05),IND使caspase 3、8蛋白表达呈中等至强阳性,明显高于对照组(P<0 05),IND各剂量组caspase 3     

温热刺激致bcr/abl融合基因阳性的CML细胞表面形态的改变

肿瘤热疗（hyperthermia）是通过多种方式提高人体中的恶性组织细胞的温度并维持一定时间,导致癌细胞功能不可逆损伤或增加细胞对药物的敏感性,从而加速癌细胞的凋亡或死亡。我们在前期研究中发现,温热刺激K562细胞可增高细胞热休克蛋白和胱天蛋白酶（caspase）-3的表达,提示温热刺激可能诱导K562细胞凋亡[4,5]。     

Immunohistochemical analysis of vimentin expression in myocardial tissue from autopsy cases of ischemic heart disease

Vimentin is a type III intermediate filament cytoskeletal protein that is expressed mainly in cells of mesenchymal origin and is involved in a plethora of cellular functions. In this study, myocardial tissues from patients with ischemic heart disease and a mouse model of acute myocardial infarction were subjected to immunohistochemistry for vimentin. We first examined 26 neutral formalin-fixed, paraffin-embedded myocardial tissue samples from autopsies of patients that were diagnosed with ischemic heart disease within 48 h postmortem. Myocardial cells were negative for vimentin, whereas non-myocardial cells, including vascular endothelium, vascular smooth muscle, fibroblasts, nerve fibers, adipocytes and mesothelial cells, showed positivity. Elevated vimentin expression was observed around myocardial cells undergoing remodeling, suggesting fibroblastic and endothelial proliferation in these locations. By contrast, myocardial foci that were completely fibrotic did not show upregulated vimentin expression. Inflammatory foci including macrophages and neutrophils were clearly visualized with vimentin immunostaining. The same vimentin expression phenomena as those found in human samples were observed in the mouse model. Our study indicates that immunostaining of vimentin as a marker for myocardial remodeling and the dynamics of all non-myocardial cell types may be useful for supplementing conventional staining techniques currently used in the diagnosis of ischemic heart disease.     

Protein synthesis-dependent apoptotic signalling pathway in X-irradiated MOLT-4 human leukaemia cell line.

PURPOSE: To demonstrate whether protein synthesis is required for ionizing radiation-induced apoptosis through activation of caspases in human leukaemia cell line MOLT-4, the effects of a protein synthesis inhibitor, cycloheximide, on the apoptotic signalling pathway including the activation of caspase family and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and the expression of Fas/CD95/APO-1 (Fas) were examined in X-irradiated MOLT-4 cells. MATERIALS AND METHODS: MOLT-4 cells pretreated with 0.5 microg/ml cycloheximide for 1h were exposed to 7.5Gy of X-rays. The appearance of apoptosis, expression of Fas, activation of caspases-3, -8, -9, SAPK/JNK and AP-1, the release of mitochondrial cytochrome-C and the formation of death-induced signalling complex (DISC) between Fas and the Fas-associated death domain (FADD) were measured by fluorescence microscopy, Western blotting, flow cytometry, gel shift assay and immunoprecipitation, respectively. RESULTS: Nuclear fragmentation and chromatin condensation were observed at 6 h after X-irradiation and gradually increased up to 12 h. These phenomena were significantly attenuated by cycloheximide. Cycloheximide also inhibited the activation of caspases and AP-1, the expression of Fas, the formation of DISC and the release of cytochrome-C, but not the activation of SAPK/JNK in X-irradiated MOLT-4 cells. CONCLUSION: These results indicate that apoptosis of X-ray-induced MOLT-4 cells is dependent on the activation of caspases regulated by de novo protein synthesis through SAPK/JNK activation.     

肿瘤辐射敏感性与细胞凋亡

辐射敏感性是肿瘤细胞复杂的生物学现象,在辐射诱导的细胞反应中具有不同的组织细胞类型特异性。肿瘤瘤射敏感性与辐射诱导的细胞凋亡密切相关,并受P53及其相关蛋白、Bcl-2基因家族蛋白、Fas及神经鞘磷脂-神经酰胺介导的信号途径、caspases级联反应等调控。     

黄芪皂甙、丹参酮IIA注射液对大鼠骨骼肌急性钝挫伤后胚胎型肌球蛋白重链及波形蛋白mRNA表达的影响

目的:观察黄芪皂甙、丹参酮IIA注射液对大鼠骨骼肌钝挫伤后胚胎型肌球蛋白重链(embryonicmyosin heavy chain,MHC-emb)及波形蛋白(vimentin)mRNA表达的影响。方法:用打击装置将120只雄性Wistar大鼠右侧腓肠肌中段造成钝挫伤模型,随机分为5组,在损伤局部分别注射黄芪皂甙、丹参酮IIA、黄芪皂甙丹参酮IIA混合液、黄芪丹参混合液及生理盐水。以后每3天注射1次,直到损伤后第27天。各组分别于损伤后第1、4、7、14、28和42天取损伤处骨骼肌标本。采用荧光定量聚合酶链式反应(realtime quantitativePCR)方法分析MHC-emb及波形蛋白mRNA表达。结果:(1)在骨骼肌急性钝挫伤修复早期,各组MHC-embmRNA和波形蛋白mRNA表达量均明显升高,分别于第7～14天和第4天达到高峰,然后下降。(2)从第4天至42天,各干预组MHC-emb mRNA表达水平显著高于生理盐水组,黄芪皂甙丹参酮IIA混合组和黄芪丹参组显著高于黄芪皂甙组和丹参酮IIA组。(3)从第4天至28天,各干预组波形蛋白mRNA表达水平显著低于生理盐水组,黄芪皂甙丹参酮IIA混合组和黄芪丹参组显著低于黄芪皂甙组和丹参酮IIA组。结论:在急性骨骼肌钝挫伤修复过程中,黄芪皂甙、丹参酮IIA能促进MHC-emb mRNA表达,抑制波形蛋白mRNA表达,但其单独使用时的作用均弱于黄芪丹参,混合使用效果与黄芪丹参基本一致。     

+Gz暴露对大鼠脑星形胶质细胞波形蛋白表达的影响

目的观察＋Gz暴露后大鼠脑星形胶质细胞中波形蛋白表达的改变.方法雄性SD大鼠44只,随机分为对照组、＋6 Gz/3 min暴露组及＋10Gz/3 min暴露组.分别于＋Gz暴露后不同时间灌注取脑,免疫组织化学染色观察脑波形蛋白的表达情况.结果对照组波形蛋白反应呈阴性,未见波形蛋白阳性细胞.＋6 Gz组和＋10 Gz组暴露后6 h,顶叶皮层、海马均可见波形蛋白呈弱阳性反应和波形蛋白阳性细胞;暴露后1 d,波形蛋白阳性反应进一步增强,阳性细胞数较6 h显著增多（P＜0.01）并达高峰;暴露后2 d,阳性反应有所减弱,阳性细胞数较前有所减少;暴露后4 d和6 d,波形蛋白反应呈阴性,未见阳性细胞.＋10 Gz暴露后6 h、1d和2d,顶叶皮层、海马波形蛋白阳性细胞数均显著多于＋6 Gz组（P＜0.01）.结论 ＋Gz暴露可引起大鼠顶叶皮层、海马波形蛋白表达,而＋10 Gz/3 min比＋6Gz/3 min暴露引起脑组织波形蛋白的表达增加更明显.     

The cellular energy crisis: mitochondria and cell death

Exploding nuclear reactors, environmental destruction, and global warming; the danger of energy production is clear. It is quite remarkable that in this modern age, where power usage is at a premium, we find that even on a cellular level, generation of large quantities of power comes at a cost. Mitochondria, which produce the majority of cellular energy in the form of ATP, have recently been shown to play an essential role in the death of a cell by a process known as apoptosis. During apoptosis, the integrity of mitochondria is compromised and various pro-apoptotic proteins are released into the cytoplasm. This results in activation of caspases, proteases that orchestrate the death of the cell. Cells in which apoptosis is inhibited upstream of mitochondria generally maintain the potential to proliferate, whereas inhibition of caspases downstream of mitochondria generally only delays cell death. Although breaches of the mitochondrial outer membrane result in the release of proteins that are important for respiration, mitochondria appear capable of maintaining at least some of their functions, including ATP production, even after this event. This has important implications both for the mechanism of outer-membrane permeabilization and the mechanism by which the cells eventually die in the absence of caspase activity. The events surrounding the breach of the mitochondrial outer membrane during apoptosis have therefore received much interest over the past few years.     

Beta-irradiation used for systemic radioimmunotherapy induces apoptosis and activates apoptosis pathways in leukaemia cells

Beta-irradiation used for systemic radioimmunotherapy (RIT) is a promising treatment approach for high-risk leukaemia and lymphoma. In bone marrow-selective radioimmunotherapy, beta-irradiation is applied using iodine-131, yttrium-90 or rhenium-188 labelled radioimmunoconjugates. However, the mechanisms by which beta-irradiation induces cell death are not understood at the molecular level. Here, we report that beta-irradiation induced apoptosis and activated apoptosis pathways in leukaemia cells depending on doses, time points and dose rates. After beta-irradiation, upregulation of CD95 ligand and CD95 receptor was detected and activation of caspases resulting in apoptosis was found. These effects were completely blocked by the broad-range caspase inhibitor zVAD-fmk. In addition, irradiation-mediated mitochondrial damage resulted in perturbation of mitochondrial membrane potential, caspase-9 activation and cytochrome c release. Bax, a death-promoting protein, was upregulated and Bcl-x_L, a death-inhibiting protein, was downregulated. We also found higher apoptosis rates and earlier activation of apoptosis pathways after gamma-irradiation in comparison to beta-irradiation at the same dose rate. Furthermore, irradiation-resistant cells were cross-resistant to CD95 and CD95-resistant cells were cross-resistant to irradiation, indicating that CD95 and irradiation used, at least in part, identical effector pathways. These findings demonstrate that beta-irradiation induces apoptosis and activates apoptosis pathways in leukaemia cells using both mitochondrial and death receptor pathways. Understanding the timing, sequence and molecular pathways of beta-irradiation-mediated apoptosis may allow rational adjustment of chemo- and radiotherapeutic strategies.     

Ceramide induces activation of the mitochondrial/caspases pathway in Jurkat and SCC61 cells sensitive to γ-radiation but activation of this sequence is defective in radioresistant SQ20B cells

Purpose : To clarify the molecular mechanisms leading to radiation-induced apoptosis or resistance, the kinetics (1-48 h) and sequence of events triggered in response to 10 Gy irradiation were investigated in three cell lines displaying a gradient of sensitivity to γ-rays. Materials and methods : Ceramide levels were measured by high performance liquid chromatography (HPLC). Mitochondrial function was evaluated in terms of transmembrane potential (ΔΨm) , reactive oxygen species (ROS) and glutathione levels analysed by flow cytometry or HPLC. Caspase activation was assessed by immunoblotting, and apoptosis by flow cytometry. Results : In Jurkat radiosensitive cells and SCC61 adherent cells with intermediate radiosensitivity, the degree of delayed ceramide release was directly related to their propensity to undergo apoptosis. Transduction of the death signal was mediated by a drop in ΔΨm and glutathione levels, ROS accumulation and activation of effector caspases. Experiments conducted with caspase inhibitors, bongkrekic acid, or DL-PDMP indicated that ceramide triggers mitochondrial collapse, followed by the activation of caspases-9, -8 and -3, and poly(ADP-ribose)polymerase cleavage. In SQ20B radioresistant cells, γ-radiation did not induce ceramide generation or subsequent activation of the mitochondrial/caspase apoptotic pathway. Conclusions : Ceramide appears to be a determining factor in the commitment phase of radiation-induced apoptosis. When ceramide is not generated, the whole pathway is ineffective and resistance to apoptosis may result.     

Ceramide induces activation of the mitochondrial/caspases pathway in Jurkat and SCC61 cells sensitive to gamma-radiation but activation of this sequence is defective in radioresistant SQ20B cells

PURPOSE: To clarify the molecular mechanisms leading to radiation-induced apoptosis or resistance, the kinetics (1-48 h) and sequence of events triggered in response to 10 Gy irradiation were investigated in three cell lines displaying a gradient of sensitivity to 7-rays. MATERIALS AND METHODS: Ceramide levels were measured by high performance liquid chromatography (HPLC). Mitochondrial function was evaluated in terms of transmembrane potential (delta(psi)m), reactive oxygen species (ROS) and glutathione levels analysed by flow cytometry or HPLC. Caspase activation was assessed by immunoblotting, and apoptosis by flow cytometry. RESULTS: In Jurkat radiosensitive cells and SCC61 adherent cells with intermediate radiosensitivity, the degree of delayed ceramide release was directly related to their propensity to undergo apoptosis. Transduction of the death signal was mediated by a drop in delta(psi)m and glutathione levels, ROS accumulation and activation of effector caspases. Experiments conducted with caspase inhibitors, bongkrekic acid, or DL-PDMP indicated that ceramide triggers mitochondrial collapse, followed by the activation of caspases-9, -8 and -3, and poly(ADP-ribose)polymerase cleavage. In SQ20B radioresistant cells, gamma-radiation did not induce ceramide generation or subsequent activation of the mitochondrial/caspase apoptotic pathway. CONCLUSIONS: Ceramide appears to be a determining factor in the commitment phase of radiation-induced apoptosis. When ceramide is not generated, the whole pathway is ineffective and resistance to apoptosis may result.     

Staurosporine modulates radiosensitivity and radiation-induced apoptosis in U937 cells

PURPOSE: The present study aims at investigating the involvement of several genes in the cell cycle distribution and apoptosis in U937 cells, a cell line lacking functional p53 protein, after combined treatment with staurosporine and irradiation. MATERIALS AND METHODS: Using a DNA fragmentation assay, flow cytometry and western blot analysis, the molecular basis for the effects of staurosporine in combination with the irradiation of leukemia cells was investigated. RESULTS: Our results indicated that combined treatment led to an increased apoptotic cell death in U937 cells, which is correlated with the phosphorylation of the V-Jun sarcoma virus 17 oncogene homolog (c-JUN) NH(2)-terminal kinase protein (JNK), the activation of caspases, the increase in B cell leukemia/lymphoma 2 (Bcl-2) associated X protein (Bax), the decrease in Bcl xL protein (Bcl-XL) levels, the loss of mitochondria membrane potential and the release of cytochrome c. CONCLUSIONS: Abrogation of the G2 checkpoint should be an effective strategy against p53-deficient leukemia cells to irradiation-induced cell killing.