羊栖菜多糖诱导lovo细胞凋亡及其机理探讨

本课题研究羊栖菜多糖(Sargassum Fusiforme Polysaccharides,SFPS)诱导人大肠癌lovo细胞凋亡及凋亡过程中Caspase-3的变化及其意义。MTT法检测SFPS对大肠癌细胞增殖的抑制率;通过电镜、琼脂糖凝胶电泳、流式细胞术鉴定细胞凋亡;应用Western blot法测定Caspase-3酶原的变化; RT-PCR检测Caspase-3 mRNA表达。结果显示:SFPS作用lovo细胞24,36,48和72h的IC_(50)分别为375,355,178和60mg/L,表明对lovo细胞具有显著生长抑制作用。在电镜下,可见明显的细胞凋亡特征:细胞膜表面微绒毛减少、染色质固缩、边集,凋亡小体形成。在琼脂糖凝胶电泳中,药物浓度为5-300mg/L作用24h后,显示有凋亡细胞特有的DNA梯状条带;而500mg/L处理后梯状条带模糊,开始出现“涂片状”,表明在高药物浓度的作用下,细胞有坏死。流式细胞仪测得细胞凋亡率有剂量的依赖性;DNA直方图出现亚G1峰,但细胞周期时相的分布无明显改变。SFPS处理lovo细胞后,发现Caspase-3酶原蛋白表达降低,Caspase-3的mRNA高表达,并具有剂量和时间的依赖性。实验结果提示,SFPS在体外能够诱导lovo细胞凋亡,这可能是SFPS抑制肿瘤增殖的机制之一,而Caspase-3的活化参与了SFPS诱导lovo细胞凋亡的调控。     

镉诱导HEK293细胞凋亡及其线粒体凋亡途径

本课题研究了氯化镉(CdCl_2)诱导HEK293细胞(人胚胎肾细胞系)的凋亡,初步探讨了凋亡过程中Caspase-3、Bcl-2的变化和凋亡诱导因子(AIF)的转移以及它们的意义。MTT法检测CdCl_2对HEK293细胞增殖的抑制作用;通过倒置显微镜、电镜、琼脂糖凝胶电泳、流式细胞术、激光共聚焦观察细胞凋亡;应用Western blot法和荧光免疫法测定Caspase-3酶原、Bcl-2蛋白的变化以及检测AIF蛋白在细胞中的定位。结果显示:CdCl_2对HEK293细胞具有显著的生长抑制作用,并呈明显的剂量和时间依赖性。在琼脂糖凝胶电泳中,显示有凋亡细胞特有的DNA梯状条带,其中30μmol/L作用6-9h梯状条带最为清晰,时间过长或浓度过高则梯状条带逐渐模糊,表明镉浓度过高或处理时间过长,细胞有坏死。流式细胞仪检测也印证了这一结果。形态学观察可见明显的细胞凋亡特征。同时线粒体膜电位明显下降,发现Caspase-3酶原蛋白、Bcl-2蛋白含量减少,并具有时间依赖性;另外检测到线粒体AIF向细胞核转移。而Bcl-2转染后有一定的抑制凋亡作用。实验结果提示,CdCl_2能够诱导HEK293细胞凋亡,线粒体损伤导致AIF转移与细胞色素c释放,从而引发的非Caspases与Caspases凋亡途径可能在镉引发的细胞凋亡过程中起重要作用,而Caspase-3, Bcl-2起着重要的调控作用。     

大剂量地塞米松快速高效诱导巨噬细胞凋亡

运用透射电镜、DNA琼脂糖凝胶电泳、流式细胞术、原位末端标记法(TUNEL)染色等技术,检测了大剂量地塞米松处理小鼠腹腔巨噬细胞的各种变化.结果显示,大剂量地塞米松处理的巨噬细胞发生胞体皱缩、染色质凝聚、胞质浓缩;TUNEL染色呈阳性;0.5 h后DNA凝胶电泳即呈梯状条带;流式细胞术作周期分析出现凋亡峰等明显的凋亡特征.并且随处理时间延长凋亡率升高.结果表明,大剂量地塞米松快速、高效诱导小鼠腹腔巨噬细胞凋亡.     

大蒜素对全脑缺血/再灌注诱导的海马神经元凋亡的影响

目的:观察大蒜素对全脑缺血/再灌注诱导的海马神经元凋亡的影响。方法:采用大鼠全脑缺血/再灌注模型;应用DNA琼脂糖凝胶电泳、透射电镜和流式细胞仪检测海马神经元凋亡情况。结果:缺血/再灌注大鼠海马DNA电泳呈现细胞凋亡特有的"梯状条带",大蒜素预处理组未出现"梯状条带";透射电镜观察到缺血/再灌注海马部分神经元超微结构呈现明显的凋亡特征,大蒜素预处理可改善神经元超微结构;缺血/再灌注海马神经元凋亡率较假手术组明显增加,大蒜素预处理可明显降低缺血/再灌注大鼠海马神经元凋亡率。结论:大蒜素可抑制全脑缺血/再灌注诱导的海马神经元凋亡。     

大蒜素对全脑缺血侑灌注诱导的海马神经元凋亡的影响

的：观察大蒜素对全脑缺血／再灌注诱导的海马神经元凋亡的影响：方法：采用大鼠全脑缺血／再灌注模型;应用DNA琼脂糖凝胶电泳、透射电镜和流式细胞仪检测海马神经元凋亡情况结果：缺血／再灌注大鼠海马DNA电泳呈现细胞凋亡特有的“梯状条带”,大蒜素预处理组未出现“梯状条带”;透射电镜观察到缺血／再灌注海马部分神经元超微结构呈现明显的凋亡特征,大蒜素预处理可改善神经元超微结构;缺血／再灌注海马神经元凋亡率较假手术组明显增加,大蒜素预处理可明显降低缺血／再灌注大鼠海马神经元凋亡率,结论：大蒜素可抑制全脑缺血／再灌注诱导的海马神经元凋亡、     

羊栖菜多糖诱导lovo细胞凋亡及其机理探讨

本课题研究羊栖菜多糖（Sargassum Fusforme Polysaccharides．SFPS）诱导人大肠癌lovo细胞凋亡及凋亡过程中Caspase-3的变化及其意义。MTT法检测SFPS对大肠癌细胞增殖的抑制率：通过电镜、琼脂糖凝胶电泳、流式细胞术鉴定细胞凋亡：应用Westernblot法测定Caspase-3酶原的变化：RT—PCR检测Caspase-3mRNA表达。结果显示：SFPS作用lovo细胞24,36,48和72h的IC50分别为375,355,178和60mg／L,表明对lovo细胞具有显著生长抑制作用。在电镜下,可见明显的细胞凋亡特征：细胞膜表面微绒毛减少、染色质固缩、边集,凋亡小体形成。在琼脂糖凝胶电泳中,药物浓度为5—300mg／L作用24h后,显示有凋亡细胞特有的DNA梯状条带：而500mg／L处理后梯状条带模糊,开始出现“涂片状”,表明在高药物浓度的作用下,细胞有坏死。流式细胞仪测得细胞凋亡率有剂量的依赖性;DNA直方图出现亚G1峰,但细胞周期时相的分布无明显改变。SFPS处理lovo细胞后,发现Caspase-3酶原蛋白表达降低,Caspase-3的mRNA高表达,并具有剂量和时间的依赖性。实验结果提示,SFPS在体外能够诱导lovo细胞凋亡,这可能是SFPS抑制肿瘤增殖的机制之一．而Caspase-3的活化参与了SFPS诱导lovo细胞凋亡的调控。     

羊栖菜多糖诱导HL-60细胞凋亡的研究

用MTT法观察羊栖莱多糖(SFPS)在体外抗人白血病HL-60细胞增殖作用;扫描电镜、透射电镜、DNA电泳和流式细胞仪检测HL-60细胞凋亡。结果表明SFPS对HL-60细胞具有显著生长抑制作用,并呈量效和时效关系,药物作用24,36,48,72h的IC50分别为390,362,402,421mg／L;药物浓度为300mg／L和500mg／L作用HL-60细胞后,琼脂糖凝胶电泳显示有凋亡细胞特有的DNA梯状条带,细胞微绒毛减少、染色质固缩、边集,凋亡小体形成;DNA直方图出现亚G1峰。在一定浓度范围内,SFPS诱导细胞凋亡的作用呈现浓度和时间依赖性,同时G2／M期细胞比例增多。因此,SFPS抗肿瘤作用与诱导细胞凋亡和G2／M期细胞阻滞有关。     

胡桃楸提取液诱导Hela细胞凋亡的研究

目的：初步探讨胡桃揪提取液对Hela细胞的凋亡诱导作用。方法：通过形态学观察,琼脂糖凝胶电泳及流式细胞术（FCM）检测凋亡细胞。结果：形态学观察发现核固缩、出现凋亡小体。琼脂糖凝胶电泳可见DNA ladder条带。流式细胞仪检测有凋亡峰。结论：胡桃揪提取液可诱导Hela细胞凋亡。     

姜黄素诱导沉默MDR1基因的SGC7901/ADM细胞凋亡

为探讨MDR1基因沉默对姜黄素诱导人胃癌SGC7901/ADM细胞凋亡的影响,将已构建的靶向MDR1基因的RNAi表达载体转染SGC7901/ADM细胞,建立转染细胞单克隆,流式细胞术检测细胞外排功能。姜黄素处理SGC7901/ADM细胞48 h后,通过激光共聚焦显微镜下观察细胞形态结构,琼脂糖凝胶电泳检测DNA片段化。结果显示,各干扰载体转染组细胞内Rho-123荧光强度不同程度增强,细胞经姜黄素处理48 h后,激光共聚焦显微镜下呈明显的凋亡形态结构,DNA琼脂糖凝胶电泳呈梯状条带,说明MDR1基因沉默能促进姜黄素诱导SGC7901/ADM细胞凋亡。     

羊栖菜多糖诱导HL-60细胞凋亡的研究

用MTT法观察羊栖菜多糖(SFPS)在体外抗人白血病HL-60细胞增殖作用;扫描电镜、透射电镜、DNA电泳和流式细胞仪检测HL-60细胞凋亡。结果表明SFPS对HL-60细胞具有显著生长抑制作用,并呈量效和时效关系,药物作用24,36,48,72h的IC_(50)分别为390,362,402,421mg/L;药物浓度为300mg/L和500mg/L作用HL-60细胞后,琼脂糖凝胶电泳显示有凋亡细胞特有的DNA梯状条带,细胞微绒毛减少、染色质固缩、过集,凋亡小体形成;DNA直方图出现亚G_1峰。在一定浓度范围内,SFPS诱导细胞凋亡的作用呈现浓度和时间依赖性,同时G_2/M期细胞比例增多。因此,SFPS抗肿瘤作用与诱导细胞凋亡和G_2/M期细胞阻滞有关。     

TRAIL causes cleavage of bid by caspase-8 and loss of mitochondrial membrane potential resulting in apoptosis in BJAB cells.

A new member of the TNF family, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), has been shown to induce apoptosis. However, the mechanism for TRAIL-induced apoptosis remains to be clarified. SDS-PAGE and Western blot analysis showed that cleavage of Bid was induced by a 1-h incubation of BJAB cells with TRAIL and was blocked by a caspase-8 inhibitor. Flow cytometry demonstrated that loss of mitochondrial membrane potential in BJAB cells began about 1.5 h after the treatment with TRAIL and was apparent at 2 h in comparison with the control. DNA ladder formation, which is characteristic for apoptosis, in the cells treated with TRAIL was detected at 2 h and observed most effectively at 3 h. The time course study suggests that TRAIL causes cleavage of Bid via activation of caspase-8, subsequently the loss of mitochondrial membrane potential, resulting in apoptosis in BJAB cells.     

Doxorubicin treatment activates a Z-VAD-sensitive caspase, which causes deltapsim loss, caspase-9 activity, and apoptosis in Jurkat cells

Doxorubicin induces caspase-3 activation and apoptosis in Jurkat cells but inhibition of this enzyme did not prevent cell death, suggesting that another caspase(s) is critically implicated. Western blot analysis of cell extracts indicated that caspases 2, 3, 4, 6, 7, 8, 9, and 10 were activated by doxorubicin. Cotreatment of cells with the caspase inhibitors Ac-DEVD-CHO, Z-VDVAD-fmk, Z-IETD-fmk, and Z-LEHD-fmk alone or in combination, or overexpression of CrmA, prevented many morphological features of apoptosis but not loss of mitochondrial membrane potential (delta(psi)m), phospatidilserine exposure, and cell death. Western blot analysis of cells treated with doxorubicin in the presence of inhibitors allowed elucidation of the sequential order of caspase activation. Z-IETD-fmk or Z-LEHD-fmk, which inhibit caspase-9 activity, blocked the activation of all caspases studied, lamin B degradation, and the development of apoptotic morphology, but not cell death. All morphological and biochemical features of apoptosis, as well as cell death, were prevented by cotreatment of cells with the general caspase inhibitor Z-VAD-fmk or by overexpression of Bcl-2. Doxorubicin cytotoxicity was also blocked by the protein synthesis inhibitor cycloheximide. Delayed addition of Z-VAD-fmk after doxorubicin treatment, but prior to the appearance of cells displaying a low delta(psi)m, prevented cell death. These results, taken together, suggest that the key mediator of doxorubicin-induced apoptosis in Jurkat cells may be an inducible, Z-VAD-sensitive caspase (caspase-X), which would cause delta(psi)m loss, release of apoptogenic factors from mitochondria, and cell death.     

Galectin-1 induced activation of the mitochondrial apoptotic pathway: evidence for a connection between death-receptor and mitochondrial pathways in human Jurkat T lymphocytes

Galectin-1 (gal-1) triggers T cell death by several distinct intracellular pathways including the activation of the death-receptor pathway. The aim of this study was to investigate whether gal-1 induced activation of the death-receptor pathway in Jurkat T lymphocytes mediates apoptosis via the mitochondrial pathway linked by truncated Bid (tBid). We demonstrate that gal-1 induced proteolytic cleavage of the death agonist Bid, a member of the Bcl-2/Bcl-xL family and a substrate of activated caspase-8, was inhibited by caspase-8 inhibitor II (Z-IETD-FMK). Downstream of Bid, gal-1 stimulated mitochondrial cytochrome c release as well as the activation and proteolytic processing of initiator procaspase-9 were effectively decreased by caspase-8 inhibitor II. Blocking of gal-1 induced cleavage of effector procaspase-3 by caspase-8 inhibitor II as well as by caspase-9 inhibitors I (Z-LEHD-FMK) and III (Ac-LEHD-CMK) indicates that receptor and mitochondrial pathways converged in procaspase-3 activation and contribute to proteolytic processing of effector procaspase-6 and -7. Western blot analyses and immunofluorescence staining revealed that exposure of Jurkat T cells to gal-1 resulted in the cleavage of the DNA-repair enzyme poly (ADP-ribose) polymerase, cytoskeletal α-fodrin, and nuclear lamin A as substrates of activated caspases. Our data demonstrate that Bid provides a connection between the death receptor and the mitochondrial pathway of gal-1 induced apoptosis in human Jurkat T lymphocytes.     

水飞蓟宾诱导肺腺癌Anip973细胞凋亡的分子机制研究

目的：探讨水飞蓟宾诱导肺腺癌Anip973细胞系细胞凋亡的分子机制。方法：采用MTT法、倒置显微镜和电子显微镜等形态学检测以及流式细胞仪（FCM）技术检测、DNALadder分析、凋亡分子PARP的表达检测细胞凋亡,同时进行凋亡相关蛋白Bax、Bcl-2、caspase-3和caspase-9表达活性分析。结果：（1）水飞蓟宾对人肺腺癌Anip973细胞系细胞的增殖有显著抑制作用;（2）水飞蓟宾作用Anip973细胞48h后,随着浓度的增加,倒置显微镜下可见细胞数目减少,胞体变小、变圆,到高浓度时出现较多的死亡细胞;（3）扫描电镜观察发现,随着水飞蓟宾作用浓度的增加,Anip973细胞中出现增多的凋亡细胞,凋亡细胞表现出典型的超微结构特征;（4）流式细胞仪检测的结果发现,随着药物作用时间的延长,Anip973细胞的G1期细胞比例增多,S期细胞明显减少,G2期细胞略有减少,并出现明显的凋亡峰。（5）水飞蓟宾作用后的Anip973细胞出现明显的DNALadder和PARP降解增加等凋亡特征;（6）水飞蓟宾作用后,Anip973细胞中的凋亡相关蛋白Bax表达增加、caspase-3和caspase-9酶活性增加,而Bcl-2表达降低。结论：水飞蓟宾在体外有抑制人肺腺癌细胞Anip973的增殖作用,并通过激活线粒体依赖的caspase凋亡通路,诱导其凋亡。     

水飞蓟宾诱导肺腺癌Anip973 细胞凋亡的分子机制研究

目的:探讨水飞蓟宾诱导肺腺癌Anip973细胞系细胞凋亡的分子机制。方法:采用MTT法、倒置显微镜和电子显微镜等形态学检测以及流式细胞仪(FCM)技术检测、DNALadder分析、凋亡分子PARP的表达检测细胞凋亡,同时进行凋亡相关蛋白Bax、Bcl-2、caspase-3和caspase-9表达活性分析。结果:(1)水飞蓟宾对人肺腺癌Anip973细胞系细胞的增殖有显著抑制作用;(2)水飞蓟宾作用Anip973细胞48h后,随着浓度的增加,倒置显微镜下可见细胞数目减少,胞体变小、变圆,到高浓度时出现较多的死亡细胞;(3)扫描电镜观察发现,随着水飞蓟宾作用浓度的增加,Anip973细胞中出现增多的凋亡细胞,凋亡细胞表现出典型的超微结构特征;(4)流式细胞仪检测的结果发现,随着药物作用时间的延长,Anip973细胞的G1期细胞比例增多,S期细胞明显减少,G2期细胞略有减少,并出现明显的凋亡峰。(5)水飞蓟宾作用后的Anip973细胞出现明显的DNALadder和PARP降解增加等凋亡特征;(6)水飞蓟宾作用后,Anip973细胞中的凋亡相关蛋白Bax表达增加、caspase-3和caspase-9酶活性增加,而Bcl-2表达降低。结论:水飞蓟宾在体外有抑制人肺腺癌细胞Anip973的增殖作用,并通过激活线粒体依赖的caspase凋亡通路,诱导其凋亡。     

Caspase-3 mRNA反义核苷酸对γ-射线诱导的HL-60细胞凋亡的影响

目的观察caspase-3 mRNA反义寡核苷酸(ASODN)对HL-60细胞凋亡的抑制作用,筛选有效ASODN。方法用脂质体介导法将针对caspase-3 mRNA不同序列的4条ASODN导入HL-60细胞中,γ-射线照射。应用电泳法检测DNA梯状条带;Hoechst 33258-碘化丙啶染色,荧光显微镜分析凋亡细胞百分率;流式细胞术进行细胞凋亡定量。结果以caspase-3 mRNA5’非编码区(-62至-46位)与编码起始区(-1至16位)ASODN转染,当转染终浓度≥3μmol/L时,DNA电泳梯状条带消失,流式细胞术亦未见明显的亚二倍体峰;荧光染色分析,凋亡细胞百分率比未转染对照组和错配寡核苷酸对照组显著降低(P<0.01),且随转染终浓度的增加,凋亡抑制率显著增加。另外,5’非编码区ASODN的抑制作用显著强于编码起始区ASODN(P<0.05)。结论caspase-3 mRNA ASODN可抑制γ-射线诱导的HL-60细胞凋亡,作用有序列特异性及剂量依赖性。该结果为细胞过度凋亡相关疾病的基因治疗提供了实验基础。     

Apoptosis induced by cadmium in human lymphoma U937 cells through Ca2+-calpain and caspase-mitochondria- dependent pathways

Apoptosis induced by cadmium has been shown in many tissues in vivo and in cultured cells in vitro. However, its molecular mechanism is not fully understood. When the human histiocytic lymphoma cell line U937 was treated with cadmium for 12 h, evidence of apoptotic features, including change in nuclear morphology, DNA fragmentation, formation of DNA ladder in agarose gel electrophoresis, and phosphatidylserine externalization, were obtained. Moreover, loss of the mitochondrial membrane potential (Deltapsi(m)) was observed in the cadmium-treated cells and was inhibited by a broad caspase inhibitor (Z-VAD-FMK). Caspase inhibitors suppressed the DNA fragmentation in the order of Z-VAD-FMK > caspase-8 inhibitor > caspase-3 inhibitor. Expression of Bcl-x(L) and Bid decreased significantly in the cadmium-treated cells, although no apparent change in Bcl-2 and Bax expression was found. Tetrakis-(2-pyridylmethyl) ethylendiamine, a cell-permeable heavy metal chelator, partially reversed the increase of fluorescence of Fura-2 in the cadmium-treated cells. In addition, verapamil (70 microm), a voltage-dependent Ca(2+) channel blocker, inhibited the DNA fragmentation induced by cadmium less than 100 microm and decreased the fluorescence of Fura-2. Cadmium up-regulated the expression of type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R) but not type 2 or type 3 IP(3)R. Calpain inhibitors I and II partially prevented DNA fragmentation. No effects of Z-VAD-FMK on the expression of type 1 IP(3)R or of calpain inhibitors on the loss of Deltapsi(m) were observed. These results suggest that cadmium possibly induced apoptosis in U937 cells through two independent pathways, the Ca(2+)-calpain-dependent pathway and the caspase-mitochondria-dependent pathway.     

Reconstitution of caspase-8 sensitizes JB6 cells to TRAIL

TRAIL induces apoptosis in various tumor cells. We report here that caspase-8 is required in TRAIL-induced cell death. Western blot analyses and enzyme assays showed that exposing Jurkat cells to TRAIL resulted in activation of caspases-8 followed by caspase-3 and -9. Acetyl-IETD-fluoromethylketone, a caspase-8 inhibitor, potently suppressed TRAIL-induced cell death compared to acetyl-DEVD-fluoromethylketone and acetyl-LEHD-fluoromethylketone, inhibitors of caspase-3 and caspase-9, respectively. JB6 cells, a caspase-8-deficient Jurkat variant, were completely resistant to TRAIL. However, reconstitution with a caspase-8, but not with caspase-2 or -3, sensitized JB6 cells to subsequent exposure to TRAIL. These results are indicative of the crucial function of caspase-8 in TRAIL-induced apoptosis in Jurkat cells.     

Caspase independent/dependent regulation of K(+), cell shrinkage, and mitochondrial membrane potential during lymphocyte apoptosis.

The loss of cell volume is a fundamental feature of apoptosis. We have previously shown that DNA degradation and caspase activity occur only in cells which have shrunken as a result of potassium and sodium efflux (Bortner, C. D., Hughes, F. M., Jr., and Cidlowski, J. A. (1997) J. Biol. Chem. 272, 32436-32442). Furthermore, maintaining a normal intracellular potassium concentration represses the cell death process by inhibiting the activity of apoptotic nucleases and suppressing the activation of effector caspases (Hughes, F. M., Jr., Bortner, C. D. Purdy, G. D., and Cidlowski, J. A. (1997) J. Biol. Chem. 272, 30567-30576). We have now investigated the relationship between cell shrinkage, ion efflux, and changes in the mitochondrial membrane potential, in addition to the role of caspases in these apoptotic events. Treatment of Jurkat cells with a series of inducers which act via distinct signal transduction pathways, resulted in all of the cell death characteristics including loss of cell viability, cell shrinkage, K(+) efflux, altered mitochondrial membrane potential, and DNA fragmentation. Interestingly, only cells which shrunk had a loss of mitochondrial membrane potential and the other apoptotic characteristics. Treatment of Jurkat cells with an anti-Fas antibody in the presence of the general caspase inhibitor z-VAD, abrogated these features. In contrast, when Jurkat cells were treated with either the calcium ionophore A23187 or thapsigargin, z-VAD failed to prevent cell shrinkage, K(+) efflux, or changes in the mitochondrial membrane potential, while effectively inhibiting DNA degradation. Treatment of Jurkat cells with various apoptotic agents in the presence of either the caspase-3 inhibitor DEVD, or the caspase-8 inhibitor IETD also blocked DNA degradation, but failed to prevent other characteristics of apoptosis. Together these data suggest that the cell shrinkage, K(+) efflux, and changes in the mitochondrial membrane potential are tightly coupled, but occur independent of DNA degradation, and can be largely caspase independent depending on the particular signal transduction pathway.