三氧化二砷诱导Hela细胞凋亡的机制研究

目的:了解三氧化二砷(As2O3)诱导的细胞凋亡是否与线粒体跨膜电位(Δψm)改变有关。方法:以Hela细胞为体外模型,应用碘化丙啶(PI)/Rhodamine123(Rh123)双重染色检测Δψm,通过测定细胞活力、流式细胞仪、HE染色及DNA电泳检测细胞凋亡。结果:As2O3处理Hela细胞后。台盼蓝拒染法测定细胞活力降低,流式细胞仪检测发现凋亡细胞明显增多,HE染色可见明显的细胞凋亡形态特征,琼脂糖凝胶电泳出现DNA梯形条带;As2O3使线粒体膜电位降低(P<0.01)。结论:As2O3在体外诱导Hela细胞凋亡的机制可能与降低线粒体膜电位有关。     

氯苄四氢小檗碱对抗H2O2引起无血清培养的血管内皮细胞凋亡及坏死

目的：研究氯苄四氢小檗碱对过氧化氢（H2O2）引起无血清培养的小牛主动脉血管内皮细胞的凋亡、增殖、坏死的影响。方法：通过体外细胞培养，以噻唑兰（MTT）法测定细胞存活率；用流式细胞术检测细胞DNA含量及凋亡细胞百分率、增殖率；DNA琼脂糖凝胶电泳法观察细胞凋亡过程中DNA断裂程度。结果：H2O2诱导无血清培养的内皮细胞凋亡，氯苄四氢小檗碱可显降低内皮肌细胞中凋亡细胞百分率，并抑制其增殖，也减少凋亡细胞DNA断裂，同时也减少其引起的平滑肌细胞坏死。结论：氯苄四氢小檗碱可对抗H2O2引起的无血清培养的血管平滑肌细胞凋亡、增殖及坏死。     

三氧化二砷诱导喉癌细胞凋亡的体外研究

目的探讨三氧化二砷(As2O3)诱导喉癌Hep-2细胞株凋亡的作用。方法体外培养的Hep-2细胞与不同浓度的As2O3作用不同时间,用流式细胞仪、DNA琼脂糖凝胶电泳研究细胞凋亡的发生及对细胞周期的影响。结果流式细胞仪检测发现不同药物浓度和作用时间下均有细胞凋亡发生。2滋mol/LAs2O3引起细胞周期明显变化,作用24h后S期细胞比例增高,72h后S期细胞明显下降,细胞大量凋亡。结论As2O3可有效抑制人喉癌Hep-2细胞的体外生长,具有时间、浓度依赖性。As2O3诱导Hep-2细胞凋亡主要是S期的细胞。     

砷诱导的肝癌细胞凋亡及端粒酶活性改变

目的：研究三氧化二砷（As2O3)对肝癌细胞凋亡的诱导作用及其机制。方法：1μmol/L As2O3处理培养的人肝癌HCC－9204细胞48h，用形态学观察、细胞超微结构分析和流式细胞仪检测等方法鉴定细胞凋亡。用噻唑蓝（MTT）比色试验观察蛋白质全盛抑制剂放线菌素D对As2O3凋亡诱导作用的影响。用TRAP－PCR－ELISA法检测As2O3处理前后肝癌细胞端粒酶活性的变化情况。结果：1μmol/L As2O3作用48h后，HCC－9204细胞出现了典型的凋亡形态学改变和超微结构变化；流式细胞仪检测到大量Annexin-阳性、PI－阴性细胞、放线菌素D可明显拮抗As2O3的凋亡诱导作用。细胞凋亡发生后端粒酶活性显著降低，A450/A630值从2．874降为0．767。结论：As2O3可以有效诱导肝癌细胞凋亡，端粒酶活性降低可能是其中的机制之一。     

Apoptin基因活化caspase-3诱导人黑素瘤细胞A375凋亡

为研究肿瘤细胞凋亡基因（apoptingene）诱导人黑素瘤细胞系A375凋亡时是否通过活化easpase-3发挥作用，用含有apoptin基因的真核表达载体短时转染体外培养的人黑素瘤细胞A375；采用RT-PER、DNA凝胶电泳、流式细胞术检测A375细胞的凋亡；以比色法检测easpase-3的相对活性。结果表明，apoptin基因短时转染的A375细胞可出现典型的细胞凋亡所具有的DNA梯状带；流式细胞术显示实验组细胞凋亡率明显高于其它各组（P〈0．01）；转染后24h实验组caspase-3的活性开始升高，72h达高峰，明显高于其它各组（P〈0．01）。结论：apoptin基因可活化caspase-3诱导人黑素瘤细胞A375凋亡。     

三氧化二砷和维生素C联合诱导肝癌细胞凋亡的作用研究

目的探讨维生素C(Vit C)与三氧化二砷（As2O3）促肝癌细胞株（HepG2）凋亡的协同效应。 方法体外培养人肝癌细胞株HepG2，以As2O3，Vit C，以及它们不同浓度组合孵育细胞，采用四甲基偶氮唑蓝（MTT）法，Annexin V/PI双染色流式细胞术来观察各组的细胞凋亡情况，并通过流式软件分析细胞周期变化。结果As2O3与Vit C联用能显著提高单用As2O3的细胞抑制率和细胞凋亡率(P＜0.05)，Vit C能显著增强As2O3诱导细胞集中于S期的作用，从而增强其促细胞凋亡作用。结论Vit C增强三氧化二砷诱导细胞凋亡的作用，该作用可能与影响细胞周期有关。     

槲寄生凝集素对人结肠癌HT-29细胞增殖及凋亡的影响

目的：研究槲寄生凝集素对人结肠癌HT-29细胞增殖和凋亡的影响。方法：采用MTT法观察槲寄生凝集素对体外培养的人结肠癌HT-29细胞增殖的影响,运用基因组DNA电泳观察凋亡特征性“梯状”条带及原位末端标记法检测槲寄生凝集素诱导HT-29细胞凋亡。结果：槲寄生凝集素对体外培养的人结肠癌HT-29细胞抑制作用表现出时间和剂量依赖性。基因组DNA琼脂糖凝胶电泳显示槲寄生凝集素作用于HT-29细胞后出现凋亡细胞特有的DNA阶梯状条带。原位末端标记法检测显示槲寄生凝集素可明显诱导HT-29细胞的凋亡。结论：槲寄生凝集素可抑制人结肠癌HT-29细胞生长并诱导其凋亡。     

三氧化二砷诱导粒细胞性白血病原代细胞凋亡的研究

目的：探讨三氧化二砷（As2O3)对慢性粒细胞性白血病（CML）慢性期原代细胞生长的影响。方法：通过细胞增殖,活力检测,形态学观察,粒系髓系集落形成（CFU－GM）集落培养,凝胶电泳等方法检测。结果：As2O3可抑制CML原代细胞增殖,经平均浓度5．0umol.L^-1 As2O3处理5d时抑制率为50％,经大于等于2．6－5．0umol.L^-1 As2O3培养2－5d,可引起细胞凋亡的形态学改变及DNA片段化,As2O3浓度2．5umol.L^-1,CFU－GM的形成抑制率＞50％,结论：As2O3可以抑制CML慢性期原代细胞的增殖,并诱导凋。     

As2O3诱导K562细胞凋亡过程中细胞表面电荷变化的研究

目的 :研究三氧化二砷 (As2 O3 )诱导K562 细胞凋亡时细胞表面电荷的变化 ,阐明As2 O3 诱导K562 细胞凋亡的可能机制 ,为As2 O3 在临床上的应用提供理论依据。方法 :应用细胞电泳仪检测As2 O3 诱导K562 细胞凋亡中细胞电泳率的变化。通过细胞增殖、活力检测 ,形态学观察 ,亚G1期细胞含量和DNA凝胶电泳等鉴定细胞凋亡。结果 :1.0～ 2 0 μmol·L-1As2 O3 作用于K562 细胞 ,在细胞形态、DNA凝胶电泳、FCM显示出典型的细胞凋亡特征之前 ,试验组细胞表面电荷已在 1.6h左右开始下降 ,6h内降低最明显 ,6h后虽有下降 ,但幅度明显减弱。与对照组比较 ,低于 1.0 μmol·L-1的As2 O3 对细胞表面电荷影响不大。结论 :细胞表面电荷的下降是K562 细胞凋亡的早期事件。细胞表面电荷的下降有一定的时间范围 ,超过此范围 ,细胞表面电荷下降趋向无限大。     

三氧化二砷对肝癌细胞内活性氧水平的影响

目的探讨三氧化二砷（As2O3）对肝癌细胞增殖及细胞内活性氧（ROS）水平的影响。方法用As2O3作用于体外培养的人肝癌SMMC-7721细胞株，分别用MTT法和流式细胞仪观察SMMC-7721细胞增殖情况并检测ROS。结果MTT结果显示As2O3能明显抑制SMMC-7721细胞的增殖．并呈时间和浓度依赖性。流式细胞仪分析显示As2O3作用后的人肝癌SMMC-7721细胞内ROS水平明显增高（P〈0．01）．并也呈时间和浓度依赖性。结论As2O3可通过抑制人肝癌SMMC-7721细胞的增殖和提高细胞内ROS水平诱导细胞凋亡而发挥抗肿瘤作用，这可能也是As2O3抗肝癌的主要途径之一。     

VEGF通过对p38和p42／p44 CCDPK信号的共调节作用抑制TNF—α和H2O2诱导的牛主动脉内皮细胞凋亡

目的：研究血管内皮细胞生长因子（VEGF）对肿瘤坏死因子（TNF－α）和过氧化氢（H2O2）诱导主动脉内皮细胞（BAEC）凋亡的影响及信号机制。方法：BAEC培养并传代于DMEM。经TNF－α,或H2O2处理24h后,Hoechst 33258染色,荧光显微镜观察形态学变化及凋亡细胞计数。MTT法测定细胞活性,琼脂糖凝胶电泳DNA解,Western blot法检测磷酸化p38和p42/p44 CCDPK表达。结果：TNF－α5000kU/L和H2O300μmol/L均可诱导BAEC产生DNA断片。VEGF100μg/L显著增强TNF－α和H2O2诱导的磷酸化p42-p44 CCDPK表达,而明显抑制磷酸化p38 CCDPK的活化。对二者所致BAEC凋亡起明显的抑制作用。p42/p44 CCDPK抑制剂U0126可取消VEGF引起的磷酸化,p42/p44 CCDPK表达上调和其抗凋亡作用。结论：VEGF通过其共调节作用激活p42/p44 CCDPK,抑制p38 CCDPK信号途径而对TNF－α和H2O2所致凋亡产生的抑制效应,是内皮细胞存活的重要机制。     

Effects of {2-[（3-carboxy-l-oxoprogyl）amino]-2-deoxy-D-glucose} on human hepatocellular carcinoma cell line

Aim: To study the effects of {2-[(3-carboxy-1-oxoprogy 1)amino]-2-deoxy-D-glucose (COPADG) on cultured human hepatocellular carcinoma cells (HepG2).Methods: HepG2 cells were cultured in RPMI-1640 medium. Cell proliferation was determined by MTF assay. Apoptosis was determined by fluorescence microscopy,transmission electron microscopy, agarose gel electrophoresis of DNA fragmentation, and flow cytometry. Results: At the concentration ranging between 1-30μmol/L, COPADG potently inhibited the growth and induced apoptosis of HepG2 cells. Conclusion: COPADG could effectively induce apoptosis in human hepatocellular carcinoma cells. More investigations are warranted for the potential use of this compound as a new agent for the non-surgical management of human hepatocellular carcinoma.     

冬凌草甲素通过诱导人宫颈癌HeLa细胞自噬下调凋亡的机制

研究冬凌草甲素通过诱导人宫颈癌HeLa细胞自噬拮抗凋亡的机制。MTT法测定冬凌草甲素对HeLa细胞的细胞毒作用。通过相差显微镜观察细胞形态学变化，用琼脂糖凝胶电泳检测DNA片段化，用流式细胞仪检测细胞自噬和凋亡水平，用Western blotting检测分析药物对蛋白质表达的影响。冬凌草甲素明显抑制HeLa细胞的增殖，诱导HeLa细胞凋亡，同时诱导HeLa细胞发生自噬。Western blotting检测结果表明，冬凌草甲素作用24 h后，促凋亡蛋白Bax、细胞色素c和控制Bax活力的去乙酰化酶SIRT-1的表达明显改变。冬凌草甲素(64 μmol·L^-1)诱导的自噬通过影响SIRT-1和线粒体途径蛋白的表达下调凋亡。     

西红花苷对氧化甾醇诱导血管内皮细胞凋亡的影响

目的:研究西红花苷对培养的牛血管内皮细胞凋亡的影响。方法:采用3β、5α、6β三羟胆固(烷)醇(Triol)诱导内皮细胞凋亡的模型,用比色法测定丙二醛含量,光学显微镜、电子显微镜检测形态和超微结构的变化,DNA电泳检测DNAladder及流式细胞仪分析法检测凋亡率,采用逆转录聚合酶链式反应(RTPCR)法检测BaxmRNA表达的变化。结果:Triol处理后,培养液中MDA含量增加,为1.761nmol·L-1(P<0.01),细胞收缩,核浓缩,深染,线粒体肿胀空化,出现凋亡小体,出现凋亡典型的“DNAladder”,和亚二倍体峰,凋亡率为30.62%,BaxmRNA表达量增加;西红花苷(10-7,10-6mol·L-1) Triol组,MDA含量减少,内皮细胞形态结构的完整,凋亡率分别为24.4%,6.3%,BaxmRNA表达量降低。结论:西红花苷可能是抗脂质过氧化并通过调节BaxmRNA表达,减少细胞异常凋亡。     

姜黄素抑制宫颈癌细胞株增殖和诱导凋亡的体外研究

目的：探讨中药姜黄素（Cur）对子宫颈癌SiHa细胞株的增殖抑制和诱导凋亡的作用。方法：以不同浓度的姜黄素（10umol／L、20umol／L、30umol／L）,分12h、24h、48h、72h4个时间点处理SiHa细胞,光镜观察细胞形态变化;用四甲基偶氮唑蓝（MTT）法测定细胞增殖抑制率;用透射电子显微镜及琼脂糖凝胶电泳检测凋亡的发生。结果：姜黄素可抑制SiHa细胞增殖,作用呈明显的时效和量效关系,差异有非常显著性（P〈0.01）;姜黄素可诱导SiHa细胞凋亡,透射电子显微镜下可见凋亡细胞;琼脂糖凝胶电泳出现细胞凋亡典型的DNA“梯状”条带。结论：姜黄素体外对SiHa细胞具有增殖抑制作用和促进凋亡作用。     

Effects of p38 and p42/p44 CCDPK signaling on H2O2-induced apoptosis in bovine aortic endothelial cells

AIM: To investigate the effects of p38 and p42/p44 Ca(2+)-calmodulin dependent protein kinases (CCDPK) signaling on hydroperoxide (H2O2)-induced apoptosis in cultured bovine aortic endothelial cells (BAEC). METHODS: Morphologic changes and quantification of apoptotic cells were determined under fluorescence microscope after a 24-h treatment of BAEC by H2O2. Cell viability was determined with MTT method. DNA fragmentation was visualized by agarose gel electrophoresis. The expression of phospho-p38 and phospho-p42/p44 CCDPK was measured by Western blotting. RESULTS: H2O2 elicited typical apoptotic morphologic changes (chromatic condensation, nucleus fragmentation) and DNA fragmentation. At 100-500 mumol.L-1, incubation of BAEC with H2O2 for 24 h also induced phospho-p38 and phospho-p42/p44 CCDPK expression in a concentration-dependent manner. Interestingly, H2O2-induced apoptosis was markedly increased by preincubation with U0126, a specific p42/p44 CCDPK inhibitor. However, SB203580, a specific p38 CCDPK inhibitor, enhanced the expression of phospho-p42/p44 CCDPK induced by H2O2, but had no effect on BAEC survival. CONCLUSION: p42/p44 CCDPK signaling appears to play protective roles in H2O2-induced apoptosis in BAEC, whereas p38 CCDPK is not the main signaling pathway mediating H2O2-induced cellular apoptosis.     

PC-407 inhibited proliferation and induced apoptosis in human colon cancer SW-1116 cells

AIM: To study whether PC-407 [4-[5-naphthyl-3- (trifluoromethyl)-1H-pyrazol-1-yl] benzenesulfonamide] inhibits cell viability and induces apoptosis in human colon cancer SW-1116 cells. METHODS: Inhibition of SW-1116 proliferation was measured by MTT assay. Morphological assessment of apoptosis was performed with fluorescence microscope and electron microscope. DNA fragmentation was visualized by agarose gel electrophoresis. The amount of apoptotic cells was measured by flow cytometry. RESULTS: PC-407 inhibited SW-1116 cell proliferation in a concentration-dependent manner after 3 d of treatment, and the IC50 for PC-407 inhibition of cell number was 16.67±0.17 μmol/L. After incubation of SW-1116 cells with PC-407 20 μmol/L for 24 h, morphological changes of typical apoptosis were observed by AO/EB staining or transmission electron microscopy. Flow cytometry analysis showed that PC-407 induced apoptosis in SW-1116 cells in a time- and concentration-dependant manner. The agarose gel electrophoresis of DN     

Biphasic effect of aspirin on apoptosis of bovine vascular endothelial cells and its molecular mechanism

AIM: To investigate the effect of aspirin on the apoptosis of cultured bovine aortic endothelial cells (BAEC) and the signal pathways involved in this process. METHODS: BAEC were cultured and passaged in Dulbecco's modified Eagle's medium culture medium. Morphologic changes and quantification of apoptotic cells were determined using fluorescence microscope after staining the cells with Hoechst 33258. Cell viability was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) method. DNA fragmentation was visualized by agarose gel electrophoresis. Phospho-p38 mitogen-activated protein kinase (MAPK) expression was detected by Western blotting. RESULTS: Aspirin at low concentrations from 1X10( -10) mol/L to 1X10( -8) mol/L decreased the apoptosis and p38 MAPK phosphorylation induced by H2O2 in BAEC, while high doses of aspirin (1X10( -7)-1X10( -4) mol/L) induced typical apoptotic changes in BAEC and stimulated the expression of phospho-p38 MAPK in a concentration-dependent manner. SB203580, a specific p38 MAPK inhibitor, blocked such effects. CONCLUSION: Aspirin exhibits a biphasic effect on the apoptosis in BAEC, reducing apoptosis at low concentration and inducing apoptosis at high concentration. p38 MAPK may be an important signal molecule mediating the effects of aspirin.     

Diosgenin induces apoptosis in HeLa cells via activation of caspase pathway

AIM: To investigate the mechanism of diosgenin-induced HeLa cell apoptosis. METHODS: HeLa cell growth was measured by MTT method. Apoptosis was detected by electron microscopy and agarose gel electrophoresis. Ratio of apoptotic cells was measured by APO-BRDU kit. Cell cycle distribution and changes of mitochondrial membrane potential were monitored by flow cytometry. Caspase activities were assayed by caspase apoptosis detection kit. Western blot analysis was used to evaluate the level of mitochondrial Bcl-2 expression. RESULTS: Diosgenin inhibited HeLa cell growth. HeLa cells treated with diosgenin showed typical characteristics of apoptosis including the morphological changes and DNA fragmentation. Caspase family inhibitor (z-VAD-fmk), caspase-9 inhibitor (Ac-AAVALPAVLLALLAPLEHD-CHO), and caspase-3 inhibitor (z-DEVD-fmk) partially prevented diosgenin-induced apoptosis, but not caspase-8 inhibitor (z-IETD-fmk) and caspase-10 inhibitor (z-AEVD-fmk). Diosgenin caused reduction of mitochondrial membrane po     

天佛参口服液抗肿瘤机理的实验研究

目的 观察天佛参口服液对Hep-2细胞的影响以探讨其抗肿瘤的机理。方法 采用体外细胞培养实验方法，应用MTT、透射电镜，DNA琼脂糖凝胶电泳，TUNEL等技术，检测药物对细胞的抑制率及凋亡率。结果 MTT测得细胞抑制率随时间，剂量而增大，活细胞观察，透射电镜下见到凋亡细胞及凋亡小体形成，电泳结果显示清晰的DNAladder,TUNEL法检测出细胞凋亡率具有时间，剂量依赖性，药物组与对照组有显著性差异。结论 天佛参口服液能诱导Hep-2细胞凋亡，细胞凋亡率随时间延长增加，且呈现剂量依赖性。