DMSO诱导MCF-7细胞凋亡的研究

目的研究二甲基亚砜(DMSO)对MCF-7细胞凋亡的诱导作用。方法用不同浓度DMSO处理体外培养的MCF-7细胞,应用倒置光显微镜观察细胞形态学变化,用MTT比色法检测细胞存活率;Hoechst33258/PI荧光染色,用荧光显微镜分析凋亡细胞比率;琼脂糖凝胶电泳检测DNA梯状条带。结果在倒置光学显微镜下观察1%DMSO处理细胞12h后细胞形态发生变化。约有50%以上的细胞变圆,细胞内有多泡小体形成。随DMSO浓度增加和作用时间的延长,细胞存活率明显下降,经MTT检测其IC_(50)值为1%;荧光显微镜下可见60%以上细胞核染色质凝集,核碎裂等凋亡细胞的形态学变化;琼脂糖凝胶电泳呈现梯状条带(DNA ladder)。结论适当浓度的DMSO能够抑制乳腺癌细胞增殖并诱导其凋亡。     

survivin反义寡核苷酸在体外诱导胰癌细胞凋亡

目的：探讨survivin反义核酸对胰腺癌细胞株Panc-1细胞凋亡的影响.方法：用脂质体瞬时转染法介导survivin反义核苷酸处理胰腺癌Panc-1细胞后,MTT试验测定转染细胞的相对存活率,RT-PCR检测survivin mRNA表达,琼脂糖凝胶电泳分析其对Panc-1细胞的凋亡诱导作用.结果：转染survivin反义核酸的Panc-1细胞增殖明显受抑制,与对照进行比较,具有显著性差异（P＜0.05）;经琼脂糖凝胶电泳,转染survivin反义核酸的Panc-1细胞可见到DNA梯形条带,而对照细胞未见到.结论：survivin反义核酸能够诱导Panc-1细胞凋亡.     

伪狂犬病病毒Ea株诱导牛肾细胞凋亡及核基质蛋白的变化

目的研究伪狂犬病病毒(PrV)是否具有诱导组织培养细胞发生细胞凋亡的功能及探讨凋亡细胞核基质蛋白表达的变化。方法利用锥虫蓝(台盼蓝)染色绘制PrV在不同细胞上的增殖曲线;荧光染色观察凋亡细胞核的形态特征;琼脂糖凝胶电泳分析细胞染色体DNA的片段化;高分辨率双向电泳分析细胞凋亡前后核基质蛋白的表达差异。结果膜通透性的DNA特异性结合染料Hoechst 33342染色显示,PrV感染后36h,牛肾(MDBK)细胞核染色质开始固缩、凝聚,细胞核碎裂;DNA提取及琼脂糖凝胶电泳分析表明,细胞染色体DNA发生片段化,形成“DNA梯状”条带(DNA ladder);尽管PrV可以诱导MDBK细胞发生细胞凋亡,但不能诱导IBRS-2,BHK-21及PK-15细胞发生凋亡。MDBK细胞发生凋亡前后核基质蛋白的表达发生改变,存在表达上调、下调、诱导表达及表达遏制等情况。结论PrV诱导细胞发生细胞凋亡是其致细胞死亡的形式之一,间接反映了PrV与宿主细胞间复杂的相互作用。细胞凋亡前后核基质蛋白表达存在的差异说明PrV致MDBK细胞发生凋亡是自身基因产物或诱导或抑制宿主细胞基因表达的结果。     

鲨鱼软骨抽提物诱导人胃癌细胞凋亡并下调Bcl-2表达

目的：探讨鲨鱼软骨抽提物(SCP)诱导人胃癌MGC80—3细胞调亡的作用机制。方法：以不同浓度SCP加入体外培养的MGC80—3细胞中，用MTT比色法检测细胞存活率；Hoeehst33342／PI荧光染色，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；琼脂糖凝胶电泳检测DNA梯状条带；免疫细胞化学染色法检测Bel-2蛋白的表达。结果：SCP明显抑制(MGC80—3细胞生长，IC50值为1mg／ml；荧光显微镜下可见60％以上细胞为凋亡细胞的形态学改变；琼脂糖凝胶电泳呈现梯状条带(DNA ladder)；免疫细胞化学检测显示Bcl-2表达明显降低。结论：SCP诱导人胃癌MGC80—3细胞凋亡，其作用机制可能与下调Bcl-2蛋白的表达水平有关。     

顺铂诱导大鼠C6胶质瘤细胞凋亡

为深入进行胶质瘤细胞凋亡的分了生物学研究及提高胶质瘤辅助化疗疗效打下基础。通过光镜,电镜,荧光显微镜分析,DNA断裂分析及流式细胞仪分析,进行顺铂诱导C6胶质瘤细胞调亡研究。本研究证实在3μg／mLCDDP作用72h,C6细胞出观细胞凋亡;光镜和电镜可见细胞形态学上出现细胞皱缩,染色质浓集贴达;流式细胞仅结果提示有凋亡峰出现,凋亡细胞占细胞总数17.3％±1.2％;荧光显微镜观察出现染色质浓集,染色质断裂：DNA电泳未表现出DNA呈梯状带型断裂。上述结果提示用顺铂成功地诱导大鼠C6胶质瘤细胞发生凋亡。     

姜黄素诱导永生化人HaCaT细胞凋亡

目的 探讨姜黄素对人永生化上皮细胞凋亡的诱导作用.方法 应用细胞计数、流式细胞术、琼脂糖凝胶电泳、Hoechst33258染色、苏木素-伊红(HE)染色和透射电镜检测经姜黄素诱导处理后人永生化上皮HaCaT细胞的凋亡.结果经7.5mg/L姜黄素诱导处理后,人永生化上皮HaCaT细胞的增殖活动受到明显的抑制,细胞生长抑制率达83.03%;细胞周期检测出现亚二倍体(亚G1期)细胞峰值,细胞凋亡率达13.1%,并发生G2/M期阻滞;琼脂糖凝胶电泳出现细胞凋亡典型的DNA"梯状"条带;细胞核经Hoechst33258染色出现浓染致密的固缩形态和颗粒状荧光;光镜和电镜观察结果显示,姜黄素处理组细胞体积缩小,细胞核固缩,染色质凝聚,线粒体肿胀,有凋亡小体形成等显著的凋亡特征.结论姜黄素对人永生化上皮HaCaT细胞凋亡有显著的诱导作用,从而为进一步研究表皮细胞衰老、凋亡机理提供了重要基础和研究依据.     

含蟾酥胶囊血清诱导人肝癌BEL-7402细胞株凋亡的机制

目的:探讨含蟾酥胶囊血清诱导人肝癌BEL-7402细胞株凋亡的机制.方法:用血清药理学方法,把含不同浓度蟾酥胶囊的血清加入体外培养的人肝癌BEL-7402细胞,分别孵育24、48 h,显微镜下观察细胞形态学改变;MTT比色检测细胞的存活率;流式细胞仪检测细胞的凋亡率;琼脂糖凝胶电泳测定DNA条带;免疫细胞化学显色检测Bcl-2蛋白的表达.结果:实验组部分细胞凋亡,形态学改变;细胞存活率降低,增殖受到抑制;流式细胞仪检测,可见凋亡峰;孵育48 h,琼脂糖凝胶电泳呈梯状条带(DNA Ladder);免疫细胞化学检测,Bcl-2表达明显降低.结论:蟾酥胶囊诱导人肝癌细胞BEL-7402株凋亡的作用机制可能与下调Bcl-2蛋白表达有关.     

含蟾酥胶囊血清诱导人肝癌BEL-7402细胞凋亡并下调Bcl-2的表达

目的 探讨含蟾酥胶囊血清诱导人肝癌BEL-7402细胞凋亡的作用机制.方法 用中药血清药理学方法,不同浓度含药血清加入体外培养的人肝癌BEL-7402细胞,分别孵育24h、48h,显微镜下观察细胞形态学变化,MTT比色法检测细胞存活率,流式细胞仪检测细胞凋亡率,琼脂糖凝胶电泳测定DNA梯状条带,免疫细胞化学染色检测Bc...     

8-硝基白杨素对人宫颈癌Hela细胞增殖和凋亡的影响

目的：观察8-硝基白杨素(8-NOChR)抑制体外培养人宫颈癌Hela细胞增殖和诱导凋亡作用。方法：体外培养人宫颈癌Hela细胞系细胞。MTT比色法测定Hela细胞增殖活性。软琼脂培养克隆形成法检测Hela细胞集落形成能力。AO/EB染色荧光显微镜观察Hela细胞凋亡形态学改变。DNA凝胶电泳观察梯形DNA条带。结果：MTT比色测定显示,8-NOChR抑制Hela细胞增殖,呈剂量依赖性。软琼脂培养克隆形成法检测表明,8-NOChR显著抑制Hela细胞集落形成,呈剂量依赖性。AO/EB染色荧光显微镜观察发现,8-NOChR诱导Hela细胞呈现典型凋亡细胞形态特征。8-NOChR（30μmol/L）处理Hela细胞72h,琼脂糖凝胶电泳出现“梯形”DNA条带。结论：8-NOChR具有抑制人宫颈癌Hela细胞增殖和诱导细胞凋亡作用     

无血清培养体系中CD3AK细胞诱导HL—60细胞凋亡的研究

目的：采用无血清培养体系,研究CD3单克隆抗体激活的杀伤（CD3AK）细胞诱导HL－60细胞凋亡情况。方法：用血清代用品代替人血清培养CD3AK细胞,将诱导生成的CD3AK细胞与HL－60细胞共同孵育后,通过观察细胞形态、琼脂糖凝胶电泳检测DNA条带及充式细胞仪（FCM）检测分析HL－60细胞凋亡情况。结果：与CD3AK细胞孵育7h后的HL－60细胞出现凋亡细胞形态特征,DNA凝胶电泳典型梯状电泳带,FCM检测凋亡细胞比例为24.59%。结论：无血清培养条件下CD3AK细胞可诱导HL－60细胞凋亡。     

胸腺嘧啶激酶/丙氧鸟苷体外杀伤携带胸腺嘧啶激酶基因的C6胶质瘤细胞时细胞凋亡的检测

目的检测胸腺嘧啶激酶基因转染C6胶质瘤细胞后用丙氧鸟苷处理时细胞凋亡的发生。方法用脂质体介导的基因转染将单纯疱疹病毒胸腺嘧啶激酶基因转入C6胶质瘤细胞中,用G418筛选,建立稳定表达胸腺嘧啶激酶的C6细胞株;加入丙氧鸟苷作用不同时间,用原位末端标记、透射电镜和DNA凝胶电泳法检测。结果用丙氧鸟苷作用72h发现有大量原位末端标记阳性的细胞,阳性信号呈块状或半月形位于核膜内侧;透射电镜发现肿瘤细胞核内染色质呈块状或帽状位于核膜内侧,非染色质部分呈空泡状,细胞质有的固缩,细胞器基本保持完整;有的则高度肿胀,细胞内亚单位遭到破坏;DNA凝胶电泳发现用丙氧鸟苷作用72h细胞基因组DNA呈梯状带。结论实验结果提示胸腺嘧啶激酶/丙氧鸟苷系统作用于表达胸腺嘧啶激酶的C6细胞时该细胞主要是以凋亡的方式死亡的。     

Simvastatin induces proliferation inhibition and apoptosis in C6 glioma cells via c-jun N-terminal kinase

The lipid-lowering drugs, statins, induce apoptosis in a variety of tumor cells. Here we investigated the apoptotic effect of the lipophilic statin, simvastatin, in C6 glioma cells and the underlying effects on intracellular signal transduction. Simvastatin inhibited cell proliferation totally after 20 h of treatment as shown by the decrease in proliferating cell nuclear antigen expression in the nucleus. Subsequently, simvastatin caused apoptotic cell death by shrinkage of cytoplasm and condensation of chromatin, and DNA fragmentation. The features of apoptosis were visible only after 48 h of treatment, possibly reflecting a requirement for cell commitment to growth arrest. In immunocytochemical and immunoblotting experiments we have shown that simvastatin markedly increased the phosphorylation of ATF-2 and c-jun in the nucleus of the C6 glioma cells at early time points which was preserved even 24 h after treatment. In contrast, activities of protein kinases Erk1/2 and AKT in the cell survival pathway remained unchanged throughout the treatment. Selective inhibitor of JNK, but not p38 kinase, reduced simvastatin-induced cell death and ATF-2 and c-jun phosphorylation suggesting that JNK-dependent activation of ATF-2 and c-jun may play an important role in simvastatin-induced proliferation inhibition and apoptosis in C6 glioma cells. These observations suggest that statins may have clinical significance in the prevention of glial tumors beyond their cholesterol-lowering effect and JNK may be a rational target for sensitizing glioma cells to chemotherapeutic agents.     

用反义寡核苷酸阻断热休克蛋白70表达诱导卵巢癌细胞凋亡

目的 观察特异性热体克蛋白７０（ｈｅａｔ－ｓｈｏｃｋ ｐｒｏｔｅｉｎ,ＨＳＰ）反义寡核苷酸阻断卵巢癌细胞的ＨＳＰ７０表达,及其对卵巢癌细胞生长和增殖的影响。方法 用胎盘蓝拒染法计算卵巢癌细胞的生长抑制率。Ｇｉｅｎｓａ染色法从形态上了解凋亡的发生,用琼脂糖凝胶电脉进一步检测发生凋亡的特征性ＤＮＡ降解,流式细胞仪定量分析凋亡发生率及周期性异性。结果 用ＨＳＰ７０反义寡核苷酸处理的卵巢细胞表现有显的生长     

Apoptosis induced by generated OH radicals inside cells after irradiation

OH radicals play a major role in radiation-induced DNA and cell membrane damage. These types of damage can also induce death by apoptosis through activation of a pro-apoptosis pathway. We attempted to detect OH radicals inside human promyelocytic leukemia (HL60) cells and estimate the relationship between radiation-induced apoptosis and OH radicals generated inside the cells. Electron spin resonance spectroscopy showed that OH radicals were generated by X-rays within irradiated cell pellets and the relative signal intensities of OH radicals increased with the radiation dose. Agarose gel electrophoresis revealed that the death of HL60 cells by apoptosis was accompanied by internucleosomal DNA fragmentation at 2 h after irradiation with 10-30 Gy. On ultrastructure evaluation by transmission electron microscopy, certain irradiated HL60 cells demonstrated condensed chromatin forms at the nuclear membrane and nuclear fragmentation. The frequency of apoptotic cells with condensation and fragmentation of nuclear chromatin increased with radiation dose in semithin sections. The increase of quantitative DNA fragmentation and percentage of non-living cells also correlated with radiation dose. These results suggest that OH radicals are generated inside cells before apoptosis occurs. The amount of OH radicals generated correlates with apoptotic cell death.     

Oridonin-induced apoptosis of Jurkat cells and its mechanism

Jurkat cells in culture medium in vitro were exposed to different concentrations of oridonin. The proliferation rate of the cells was measured by MTT assay, cell apoptotic rate was detected by flow cytometry, morphology of cell apoptosis was observed by Hoechst 33258 fluorescence staining, DNA fragmentation was assayed by agarose gel electrophoresis, caspase-3 and poly(ADP-ribose) polymerase (PARP) expressions were detected by Western blotting using polyclonal anti-caspase-3 antibody and mouse anti-human PARP monoclonal antibodies, and caspase-3 activity was assayed with a colorimetric assay kit before and after apoptosis had occurred. Oridonin (over 32 mol/l) inhibited the growth of Jurkat cells and caused significant apoptosis. The suppression was both time-dependent and dose-dependent. Marked morphological changes of cell apoptosis, including condensation of chromatin and nuclear fragmentation, were clearly observed by Hoechst 33258 fluorescence staining, as well as by agarose gel electrophoresis. Western blotting showed cleavage of the caspase-3 zymogen protein (32 kDa), with the appearance of its 17 kDa subunit, and a cleaved 89-kDa fragment of 116 kDa PARP was also found, together with a concurrent increase in caspase-3 apoptotic activity. Oridonin can induce apoptosis in Jurkat cells via activation of caspase-3; the results indicate that oridonin might be an important potential anti-leukaemia reagent.     

人endostatin体外诱导血管内皮细胞凋亡的实验研究

目的 探讨人endostatin抑制血管内皮细胞增殖的作用机制。方法 在含重组人endostatin蛋白和10%小牛血清的DMEM培养基中，培养人脐静脉内皮细胞ECV304。72h后，采用透射电镜，流式细胞术细胞周期分析和细胞核DNA的1.5%琼脂糖凝胶电泳，检测重组人endostatin蛋白作用后，血管内皮细胞的凋亡。结果 透射电镜下可见实验组ECV304细胞核染色质浓缩，边集，核碎裂及胞浆浓缩等，呈典型的凋亡细胞的形态学表现，流式细胞术细胞周期分析显示，在G1期峰前存在1个凋亡峰（20.6%)，1.5%琼脂糖凝胶电泳显示，细胞核DNA呈梯状，对照组ECV304细胞表达正常。结论 重组人endostatin蛋白可诱导血管内皮细胞凋亡，是其抑制血管内皮细胞增殖的原因之一。     

Induction of apoptosis in a carp leucocyte cell line infected with turbot (Scophthalmus maximus L.) rhabdovirus

A rhabdovirus was observed from the diseased turbot (Scophthalmus maximus L.) with lethal syndrome. In this study, a carp leucocyte (CLC) cell line was used to investigate the infection process and cell death mechanism occurring during the virus infection. Strong cytopathogenic effect (CPE) and the morphological changes, such as extreme chromatin condensation, nucleus fragmentation, and apoptotic body formation, were observed under fluorescence microscopy after DAPI staining in the infected CLC cells. Transmission electron microscopy analysis showed cell shrinkage, plasma membrane blebbing, cytoplasm vacuolization, chromatin condensation, nuclear breakdown and formation of discrete apoptotic bodies. The bullet-shaped nucleocapsids were measured and ranged in size from 110 to 150 nm in length and 40 to 60 nm in diameter. And therefore the virus is called Scophthalmus maximus rhabdovirus (SMRV). Agarose gel electrophoresis analysis of the DNA extracted from infected cells showed typical DNA ladder in the course of SMRV infection. Flow cytometry analysis of SMRV infected CLC cells detected apoptotic peak in the virus infected CLC cells. Virus titre analysis and electron microscopic observation revealed that the virus replication fastigium was earlier than that of the apoptosis occurrence. No apoptosis was observed in the CLC infected with UV-inactivated SMRV. All these supported that SMRV infected CLC cells undergo apoptosis and the virus replication is necessary for apoptosis induction of CLC cells.     

Morphometric quantification of apoptotic stages in cell culture

Apoptosis is an active process of self-destruction, whereby cells undergo physiological cell death. It occurs during development and regulation of tissue homeostasis or as a result of changes in environmental stimuli. Chromatin condensation and nuclear fragmentation, which are typical features of apoptotic nuclei, are usually quantified by fluorescent DNA dyes. The present study reports a reliable method to analyze morphological apoptotic stages in cultured cells, using light microscopy. We used the human neuroblastoma cell line SK-N-BE as a model to study apoptosis induced by inadequate cell-matrix interactions. Apoptosis was detected on cells cultured for different time intervals on polyHEMA, poly-L-lysine or collagen I. Quantitative morphometric and densitometric analysis after hematoxylin nuclear staining and caspase-3 immunocytochemistry, as markers of occurring apoptosis, were performed. Our method identifies different stages of caspase-3 activation and the subsequent DNA fragmentation and condensation. This experimental procedure enables us to detect slight differences in apoptosis progression by morphological analysis.     

全硫代反义寡核苷酸对脑胶质瘤细胞端粒酶活性作用

目的本研究采用针对人端粒酶hTR基因的反义寡聚脱氧核苷酸,探讨端粒酶反义寡聚脱氧核苷酸(antisense oligodeoxy nucleotides,ASODN)对脑胶质瘤HO-8910细胞端粒酶活性及细胞增殖的影响。方法本实验将互补于hTR模板区的全硫代修饰型反义寡核苷酸(PS-ASODN)经lipotap脂质体转染作用于脑胶质瘤细胞系C6,然后分别采用四甲基偶氮唑蓝(MTT)比色实验、酶联免疫吸附试验(ELISA)法和流式细胞术检测C6脑胶质瘤细胞的体外增殖、端粒酶活性、细胞凋亡和细胞周期的改变。结果经MTT法检测,PS-ASODN对C6细胞生长具有明显的抑制作用和诱导细胞凋亡,1.0μMPS-ASODN处理后24h细胞生长抑制率为12.93%,且随着时间的延长,抑制作用逐渐增强;给药72h对细胞生长抑制率达到最高,为50.81%,有相应的浓度依赖关系。ELISA法检测结果 ,0.5～1.5μM的PS-ASODN对C6细胞作用72h后端粒酶皆为阴性,表明PS-ASODN能够抑制端粒酶活性,对照组端粒酶皆为阳性。结论端粒酶PS-ASODN对体外培养的脑胶质瘤C6细胞的增殖有明显的浓度、时间依赖性抑制作用;抑制脑胶质瘤C6的端粒酶活性和诱导C6脑胶质瘤细胞发生凋亡,可能是端粒酶PS-ASODN抑制C6细胞增殖的主要机制。