右旋柠烯诱导人胃癌细胞凋亡

目的　探讨右旋柠烯 (D limonene)诱导人胃癌细胞株凋亡作用的机制。方法　采用噻唑蓝 (MTT)比色法、电镜、流式细胞术以及免疫细胞化学法 ,对p5 3和bcl 2在肿瘤细胞内的表达以及细胞凋亡的定性与定量指标进行检测。结果　经D limonene处理的BGC 82 3细胞出现核固缩 ,染色质边集 ,凋亡小体形成。凋亡细胞发生率与药物浓度呈正相关 ,即药物浓度为 0 .2 5 μg/ml(4 8h) ,凋亡细胞的发生率从 (2 .71± 0 .78) %上升至 (31.6 2± 7.81) %。经D limonene处理的BGC 82 3细胞内p5 3蛋白表达较对照组明显增加 ,bcl 2蛋白表达较对照组降低。结论　D limonene对人胃癌细胞的杀伤主要是通过诱导细胞凋亡 ,升提p5 3及降低bcl 2的蛋白表达为其诱发肿瘤细胞凋亡的机制之一。     

宫颈癌放疗前后肿瘤细胞凋亡及其相关基因的变化

目的:探讨宫颈癌组织放疗前后肿瘤细胞凋亡及凋亡调节基因p53、bcl-2和bax蛋白表达的变化及意义.方法:选择未经治疗的宫颈癌患者20例为试验对象,采集放疗前和放疗10Gy后宫颈癌组织标本,用原位DNA切口末端标记(TUNEL)法检测凋亡细胞;单克隆抗体免疫组化SABC法检测细胞凋亡相关基因p53、bcl-2和bax的蛋白表达水平.结果:1)在宫颈癌放疗前后,肿瘤细胞凋亡阳性率和平均凋亡指数分别为25.00%和0.11%、75.00%和3.40%,放疗前后有显著性差异(P＜0.01);2)放疗前肿瘤细胞凋亡相关基因p53、bax和bcl-2蛋白表达阳性率分别为55%、10%和20%,而放疗后分别为25%、60%和25%,放疗后p53蛋白表达减少,bax蛋白明显增加,bcl-2蛋白无明显变化.结论:放射治疗诱导宫颈癌肿瘤细胞凋亡,可能与凋亡调节基因bax基因表达的诱导密切相关.     

三氧化二砷诱导鼻咽癌CNE1细胞凋亡及其作用机制的实验研究

 目的研究三氧化二砷（As₂O₃）诱导人鼻咽癌细胞株凋亡作用及其相关机制。方法采用流式细胞术，电镜、TUNEL方法检测As₂O₃诱导CNE1细胞凋亡作用，免疫组织化学法检测As₂O₃对CNE1细胞p53、bcl-2和bax蛋白表达的影响。结果经As₂O₃处理的CNE1细胞发生凋亡，表现为FCM可检测到“亚二倍体峰”，形态学上可见核固缩，染色质边集，凋亡小体形成等改变；TUNEL法可检测到细胞内呈棕色颗粒的凋亡细胞，随着药物浓度升高，凋亡细胞发生率逐渐增多，As₂O₃目浓度为0．5mg／L、1．0mg／L和2．0mg／L作用48h后，CNE1细胞的凋亡指数分别为2．66±0．64、8．15±0．96和11．59±0．68，显著高于非药物处理组（0．43±0．43，P〈0．05）。经As2O3处理的CNE1细胞内p53和bax蛋白表达较对照组明显增加，与凋亡指数呈正相关关系（r=0．554，P=0．011；r=0．891，P=0．000…）；p53和bax蛋白表达也呈正相关关系（r=0．626，P=0．003）。结论As₂O₃在体外可诱导鼻咽癌CNE1细胞凋亡，其机制可能与上调p53、bax基因表达有关。     

熊果酸诱导人胃癌BGC823细胞凋亡及其作用机制初探

背景与目的：熊果酸（ursolic acid,UA）广泛存在于夏枯草、白花蛇舌草等清热解毒药中,可抑制多种肿瘤细胞增殖并诱导其凋亡,具有广泛生物学活性。本研究将UA作用于人胃癌BGC823细胞,观察其对细胞凋亡的影响,并探讨其可能的作用机制。方法：MTT法检测UA对BGC823细胞增殖的影响;流式细胞术检测细胞周期与凋亡的变化;Realtime-PCR检测细胞bax、bcl-2mRNA表达的变化。结果：UA呈时间-剂量依赖性抑制BGC823细胞增殖,分别作用24、48、72h后半数抑制浓度依次为36.88、34.72、32.18μmol/L;UA能诱导BGC823细胞凋亡,并阻滞细胞于G2/M期;此外,其还可上调BGC823细胞bax及下调bc1-2mRNA表达,且作用随剂量的增加而加强。结论：UA呈时间-剂量依赖性抑制BGC823细胞增殖,并诱导其凋亡,阻滞细胞于G2/M期;其凋亡机制可能与上调bax及下调bcl-2基因的表达有关。     

胃癌p53基因与细胞凋亡的检测及其关系的研究

目的：研究p53基因与细胞凋亡在胃癌中的检测及其之间的关系。方法：采用p53原因杂交、原位脱氧核糖核酸末端转移酶标记法（TUNEL）和免疫组织化学技术对56例胃癌进行检测。结果：56例胃癌p53蛋白表达48．2％,p53原位杂交567．9％,二者的检测结果不完全一致,但均与胃癌的分期有关。晚期胃癌高于早期胃癌,差异有显著性（P＜0．01）。56例胃癌抗凋亡基因bcl－2阳性率为58．9％。TUNEL标记细胞凋亡指数平均为4．78,均与组织学分型和胃癌的分期有关,但又有相反的表达。未发化型腺癌和早期胃癌抗凋亡基因bcl－2高表达,而分化型腺癌和晚期胃癌低表达;TUNEL显示未分化型腺癌和早期胃癌凋亡细胞指数明显高地分化型腺癌和晚期胃癌,差异有显著性（P＜0．01）。p53基因与抗凋亡基因bcl－2呈负相关,与细胞凋亡指数呈正相关。结论：胃癌p53基因和细胞凋亡共同参与调节细胞的生存与死亡;二者均与胃癌的分期有关。     

叶酸诱导人肝癌细胞凋亡及其机制的研究

目的：研究叶酸诱导人肝癌细胞凋亡的作用及其机制。方法：应用叶酸连续处理体外培养的人肝癌细胞（SMMC－7721）,4天后光镜及电镜观察细胞形态改变,细胞计数测定细胞生长及增殖情况,DNA凝胶电泳分析DNA改变,流式细胞仪检测细胞周期分布,细胞凋亡指数及凋亡相关基因及其表达的改变。结果：10ug/ml叶酸可诱导SMMC－7721细胞出现凋亡特征性形态改变,其细胞NDA电一现“梯状”断裂现象,细胞凋亡指数为16．8％（空白对照组为6．9％）,S期细胞减少,细胞生长和增殖被明显抑制,且凋亡相关基因fas 和p53表达增加,而bcl-2和TGF－β1表达降低。结论：叶酸具有诱导人肝癌细胞凋亡的作用,其机制可能与凋控细胞凋亡相关基因的表达有关。     

p53/bcl-2与放疗诱导的宫颈癌细胞凋亡关系的研究

目的探讨宫颈癌放疗过程中,放疗诱导的细胞凋亡与p53、bcl-2的相关性.方法选择未经治疗的宫颈癌病人20例为实验对象,搜集分割放疗前后宫颈癌组织标本,用TDT-mediated dUTP-biotin nick end labeling(TUNEL)方法检测凋亡细胞;采用单克隆抗体免疫组化ABC法检测细胞凋亡相关基因p53、bcl-2的蛋白表达水平.结果(1)在宫颈癌放疗前后,细胞凋亡阳性率和平均凋亡指数分别为25%和0.11%、75%和2.8%,放疗前后有显著差异(P＜0.001);(2)放疗后p53蛋白表达显著减少,bcl-2蛋白无显著变化;(3)放疗前后,p53基因的表达与细胞凋亡呈有意义的相关性变化.结论放射治疗诱导了宫颈癌细胞凋亡的发生,并与凋亡调节基因p53及其表达呈间接有关.     

细胞凋亡调节与宫颈癌发生

目的：探讨子宫颈癌发生中的影响细胞凋亡的调节因素。方法：检测对象为194例经福尔马林固定石蜡包埋的手术切除标本，包括上皮内瘤样病变（CIN）78例[其中重度非典型增生（SD）41例；原位癌（CIS）37例]早期浸润癌（MIC）35例；大细胞非角化型浸润癌（IC）40例及正常宫颈鳞状上皮（NE）41例。凋亡细胞的检出使用TDT－mediated dUTP-biotin nick end labeling(TUNEL)方法；细胞凋亡相关基因蛋白p53、bcl-2、bax的表达采用单克隆抗体免疫组化ABC染色方法；HPV16、18型E6 DNA感染使用PCR方法进行检测。结果：1）凋亡细胞标记率仅在CIN呈现有意义地进行性减少（P＜0．01），癌变以后不再继续下降；2）在SD、CIS组，p53、bcl-2及bax基因蛋白的表达与TUNEL标记率呈现有意义地相关性变化（P＜0．05）；3）HPV16、18型E6 DNA感染与凋亡细胞及相关基因蛋白表达未呈现相关性改变。结论：以上结果提示细胞凋亡的改变与宫颈癌发生的早期过程有关，p53、bcl-2、bax基因蛋白参与其调节，而HPV16、18型E6 DNA感染与其未呈现直接的相关性。     

蛴螬粗提物对人宫颈癌HeLa细胞诱导凋亡作用及其机制

 目的 研究蛴螬粗提物对人宫颈癌HeLa细胞诱导凋亡作用，并探讨其作用机制。方法 采用流式细胞仪检测HeLa细胞的细胞周期分布相和细胞凋亡率；末端脱氧核苷酸转移酶（TdT）介导的脱氧核苷酸缺口末端标记（TdT mediated dUTP nick end labeling）TUNEL法检测凋亡细胞；通过免疫细胞化学SP法测定凋亡相关基因产物p53、Fas、bcl 2、Bax蛋白的表达情况。结果 (1)蛴螬粗提物可以诱导HeLa细胞发生凋亡，加药组出现亚二倍体峰，并且G0/G1期细胞比例显著增加（P＜0.01），而S期和G2/M期细胞比例则明显下降,将细胞阻滞在G0/G1期；（2）蛴螬粗提物作用细胞后凋亡细胞数目随时间延长而增多，凋亡指数与药物处理呈时间依赖性和剂量依赖性；(3)蛴螬粗提物作用后bcl 2、p53蛋白随提取物浓度表达均下降，Bax、Fas蛋白表达上升，四种蛋白表达各组间比较差异均有具有统计学意义（P<0.01）。结论 (1) 蛴螬粗提物对人宫颈癌HeLa细胞具有诱导凋亡作用；(2)蛴螬粗提取物诱导凋亡作用机制可能与细胞周期发生G0/G1期阻滞有关；并且通过下调bcl 2、p53蛋白表达,上调Bax、Fas蛋白表达，经由细胞凋亡的死亡受体通路和线粒体通路完成凋亡的启动和执行。     

bcl-2、bax、p53蛋白表达与外阴鳞癌细胞凋亡的相关性研究

目的　探讨bcl 2、bax、p5 3基因与外阴鳞癌细胞凋亡的关系。方法　应用原位末端标记及免疫组化S P法检测 30份外阴鳞癌石蜡包埋组织中的细胞凋亡API和bcl 2、bax、p5 3的表达情况。 结果　外阴鳞癌组织和正常外阴皮肤中均有细胞凋亡和bcl 2、bax、p5 3的表达。随着外阴鳞癌恶性程度的增高 ,API、bcl 2、bax表达降低 ;p5 3表达增高。在外阴鳞癌的部位凋亡细胞的分布与突变型 p5 3蛋白表达呈负相关 ,p5 3分别与bcl 2及bax的表达呈负相关 (P <0 .0 5 )。在正常外阴皮肤组织中 ,凋亡细胞的分布区域与表达bcl 2及bax蛋白的细胞分布区域一致 ,bcl 2与bax的表达呈正相关 (P <0 .0 5 )。结论　外阴鳞癌的发生、发展与细胞凋亡被抑制有关 ,这一过程可能主要由 p5 3基因调控 ,bcl 2可能仅在早期起作用。     

胃癌细胞MKN28(p53-/-)对NSAIDs诱导凋亡敏感性的研究

目的1.明确非甾体消炎药(NSAIDs)能否诱导p53基因突变的胃癌细胞MKN28凋亡.2.明确NSAIDs对MKN28细胞凋亡相关基因bcl-2、bax表达的调控.方法1.通过MTT比色法检测NSAIDs对细胞生长活力的影响.2.应用丫啶橙染色、Annexin-Ⅴ/PI双染色、共聚焦显微镜、流式细胞术检测细胞凋亡.3.应用RT-PCR(逆转录-聚合酶链反应)方法检测bcl-2、bax基因水平的改变.结果1.NSAIDs药物吲哚美辛(Indo)和阿斯匹林(Asp)对胃癌细胞株MKN28均有生长抑制作用,且呈时间/浓度依赖性增强.2.在Indo 800μmol/L、Asp8 mmol/L作用48～96 h后,MKN28细胞凋亡数量稍有增多,但不具有统计学意义.3.随着药物作用时间的延长,MKN28细胞的bcl-2基因mRNA表达逐渐减弱,bax基因表达逐渐增强.结论1.NSAIDs可抑制MKN28胃癌细胞株增殖.2.NSAIDs不能诱导p53基因突变的MKN28胃癌细胞株发生显著的凋亡,提示p53基因突变可能阻断了NSAIDs诱导的细胞凋亡.3.NSAIDs可使MKN28细胞凋亡相关基因bcl-2和bax的水平呈现促进凋亡倾向的改变.     

Inhibition of proteasome function induced apoptosis in gastric cancer

The ubiquitin-proteasome pathway plays a critical role in the degradation of cellular proteins and cell cycle control. Dysregulating the degradation of such proteins should have profound effects on tumor growth and causes cells to undergo apoptosis. The aims of this study are to evaluate the ubiquitin-proteasome pathway in gastric cancer and the potential role of pharmacological inhibition of proteasome on induction of apoptosis in gastric cancer cells. Gastric cancer cell lines AGS (p53 wild-type) and MKN-28 (p53 mutant) were treated with proteasome inhibitor MG132. The results showed that MG132 inhibited cell proliferation in AGS and MKN-28 cells in a time- and dose-dependent manner. The inhibition of cell proliferation was caused by apoptosis which was also time- and dose-dependent. AGS cells were more responsive to MG132 than MKN-28 cells. Induction of apoptosis was preceded by the activation of caspase-3, as measured by a colorimetric caspase-3 cellular activity and Western blotting of the cleavage of caspase-3 and its substrate PARP. Activation of caspase-7 was also exhibited. In addition, z-VAD-fmk, a broad spectrum caspase inhibitor, reversed apoptosis induced by MG132 in AGS and MKN28 cells. Although z-DEVD-fmk, a specific caspase-3 inhibitor, suppressed MG132-induced apoptosis in MKN28 cells, it only partially rescued the apoptotic effect in AGS cells. Caspase-3 activation was the result of release of cytochrome c from mitochondria into the cytosol, as a consequence of upregulation of bax. There were overexpressions of all the proteasome-related proteins p53, p21(waf1) and p27(kip1) at 4 hr after proteasome inhibition which was identified by the accumulation of ubiquitin-tagged proteins. This was accompanied by accumulation of cells at G(1) phase. Our present study suggests that inhibition of proteasome function in gastric cancer cells induces apoptosis and proteasomal inhibitors have potential use as novel anticancer drugs in gastric cancer.     

Androgen receptor expression in relation to apoptosis and the expression of cell cycle related proteins in prostate cancer

The expression of several genes involved in the regulation of cell cycle and apoptosis may be regulated via the androgen receptor (AR) in the prostate. AR may have a role in the prognosis of prostatic carcinoma. The aim was to examine AR expression status and its relationship with markers of proliferation, apoptosis and cell cycle control in prostate cancer. Expression of AR, bcl-2, bax, Ki-67 and p53 was examined in paraffin-embedded tissues from 50 cases of prostate carcinoma by immunohistochemistry and evaluated using an index of staining. Detection of apoptotic cells was performed by TUNEL method. Correlation between AR expression and apoptosis, proliferation index, bcl-2, bax and p53 and also clinicopathological parameters including stage, pathological grade and Gleason score were determined. AR expression was observed in all cases with mean expression of 81%±15 and mean staining index of 141±65. No correlation was found between AR expression and apoptosis detected in patients. The mean AR staining index was 170±72 in bcl-2 positive tumors versus 120±53 in bcl-2 negative tumors showing a significant association between AR and bcl-2 expression (p=0.015). AR expression also showed a significant association with bcl-2/bax ratio (r=0.321, p=0.023) and Ki-67 proliferation staining index (r=0.396, p=0.004). Although a significant correlation between Ki-67 and p53 with differentiation status of the tumors was observed (p<0.004) no correlation was found with AR. AR expression showed no prognostic value regarding its correlation with stage and differentiation status of the prostate carcinoma. However, its significant correlation with Ki-67 and bcl-2 that are markers of cell survival suggest its contribution to tumor cell progression.     

Relationship between apoptosis regulator proteins (bcl-2 and p53) and Gleason score in prostate cancer

Cellular proliferation programmed cell death (apoptosis) are associated with tumor growth in general, and prostate cancer growth in particular. The aim of this study was to examine the expression of the apoptosis regulating genes bcl-2 and p53 and Gleason score in core needle biopsy specimens of prostate cancer using immunohistochemistry. We studied bcl-2 and p53 expression in 12 cases of low grade (Gleason score 2-5), 12 cases of intermediate grade (Gleason score 6-7) and 8 cases of high grade (Gleason score 8-10) prostate cancer. Overexpression of bcl-2 was noted in 3 of 32 patients (9.32%). One of them was high grade; others were intermediate grades. Expression of p53 was observed in 3 of low grades; others were high grade. The statistical analysis of present data suggest that there is no significant relation between p53 and bcl-2 expression and Gleason score in prostate cancer.     

拓扑替康促人卵巢癌顺铂耐药细胞株凋亡机制的初步研究

目的探讨拓扑替康(TPT)对人卵巢癌顺铂耐药细胞株A2780/DDP和COC1/DDP的杀伤和诱导凋亡活性及其作用机制.方法用MTT比色法测定TPT对人卵巢癌顺铂耐药细胞株A2780/DDP和COC1/DDP的杀伤效应;透射电镜和DNA凝胶电泳研究TPT对靶细胞的凋亡诱导活性;Western blot检测凋亡相关蛋白bcl-2和bax的表达.结果TPT对A2780/DDP和COC1/DDP细胞均有较强的体外杀伤作用,其IC50值分别为102.91 ng/ml和111.75 ng/ml;TPT能诱导A2780/DDP和COC1/DDP细胞产生凋亡,DNA凝胶电泳呈现典型的凋亡梯度,透射电镜观察到细胞核染色质固缩、边集,胞质浓缩,出现空泡等典型的凋亡超微结构改变;TPT不影响bcl-2蛋白表达,却能上调bax蛋白表达,增大bax/bcl-2比值,且呈剂量依赖性.结论TPT对人卵巢癌顺铂耐药细胞株A2780/DDP和COC1/DDP均有明显的杀伤和促凋亡作用,其机制可能与bax基因表达上调而提高bax/bcl-2比值有关.     

Molecular Regulation of Cell Death and Therapeutic Strategies for Cell Death Induction in Prostate Carcinoma

Many of the common molecular alterations associated with prostate cancer progression involve genes known to regulate cell death susceptibility. The significance of these molecular events is discussed in the context of developing and implementing new strategies designed to restore cell death susceptibility in prostate cancer cells and overcome therapeutic resistance.     

CIK细胞对 MGC-803胃癌细胞株的诱导凋亡作用

目的研究多种细胞因子诱导的杀伤细胞(CYtokine-induced kill cells,CIK)对MGC-803胃癌细胞株的诱导凋亡作用,并探讨其作用机制.方法应用(TdT-mediated dUTP nick end labeling)TUNEL法检测CIK细胞诱导凋亡的情况;通过免疫细胞化学法测定Bcl-2、Bax的阳性表达率,深入研究CIK细胞对MGC-803胃癌细胞株凋亡相关基因蛋白表达的影响.结果TUNEL法检测对照组成不染或均匀淡蓝色,实验组凋亡细胞缩小,核或核周成深蓝色.CIK实验组5～14小时凋亡率上调,14～24小时凋亡率下降.CIK实验组凋亡率与对照组相比有显著性差异(P＜0.01).免疫细胞化学结果表明:Bcl-2蛋白随作用时间延长表达均下降,Bax蛋白表达上调,与对照组相比均有显著性差异(P＜0.01).结论CIK细胞对MGC-803胃癌细胞早期诱导凋亡,晚期诱导肿瘤细胞坏死.其作用机制之一可能是通过上调Bax,下调Bcl-2的蛋白表达实现的.     

胃癌组织p53、p63、p73蛋白表达及细胞凋亡的研究

目的:探讨p53、p63、p73蛋白表达及细胞凋亡在胃癌发生发展中的可能机制。方法:选择57例手术切除的胃癌及胃癌旁组织,用免疫组化方法检测组织中的p53、p63、p73蛋白表达,同时用Tunel方法检测胃癌组织中的凋亡细胞。结果:胃癌组织p53、p63、p73蛋白表达率显著高于胃癌旁组织(P<0.05);低、未分化型腺癌表达率显著高于高、中分化型腺癌(P<0.05);有淋巴转移显著高于无转移组(P<0.05);p53、p63、p73高表达的癌组织,细胞凋亡指数减少,早期胃癌凋亡低于晚期胃癌(P<0.05)。结论:胃癌组织p53、p63、p73蛋白表达水平增高,可延缓细胞凋亡;细胞凋亡机制障碍可能是胃癌增生失控的基础。     

Biological markers as a predictor for response and prognosis of unresectable gastric cancer patients treated with irinotecan and cisplatin

BACKGROUND: Previously we reported that immunohistochemical examination of p53, bcl-2, glutathione S-transferase-pi (GST-pi), thymidylate synthase (TS) and vascular endothelial growth factor (VEGF) in biopsy samples was a useful method for predicting clinical outcome of gastric cancer patients treated with 5-fluorouracil and cisplatin. Here, we investigated if these biological markers can predict chemoresponse and survival of unresectable gastric cancer patients treated with irinotecan and cisplatin. METHODS: The subjects were 55 unresectable gastric cancer patients treated with irinotecan (70 mg/m(2), Days 1 and 15) and cisplatin (80 mg/m(2), Day 1). Expression of p53, bcl-2, VEGF was examined immunohistochemically in biopsy samples. RESULTS: The overall response rate and the median survival time were 55% (30/55) and 321 days, respectively. Thirty patients with intestinal-type adenocarcinoma survived longer than 25 patients with diffuse-type (median survival time: 446, 259 days, P = 0.013). The favorable phenotypes for chemoresponse were p53-negative, bcl-2-negative and VEGF-positive, which were in accordance with previous findings. The response rate was significantly correlated with the total number of these favorable phenotypes (P = 0.043). The 39 patients having 2 or 3 favorable phenotypes (p53-negative, bcl-2-negative and VEGF-positive) survived longer than the remaining 16 patients (median survival time: 444, 259 days, P = 0.021). In the Cox model, the number of the favorable phenotypes showed a tendency to correlate with survival after adjustment for potentially prognostic factors such as histological type or performance status (P = 0.070). CONCLUSIONS: Immunohistochemical examination of biological markers may be useful in predicting the clinical outcome of unresectable gastric cancer patients treated with irinotecan and cisplatin.