紫杉醇诱导胆管癌QBC939细胞凋亡的初步研究

目的:探讨紫杉醇对胆管癌QBC939细胞作用的敏感性并研究紫杉醇对胆管癌细胞周期及细胞凋亡率的影响,并初步探讨紫杉醇诱导胆管癌细胞发生凋亡的机制,为胆管癌的临床治疗寻找新的治疗药物.方法:体外培养胆管癌细胞QBC939,采用不同浓度紫杉醇予以干预,通过细胞形态观察和MTT实验,初步研究紫杉醇对人胆管癌细胞QBC939的敏感性;应用流式细胞仪检测紫杉醇诱导胆管癌细胞凋亡,同时对细胞周期的变化和动力学予以检测.结果:各浓度紫杉醇对人胆管癌细胞QBC939生长均有明显抑制作用;胆管癌细胞被紫杉醇明显阻滞在S期及G2/M期,且抑制作用呈剂量和时间依赖效应.结论:紫杉醇对人胆管癌细胞QBC939增殖的抑制作用呈剂量和时间依赖效应;紫杉醇能诱导人胆管癌QBC939细胞发生凋亡.     

紫杉醇诱导胃癌细胞凋亡的实验研究

目的 观察紫杉醇对胃癌细胞株BGC-823的体外诱导凋亡及对细胞周期的影响，探讨其治疗胃癌的作用机制。方法 利用流式细胞仪观察不同时间细胞的凋亡率以及细胞周期的变化。结果 紫杉醇诱导细胞凋亡，其凋亡率随时间的增加而增加，0，12，24，48小时细胞凋亡率分别为2．8％，6．0％，14．3％，16．0%；细胞周期也随着时间的不同而变化，与对照组相比，S期细胞减少，G2／M期细胞增多。结论 紫杉醇诱导细胞凋亡，有时间依赖性，其诱导凋亡的机制可能是通过G2／M期阻滞实现的。     

替尼泊甙对胃癌细胞凋亡及细胞周期的影响

 目的 探讨替尼泊甙体外对胃癌细胞BGC-823的作用机制。方法 体外培养胃癌细胞株BGC-823,通过MTT法,流式细胞仪,观察替尼泊甙对胃癌细胞的生长抑制作用以及对细胞凋亡,细胞周期的影响。结果 在一定浓度范围内,细胞的生长抑制率随着浓度的增加而增加,细胞的凋亡率随着时间的增加而增加,细胞周期被阻滞在G2/M期,在48小时达到高峰。结论 替尼泊甙可抑制胃癌细胞的生长,诱导细胞的凋亡,其诱导凋亡的能力可能是通过把细胞阻滞在G2/M期实现的。     

紫杉醇诱导胆管癌QBC939细胞凋亡的初步研究

目的：探讨紫杉醇对胆管癌QBC939细胞作用的敏感性并研究紫杉醇对胆管癌细胞周期及细胞凋亡率的影响,并初步探讨紫杉醇诱导胆管癌细胞发生凋亡的机制,为胆管癌的临床治疗寻找新的治疗药物。方法：体外培养胆管癌细胞QBC939,采用不同浓度紫杉醇予以干预,通过细胞形态观察和Mrrr实验,初步研究紫杉醇对人胆管癌细胞QBC939的敏感性;应用流式细胞仪检测紫杉醇诱导胆管癌细胞凋亡,同时对细胞周期的变化和动力学予以检测。结果：各浓度紫杉醇对人胆管癌细胞QBC939生长均有明显抑制作用;胆管癌细胞被紫杉醇明显阻滞在S期及G2／M期,且抑制作用呈剂量和时间依赖效应。结论：紫杉醇对人胆管癌细胞QBC939增殖的抑制作用呈剂量和时间依赖效应;紫杉醇能诱导人胆管癌QBC939细胞发生凋亡。     

三氧化二砷对胃癌BGC-823细胞增殖及细胞周期的影响

[目的]探讨三氧化二砷（AS2O3）对胃癌BGC-823细胞增殖的抑制作用及对细胞周期的影响。[方法]应用MTT方法检测三氧化二砷对胃癌BGC-823细胞增殖的抑制作用，流式细胞仪进行细胞周期解析及凋亡检测．[结果]三氧化二砷对胃癌BGC823细胞具有杀伤作用，且呈时间-浓度依赖性抑制肿瘤细胞的增殖．72h抑制细胞50％增殖的药物浓度约为3．3μmol／L，与对照组比差异显著（P=0．0048）；5μmol／LAs2O3作用24h，细胞形态学可见典型的凋亡细胞及M期阻滞；流式细胞仪进行细胞周期解析结果提示，G2/M期细胞由正常对照的25％增加到46．3％，出现了明显的G2/M期阻滞，亚二倍体凋亡细胞由4％增加到18％。[结论]三氧化二砷抑制胃癌细胞的增殖．诱导细胞周期阻滞及凋亡。     

三氧化二砷对胃癌BGC-823细胞增殖及细胞周期的影响

[目的]探讨三氧化二砷（AS2O3）对胃癌BGC-823细胞增殖的抑制作用及对细胞周期的影响。[方法]应用MTT方法检测三氧化二砷对胃癌BGC-823细胞增殖的抑制作用，流式细胞仪进行细胞周期解析及凋亡检测．[结果]三氧化二砷对胃癌BGC823细胞具有杀伤作用，且呈时间-浓度依赖性抑制肿瘤细胞的增殖．72h抑制细胞50％增殖的药物浓度约为3．3μmol／L，与对照组比差异显著（P=0．0048）；5μmol／LAs2O3作用24h，细胞形态学可见典型的凋亡细胞及M期阻滞；流式细胞仪进行细胞周期解析结果提示，G2/M期细胞由正常对照的25％增加到46．3％，出现了明显的G2/M期阻滞，亚二倍体凋亡细胞由4％增加到18％。[结论]三氧化二砷抑制胃癌细胞的增殖．诱导细胞周期阻滞及凋亡。     

糖原合酶激酶-3β磷酸化抑制紫杉醇诱导卵巢癌细胞凋亡的实验研究

目的:探讨增加糖原合酶激酶-3β(GSK3β)的p-GSK3β对紫杉醇诱导的卵巢癌细胞凋亡的作用.方法:运用GSK3β特异性抑制剂氯化锂(LiC1)增加GSK3β的磷酸化水平,应用流式细胞仪和Hochest33342染色检测细胞凋亡和细胞周期,蛋白质印迹法检测GSK3β及GSK3β磷酸化水平.结果:蛋白质印迹法显示,抑制剂LiC1作用后,GSK3β磷酸化明显增加.流式结果显示,90 nmol/L紫杉醇作用于OV2008细胞24 h,通过亚G1峰检测其凋亡卒为(29.75±1.54)0A,而应用LiC1抑制剂预孵育的细胞,凋亡率为(15.23±0.95)%,差异有统计学意义,t=46.378,P=0.002.Hochest33342染色证实LiC1作用后可明显抑制紫杉醇诱导的细胞凋亡形态学改变.流式结果周期分析显示,90 nmol/L紫杉醇作用于OV2008细胞24 h,与正常对照相比[G2M(22.83±2.40)%],可见细胞呈现明显的G2M期阻滞[G2M(69.68±1.48)%],而LiC1抑制剂的应用,可增强紫杉醇诱导的G2M期阻滞[G2M(88.49±0.86)%],差异有统计学意义,t=20.407,P=0.003.结论:GSK3β磷酸化增加可抑制紫杉醇诱导的卵巢癌细胞的凋亡并增强其周期阻滞.     

胃癌组织Tau蛋白表达与紫杉醇敏感性关系的研究

目的:研究Tau蛋白在胃癌组织中的表达情况及其与应用紫杉醇敏感性的关系.方法:免疫组化方法检测47例胃癌组织中Tau蛋白的表达水平,分析Tau蛋白的表达丰度;蛋白质印迹法检测胃癌细胞株中Tau蛋白表达水平;MTT法分析紫杉醇对胃癌细胞珠的生长抑制作用;流式细胞术检测紫杉醇对胃癌细胞珠的诱导凋亡作用.结果:免疫组化方法检测47例胃癌组织Tau蛋白(3+)表达率为12.77％(6/47),(2+)表达率51.06％(24/47),Tau蛋白高表达率为63.83％.蛋白质印迹法检测结果显示,Tau蛋白在胃癌MKN45细胞株中相对高表达,在BGC823细胞珠中相对低表达,P=0.014 7.MTT法分析发现,不同剂量紫杉醇对细胞珠MKN45和BGC823均有细胞抑制作用,但对Tau蛋白表达低的BGC823细胞株抑制作用更为明显,紫杉醇浓度6、8、10、30、50、70和90 μg/mL,其P值分别为0.051 91、0.000 50、0.021 89、0.002 28、0.002 45、0.549 39和0.022 72.流式细胞术检测结果显示,紫杉醇对两细胞珠均有诱导凋亡作用,且Tau蛋白低表达的BGC823细胞珠凋亡率更明显.结论:Tau蛋白在胃癌细胞中有较高表达,紫杉醇对Tau蛋白表达低的胃癌细胞具有更强的抑制作用,其敏感性更高.     

洛铂对人胃癌细胞BGC-823的增殖抑制及放疗增敏作用

目的:探讨洛铂对胃癌细胞株BGC-823的增殖抑制及放疗增敏作用。方法:以人胃癌细胞株BGC-823为研究对象,采用洛铂和高能射线作为干预手段,MTT法测定洛铂对胃癌细胞株BGC-823的抑制率,流式细胞术检测细胞周期及细胞凋亡率。结果:MTT显示洛铂和放疗对人胃癌细胞株BGC-823生长有显著的抑制作用,且呈现时间和剂量依赖性。洛铂对胃癌的放疗具有明显增敏作用。药物组G0/G1期细胞比例增加,S期细胞比例减少。而G2/M期变化无明显意义。照射组G2/M期细胞比例升高。联合组G0/G1期细胞比例增加,S期细胞比例减少。结论:洛铂能提高人胃癌细胞株BGC-823放疗敏感性作用。其作用可能是通过诱导细胞凋亡、改变细胞周期时相分布等机制来实现的。     

多西紫杉醇对胃癌细胞作用及其机制的研究

目的:研究多西紫杉醇对人胃癌中分化细胞株SGC7901的作用效果及机制。方法:MTT法检测多西紫杉醇对胃癌细胞株SGC7901的增殖抑制作用。AnnexinV方法检测药物作用后胃癌细胞的凋亡。PI染色法检测凋亡晚期的胃癌细胞。采用流式细胞仪检测多西紫杉醇作用前后细胞凋亡相关分子Fas蛋白、Bcl2蛋白表达水平的变化。结果:0.92、3.7、14.8、59.2μg/mL多西紫杉醇作用于SGC7901细胞72h,抑制率分别为13.3%、21.6%、57.5%、61.0%。多西紫杉醇可诱导胃癌细胞株SGC7901发生细胞凋亡。多西紫杉醇使SGC7901凋亡分子Fas表达增加,作用前后分别为(26.5±7.2)%、(37.9±7.0)%;多西紫杉醇作用SGC7901前后Bcl2表达分别为(38.9±9.1)%、(31.2±5.6)%,差异无显著性。结论:多西紫杉醇对胃癌中分化细胞株SGC7901生长有明显的抑制作用,可诱导胃癌细胞系SGC7901凋亡,凋亡作用的发生可能与多西紫杉醇诱导Fas分子表达有关。     

A model chemosensitivity test examining apoptosis in small specimens of gastric cancer

Background. Because chemosensitivity tests usually require a large amount of tissue, they are not used routinely in patients with unresectable gastric cancer. The aim of this study was to investigate whether apoptosis can be used as a sensitivity assay for chemosensitivity in small gastric cancer specimens. Methods. Apoptosis, detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick labeling (TUNEL), was investigated in small specimens of the MKN-1, MKN-45, and TMK-1 human gastric cancer cell lines as a marker of chemosensitivity following exposure to antineoplastic agents. Results. Doxorubicin (DXR), SN-38 (active metabolite of irinotecan), and paclitaxel (Taxol) induced DNA fragmentation in MKN-45 and TMK-1 cells, but not in MKN-1. In contrast, neither 5-fluorouracil (5-FU) nor cisplatin (CDDP) induced DNA fragmentation in any of the three cell lines. Small pieces cut from tumors implanted in nude mice were exposed to the antineoplastic agents in culture medium for 24 h, and the percentage of TUNEL-positive cancer cells (TUNEL positivity) was examined. TUNEL positivity in all three cancers increased after exposure to DXR, SN-38, and Taxol, but not after exposure to CDDP or 5-FU. MKN-45 showed the highest TUNEL positivity with SN-38 and Taxol, and TMK-1 TUNEL positivity was highest with DXR. MKN-45 and TMK-1 were the most sensitive to these three antineoplastic agents in vitro, while MKN-1, with the lowest TUNEL positivity, was the least sensitive to these three antineoplastic agents. TUNEL positivity after exposure to Taxol correlated with the antitumor effects of this compound in an animal model. Conclusion. These results suggest that, in small gastric cancer specimens where apoptosis is implicated, TUNEL positivity may be applicable to a chemosensitivity test. Received: January 28, 2000 / Accepted: April 14, 2000     

细胞周期蛋白依赖性激酶1在S期细胞检测点的激活

 目的 以细胞周期特异性T淋巴细胞型白血病Molt-4细胞S期凋亡为模式,检测细胞周期蛋白依赖性激酶1（CDK1）在S期细胞周期检测点蛋白磷酸化及蛋白去磷酸化,探讨细胞周期时相特异性细胞凋亡的CDK1的活性改变。 方法 对数生长期的人类急性淋巴细胞白血病细胞系Molt-4经喜树碱（CPT）诱导细胞凋亡,经不同的作用时间、剂量处理后,以API法检测细胞凋亡的细胞周期特异性,以分选后激光共聚焦荧光显微镜技术（PSC）对API结果进一步验证,从而获取最佳条件下细胞周期特异性细胞凋亡,蛋白电泳分析法检测CDK1的磷酸化、去磷酸化。结果 对数生长期的Molt-4细胞经CTP诱导后发生细胞周期特异性细胞凋亡,CPT 0.2 μg／ml处理后6 h出现S期特异性细胞凋亡。CTP诱导出现S期特异性细胞凋亡时CDK1-第15位酪氨酸（Tyr15）与对照Western blot结果条带相近,而CDK1-第161位苏氨酸（Thr161）条带较窄。结论 细胞凋亡往往伴有细胞周期特异性;经PSC形态学证实的API流式细胞术是有效、快捷的细胞周期特异性细胞凋亡分析方法;CDK1在S期特异性细胞凋亡时磷酸化活性降低。     

青蒿琥酯对人子宫内膜癌细胞体外的抑制作用

目的研究青蒿琥酯(Artesunate, Art)对人子宫内膜癌细胞Ishikawa的体外抑制作用,为进一步的临床研究及应用提供依据。方法培养人子宫内膜癌Ishikawa细胞株,利用MTT法测定不同浓度Art对Ishikawa细胞增殖的影响;流式细胞术（FCM）观察药物作用前后细胞凋亡及周期的变化。结果Art显著抑制Ishikawa细胞的增殖,IC50为45.063μmol/L;25μmol/L Art作用Ishikawa细胞24h后即能检测出典型的亚二倍体“凋亡峰”,细胞凋亡率最高达58.31%;低浓度可将细胞阻滞在G₁期,当浓度达到100μmol/L时,Art可将细胞阻滞于G₂期。结论Art能显著抑制Ishikawa细胞生长,并能调整其周期,诱导凋亡,有可能开发为有效治疗子宫内膜癌的辅助药物。     

紫杉醇诱导肝癌细胞SMMC-7721凋亡的实验研究

目的 :研究紫杉醇 (Taxol)对肝癌细胞SMMC 772 1的生长抑制和诱导凋亡的作用。方法 :应用MTT法观察紫杉醇对SMMC 772 1的生长抑制作用 ,通过瑞氏染色、荧光显微镜、透射电镜 ,流式细胞仪和TUNEL染色研究紫杉醇对SMMC 772 1诱导凋亡的作用。结果 :紫杉醇对肝癌细胞SMMC 772 1有明显的生长抑制作用 ,其IC50 为 2 1.6nmol/L。紫杉醇作用细胞后 ,可看到较为典型的细胞凋亡的形态学变化 ,细胞核固缩、染色质凝集成新月型紧贴于核膜周边 ,核碎裂、染色质片断化、凋亡小体形成等。流式细胞仪上可见S期和G2 /M期和DNA含量增加 ,并出现典型的亚二倍体“凋亡小峰”。TUNEL染色显示。紫杉醇作用于SMMC 772 1后 ,随药物浓度增加和作用时间延长 ,凋亡指数增加。结论 :紫杉醇对SMMC 772 1有明显的生长抑制作用 ;紫杉醇对SMMC 772 1有诱导凋亡的作用。     

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.     

MicroRNA-146a is down-regulated in gastric cancer and regulates cell proliferation and apoptosis

Aberrant expression of microRNA-146a (miR-146a) has been reported to be involved in development and progression in various types of cancers, but its role in gastric cancer has not been fully elucidated. The purpose of this study was to investigate the levels of miR-146a expression and its function in human gastric cancer. Quantitative real-time polymerase chain reaction was used to detect the levels of miR-146a expression in gastric cancer tissue samples and cell lines. The cell growth rate of MKN-45 gastric cancer cells transfected with miR-146a mimics was examined by MTT assay. The effects of miR-146a on cell cycle and apoptosis were assessed by FACS analyses in MKN-45 cells. Thirty-six of 43 gastric cancer tissue samples (84%) showed decreased expression of miR-146a. We found low expression of miR-146a was correlated with increased tumor size (P = 0.006) and poor differentiation (P = 0.010) in gastric cancer. Overall survival time of patients with high miR-146a expression was significantly longer than that of patients with low expression of miR-146a (P = 0.011). The MTT assay showed that introduction of miR-146a inhibited cell proliferation in MKN-45 cells (P < 0.05). The proportion of apoptotic cells induced by transfection of miR-146a mimics were greater than that induced by transfection of the negative control mimics (11.9 vs. 5.9%). Our results suggested that miR-146a has potential as a novel suppressor gene in gastric cancer and its down-regulation may promote the progression of gastric cancer.     

顺铂及增敏剂Verapamil、SDZ PSC833诱导卵巢癌细胞凋亡的研究

目的 探讨顺铂及Ｖｅｒａｐａｍｉｌ，ＳＤＺ ＰＳＣ８３３作用机制，探讨获得性耐药机制。方法 以人卵巢癌亲代细胞株ＣＯＣ１及其耐药亚株ＣＯＣ１／ＤＤＰ为材料，计算细胞生长抑制率，细胞凋亡率及进行细胞周期分析。结果 顺铂联用Ｖｅｒａｐａｍｉｌ或ＳＤＺ ＰＳＣ８３３可提高细胞生长抑制率；顺铂引起Ｓ期细胞比例增高，大剂量顺铂造成期细胞大量凋亡；     

Non-steroidal anti-inflammatory drugs induce apoptosis in gastric cancer cells through up-regulation of bax and bak

Aspirin- and non-steroidal anti-inflammatory drug (NSAID)-induced apoptosis is one of the important mechanisms for their anti-tumour effect in gastric cancer. We aimed at determining the role of bcl-2 family proteins and caspases in the apoptotic process. Gastric cancer cell lines AGS (wild-type p53) and MKN-28 (mutant p53) were used. Cell proliferation was measured by MTT assay. Apoptosis was determined by acridine orange staining. Protein expressions were determined by western blotting. Aspirin and indomethacin inhibited cell proliferation and induced apoptosis in both cells. AGS cells were more sensitive compared with MKN-28 cells. The pro-apoptotic proteins bax and bak were overexpressed after treatment, while the protein level of bcl-2 remained unchanged. Apoptosis was accompanied by an increase in caspase-3 activity and cleavage of caspase-3 and poly(ADP-ribose) polymerase. Inhibition of caspase-3 rescued aspirin-induced apoptosis. Our results suggest that one of the major pathways which mediates the anti-tumour response of aspirin and indomethacin in gastric cancer cells is through up-regulation of bax and bak and activation of caspase-3. Bax and bak are important in the chemoprevention of gastric cancer.     

藤梨根提取物对人胃癌MKN-45细胞增殖及凋亡的影响

目的:探讨太白山藤梨根提取物(Radix Actinidiae extractive,RAE)在体外对胃癌MKN-45细胞增殖与凋亡的影响。方法:用0.01-100μg/ml浓度范围内的RAE和胃癌MKN-45细胞共培养24、48、72、96小时,采用MTT法检测RAE对MKN-45细胞增殖的影响;用流式细胞仪检测其对MKN-45细胞凋亡及细胞周期分布的影响;DNA琼脂糖凝胶电泳检测细胞凋亡。结果:RAE在体外能够显著抑制胃癌MKN-45细胞的增殖,且呈现浓度和时间依赖性;RAE对胃癌细胞的作用表现为周期特异性,使细胞阻滞于G0/G1期,进一步诱导细胞凋亡。对照组凋亡率为0.53%,1μg/ml、10μg/ml、100μg/ml浓度的RAE干预胃癌细胞48小时后均出现凋亡峰,细胞凋亡率分别为3.27%、8.97%、12.6%。DNA琼脂糖凝胶电泳检测细胞凋亡,不同浓度RAE处理的细胞样品中均可看到梯形凋亡条带,并呈现浓度依赖性。结论:RAE在体外对人胃癌MKN-45细胞有显著的抑制细胞增殖及诱导细胞凋亡作用,其作用机制可能是使胃癌细胞周期阻滞于G0/G1期。     

肝细胞生长因子基因转染对阿霉素诱导胃癌细胞调亡的影响

目的研究肝细胞生长因子（HGF）基因转染对阿霉素诱导的胃癌细胞凋亡的影响。方法先构建HGF基因的真核表达质粒pIRES2-EGFP-HGF,应用pIRES2-EGFP-HGF重组质粒和pIRES2-EGFP空质粒转染人胃癌MKN-45细胞,以未转染组为对照。用转染细胞的培养液培养MDCK细胞后,以细胞形态学改变来分析目的基因mRNA、蛋白的表达,分别用RT-PCR、Western blot测定其功能。MTT法测定阿霉素对细胞生长的抑制作用,DNA凋亡条带法和PI染色法检测细胞凋亡。结果稳定转染HGF基因的MKN-45细胞株可表达HGF mRNA,其分泌的HGF蛋白具有正常功能。MTT检测表明,HGF质粒转染组活细胞数高于空质粒转染和未转染组。0．1μg／ml阿霉素作用细胞后DNA凋亡条带分析发现,空质粒转染及未转染组的MKN-45细胞出现典型阶梯状条带, HGF转染组细胞凋亡条带不显著。流式细胞术结果显示,HGF质粒转染组细胞凋亡率显著低于未转染及空质粒转染组。结论HGF基因稳定转染可显著抑制阿霉素诱导的胃癌细胞凋亡。