三氧化二砷抗胃癌作用机制的研究

目的探讨三氧化二砷(As2O3)对人胃癌细胞株MKN-28抑制生长及诱导凋亡的生物学效应。方法采用光镜观察、溴化二甲噻唑二苯四氮唑(MTT)还原法、流式细胞仪(FCM)检测法检测As2O3在不同作用时间、不同给药浓度时对MKN-28细胞生长的影响。结果MKN-28细胞经As2O3作用后,细胞增殖受到明显抑制,而且呈浓度-作用时间依赖关系。流式细胞仪检测结果显示DNA直方图上出现典型的亚二倍体凋亡峰,细胞周期分析发现药物作用后MKN-28细胞的生长周期受到明显阻滞。结论As2O3既能有效地抑制人胃癌细胞株MKN-28的生长,又能诱导该细胞凋亡,而且呈浓度依赖性和作用时间依赖性。     

胃安宁含药血清对人胃癌MGC-803细胞增殖及凋亡的影响

[目的]研究胃安宁颗粒含药血清体外对人胃癌MGC-803细胞株增殖、凋亡的影响.[方法]采用MTT比色法检测不同剂量胃安宁颗粒含药血清作用12 h、24 h、48 h后对MGC-803细胞生长增殖的影响,流式细胞仪检测细胞凋亡率.[结果]随着胃安宁颗粒含药血清浓度逐渐增加,细胞增殖率逐渐降低,细胞抑制率、凋亡率逐渐升高,且以早期凋亡为主.[结论]胃安宁颗粒能够抑制人胃癌MGC-803细胞的增殖,对MGC-803细胞有诱导其早期凋亡作用.     

延胡索总碱对6种人源胃癌细胞株的体外增殖抑制作用

[目的]研究延胡索总生物碱对6种人胃癌细胞系的增殖抑制作用及其对细胞凋亡和细胞周期的影响。[方法]应用四甲基偶氮唑蓝（MTT）法观察延胡索总碱对6种人胃癌细胞的增殖抑制作用;流式细胞仪检测延胡索总碱作用后MKN-28细胞周期变化及细胞凋亡率。[结果]延胡索总碱对6种人胃癌细胞均有显著的增殖抑制作用,尤其以对AGS、MKN-28细胞的抑制作用为著,且呈剂量-效应关系;25、100μg／ml延胡索总碱作用于MKN28细胞24h后,细胞凋亡率分别为17．2％、32．3％,差异有统计学意义（P〈0．05）;25μg／ml延胡索总碱可将细胞阻滞在S期,而100μg／ml延胡索总碱对MKN-28细胞周期无明显影响。[结论]延胡索总碱对多种胃癌细胞有显著的增殖抑制作用,其机制可能与诱导细胞凋亡、细胞周期阻滞有关。     

豆楮方对人肝癌细胞株抑制作用的体外实验研究

目的:研究豆楮方对人肝癌细胞株HepG2、SMMC-7721的生长增殖抑制作用.方法:按文献方法制备豆楮方含药血清,用MTT法观察细胞增殖情况.结果:豆楮方含药血清在体外对人肝癌细胞株HepG2、SMMC-7721均有抑制作用,对HepG2的最高抑制率为91.48%,对SMMC-7721的最高抑制率为89.42%,抑制肝癌细胞的效果优于含5-Fu血清.结论:豆楮方具有抗肝癌作用.     

YM155对人胃癌细胞株MKN28的作用及其机制研究

背景:研究发现生存素(survivin)在胃癌组织中高表达,YM155是survivin的特异性抑制剂。目的:探讨YM155对人胃癌细胞株MKN28的作用及其机制。方法:以不同浓度YM155作用于人胃癌细胞株MKN28。采用甲基噻唑基四唑(MTT)法检测细胞增殖抑制率;以原位末端标记(TUNEL法)检测细胞凋亡率;以逆转录聚合酶链反应(RTPCR)、蛋白质印迹法分别检测survivin mRNA和survivin、多聚ADP核糖聚合酶(PARP)、caspase-3蛋白表达。结果:YM155作用后,MKN28细胞增殖抑制,凋亡增加。随着YM155浓度升高,survivin mRNA和蛋白表达水平明显降低,并伴随PARP、caspase-3蛋白裂解。结论:YM155可抑制人胃癌细胞株MKN28增殖,并诱导其凋亡,此机制可能与抑制survivin表达,继而激活caspase凋亡信号通路有关。     

达沙替尼联合奥沙利铂协同抑制胃癌细胞增殖和迁移

目的:评价Src酪氨酸激酶抑制剂达沙替尼联合奥沙利铂抑制胃癌细胞体外增殖、迁移等恶性生物学行为的效应。方法:实时定量-聚合酶链反应(RT-PCR)及蛋白质印迹法(Western blot)检测Src及其活性形式p-Src在胃癌细胞株中的基础表达;蛋白质印迹技术观察不同剂量奥沙利铂作用于胃癌细胞或作用不同时间后,细胞内Src及p-Src的变化规律;细胞计数试剂盒(CCK-8)方法评价胃癌细胞暴露于不同浓度奥沙利铂和达沙替尼后的生长抑制率,分别计算2种药物的半数抑制浓度(IC50),并使用Calcusyn 2.0软件计算联合作用指数(CI);细胞集落形成实验、流式细胞术和划痕实验观察上述药物对胃癌细胞体外增殖、细胞周期、凋亡和迁移能力的影响。结果:胃癌细胞株SGC-7901、BGC-823、MKN-28的p-Src/Src基础表达比值较高;奥沙利铂可明显上调胃癌细胞SGC-7901、BGC-823、MKN-28的p-Src,并呈时间依赖性(r2=0.96、0.85、0.94);达沙替尼和奥沙利铂在胃癌细胞中的CI均     

健脾理气药诱导人肝癌细胞SMMC7721凋亡的研究

目的观察健脾理气药的诱导凋亡效应,为其临床应用进一步提供依据.方法采用血清药理学方法研究中药的体外效应.应用四甲基偶氮唑蓝(MTT)比色法检测含中药兔血清对肝癌细胞的抑制效应,以Annexin V标记法、DNA含量测定、电子显微镜方法检测含中药血清诱导凋亡及细胞周期阻滞效应.免疫组化法观察含中药血清对P53,P21WAF1/CIP1蛋白的影响,RT-PCR法观察含中药血清对P21WAF1/CIP1 mRNA表达水平的影响.结果含中药血清有一定的抑制肝癌细胞作用,其作用3 d的抑制率为6.6%,作用6 d的抑制率为36.2%.含中药血清作用2 d诱导9.8%±4.0%的肝癌细胞凋亡,使细胞周期阻滞于S期,并上调P53蛋白、P21WAF1/CIP1 mRNA及蛋白的表达.结论健脾理气药具一定的诱导凋亡及抑制肝癌细胞效应,上调p53,p21WAF1/CIP1基因的表达为分子机制.     

蛴螬提取物抗肿瘤作用的体外血清药理学实验

目的 探讨蛴螬提取物对人肺癌A549和人宫颈癌HeLa细胞株抗肿瘤作用.方法 采用血清药理学实验方法,观察含药血清体外对人肺癌A549和人宫颈癌HeLa细胞株抗增殖作用.结果 血清药理学实验结果显示,终浓度分别为20%、25%的灭活的蛴螬提取物含药大白鼠血清对A549细胞株的增殖抑制率分别为18.92%、37.54%;终浓度分别为10%、20%、25%的灭活的蛴螬提取物含药血清对HeLa细胞株的增殖抑制率分别为39.86%、24.86%及26.60%.结论 蛴螬提取物含药血清对人肺癌A549和人宫颈癌HeLa细胞株增殖有抑制作用.     

健脾理气方药物血清对肝癌细胞端粒酶活性及凋亡的影响

目的 观察健脾理气方药物血清对肝癌细胞SMMC-7721端粒酶活性的影响以及诱导肿瘤细胞凋亡的作用,探讨其抗肿瘤治疗的作用机制.方法 采用端粒重复序列扩增(TRAP)结合非变性聚丙烯酰胺凝胶电泳银染法,利用健脾理气方中药灌服新西兰兔后制备含药血清,观察健脾理气方药物血清对体外培养的人肝癌细胞株SMMC-7721端粒酶活性的影响,同时利用流式细胞仪检测和荧光显微镜观察对细胞凋亡的影响.结果 健脾理气药物血清在d 4开始对肿瘤细胞的端粒酶活性有抑制作用,电泳条带光密度明显降低;并能诱导肿瘤细胞的凋亡,凋亡的比例随作用时间的延长而升高,流式细胞术检测d2,d4,d 6的凋亡比例分别为0.81%,5.16%,8.45%.结论 对肿瘤细胞端粒酶活性的抑制和诱导肿瘤细胞凋亡是健脾理气中药抗肿瘤治疗作用机制的一部分.     

五味子多糖对人结肠癌HT29细胞增殖的抑制作用及其机制

目的探讨五味子多糖含药血清对人结肠癌细胞株HT29体外增殖的影响及作用机制。方法采用人结肠癌细胞株HT29作为研究对象,MTT法检测不同浓度的五味子多糖含药血清对细胞增殖的抑制作用;倒置显微镜和荧光显微镜观察细胞形态和细胞核的改变;流式细胞仪分析细胞凋亡的情况。结果五味子多糖含药血清对HT29细胞的增殖有抑制作用而且呈剂量和时间依赖性。五味子多糖含药血清作用72 h后,荧光显微镜可见凋亡细胞及凋亡小体;流式细胞仪检测凋亡。结论五味子多糖含药血清对HT29结肠癌细胞的增殖有抑制作用,其作用机制与诱导细胞凋亡有关。     

Induction of apoptosis by arsenic trioxide and hydroxy camptothecin in gastriccancer cells in vitro

AIM To study the effects of arsenic trioxide and HCPT on different degrees of differentiated gastric cancer cells (SGC-7901, MKN-45, MKN-28)with respect to both cytotoxicity and induction of apoptosis in vitro. ~ODS The cytotoxicity of As2O3 and HCPT on gastric cancer cells was determined by MTTassay. Morphologic changes of apoptosis of gastric cancer cells were observed by light microscopy and transmission electron microscopy. Apoptosis and cell cycle changes of gastric cancer cells induced by HCPT and As2O3 were investigated by TUNEL method and flow cytometry. RESULTS As2O3 and HCPT had remarkable cytotoxic effects on different degrees of differentiated gastric cancer cells. The IC50 of As2O3 on well differentiated gastric cancer cell MKN-28, moderately differentiated gastric cancer cell SGC-7901, and poorly differentiated gastric cancer cell MKN-28 were 8. 91 μmol/L, 10. 57 μmol/L, and 11.65 μmol/L, respectively. The IC50 of HCPT on MKN-28, SGC-7901, and MKN-45 were 9. 35 rg/L, 10. 21 rg/L, and 12. 63 mg/L respectively after 48 h treatment. After 12 h of exposure to both drugs, gastric cancer cells exhibited morphologic features of apoptosis, including cell shrinkage, nuclear condensation,and formation of apoptotic bodies. A typical subdiploid peak before G0/G1 phase was observed by flow cytometry. The apoptotic rates of SGC7901, MKN-45, and MKN-28 were 13. 84%, 22.52%, and 9. 68%, respectively after 48 h exposure to 10 μmol/L As2O3. The apoptotic rates of SGC-7901, MKN-45, and MKN-28 were 21.88%, 12.35%, and 30. 26%, respectively after 48 h exposure to 10 mg/L HCPT. The apoptotic indice were 7% - 15% as assessed by TUNEL method. The effect of As2O3 on SGC-7901 showed remarkable cell cycle specificity, which induced cell death in G1 phase, and blocked G2/M phase. HCPT also showed a remarkable cell cycle specificity, by inducing cell death and apoptosis in G1 phase and arrest of proliferation at S phase. CONCLUSION AS2O3 and HCPT exhibit significant cytotoxicity on gastric cancer cells by induction of apoptosis. As2O3 and HCPT might have a promising prospect in the treatment of gastric cancer, which needs to be further studied.     

健脾益气方促进胃黏膜修复机制研究

[目的]研究健脾益气中药对大鼠胃黏膜保护和修复的机制,观察大鼠胃黏膜表皮生长因子受体(EG-FR)和血清一氧化氮(NO)的变化.[方法]采用乙酸致溃疡大鼠模型,以SABC免疫组织化学方法观察溃疡周围胃黏膜的EGFR并进行图像分析,用镀铜镉还原比色法测定血清NO3-/NO2-代表血清NO水平.[结果]模型组胃黏膜EGFR表达较对照组增加(P＜0.01),中药组进一步升高(P＜0.01).模型组血清NO3-/NO2-明显低于对照组(P＜0.01),中药组血清NO3-/NO2-则显著高于模型组(P＜0.01).[结论]中药健脾益气方通过EGFR和NO促进胃粘膜保护和修复.     

Effect of trans-retinoic acid and folic acid on apoptosis in human gastric cancer cell lines MKN-45 and MKN-28

Induction of apoptosis has been implicated as an anticarcinogentic mechanism of both folic acid and retinoic acid. The ability of retinoic acid or folic acid to induce gastric cancer cell apoptosis was investigated in the human gastric cancer cell lines MKN-45 and MKN-28, and DNA fragmentation was studied in situ by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and DNA agarose gel electrophoresis. The rates of apoptosis in both the poorly differentiated MKN-45 and the well differentiated MKN-28 cell line were less than 5% after treatment with either retinoic or folic acid. Apoptosis may be induced by the administration of retinoic acid or folic acid, and the apoptosis indices of MKN-45 and MKN-28 cells were related to the doses of these drugs. The induction of gastric cancer cell apoptosis may play a role in the anticarcinogenetic effect of retinoic acid and folic acid, both of which are potential agents for the treatment of human gastric cancer. Received Sept. 16, 1997; accepted Mar. 17, 1998     

中药复方从脾虚论治肝癌的作用机制研究

目的:探讨脾虚方对肝癌HepG2细胞凋亡和迁移侵袭的影响。方法:体外培养HepG2细胞,加入不同中药复方含药血清干预48 h后,用流式细胞仪检测含药血清对HepG2细胞凋亡的影响;Transwell实验检测药物对细胞侵袭能力的影响。结果:干预48 h后,毒痰瘀方组、脾虚方组及索拉非尼组HepG2细胞的凋亡率分别为(11.63±0.87)%、(12.47±0.56)%、(24.20±1.61)%,脾虚方组诱导细胞凋亡效果显著,与正常对照组相比较,差异具有统计学意义(P<0.05);毒痰瘀方组、脾虚方组及索拉非尼组的迁移细胞数分别为124.6±8.4、121.0±9.6、100.2±7.1,细胞侵袭数分别为80.4±7.6、89.2±8.4、71.4±7.3,脾虚方可有效抑制HepG2细胞的迁移、侵袭能力,与正常对照组相比较,差异具有统计学意义(P<0.05)。结论:脾虚方药物血清具有促进HepG2细胞凋亡,抑制HepG2细胞迁移与侵袭的作用。     

Inhibition of human telomerase in MKN—45 cell line by antisense hTR expression vector induces cell apoptosis and growth arrest

AIM: To investigate the effects of antisense human telomerase RNA (hTR)on the biologic behavior of human gastric cancer cell line: MKN-45 by gene transfection and its potential role in the gene therapy of gastric cancer. METHODS: The hTR cDNA fragment was cloned from MKN-45 through RT-PCR and subcloned into eukaryotic expression vector (pEF6/V5-His-TOPO) in cis-direction or trans-direction by DNA recombinant methods. The constructed sense, antisense and empty vectors were transfected into MKN-45 cell lines separately by lipofectin-mediated DNA transfection technology. After drug selection, the expression of antisense hTR gene in stable transfectants and normal MKN-45 cells was detected by RT-PCR, the telomerase activity by TRAP, the apoptotic features by PI and Hoechst 33258 staining, the cell cycle distribution by flow cytometry and the population doubling time by cell counting. Comparison among the stable transfectants and normal MKN-45 cells was made. RESULTS: The sense, antisense hTR eukaryotic expression vectors and empty vector were successfully constructed and proved to be the same as original design by restriction endonuclease analysis and sequencing. Then, they were successfully transfected into MKN-45 cell lines separately with lipofectin. The expression of antisense hTR gene was only detected in MKN-45 cells stably transfected with antisense hTR vector (named as MKN-45-ahTR) but not in the control cells. In MKN-45-ahTR, the telomerase activity was inhibited by 75%, the apoptotic rate was increased to 25.3%, the percentage of cells in the G0/G1 phase was increased to 65%, the proliferation index was decreased to 35% and the population doubling time was prolonged to 35.3 hours. However, the telomerase activity, the apoptotic rate, the distribution of cell cycle, the proliferation index and the population doubling time were not different among the control cells. CONCLUSION: Antisense hTR can significantly inhibit telomerase activity and proliferation of MKN-45 cells and induce cell apoptosis. Antisense gene therapy based on telomerase inhibition can be a potential therapeutic approach to the treatment of gastric cancer.     

消瘤汤含药血清对人肝癌细胞Bel-7402增殖和凋亡的影响

[目的]观察消瘤汤含药血清对人肝癌细胞Bel-7402增殖和凋亡的影响。[方法]采用消瘤汤含药血清,应用血清药理学方法,选择不同浓度的含药血清体外培养人肝癌细胞Bel-7402 96 h。通过MTT法和细胞形态学观察检测细胞的增殖抑制率和细胞凋亡。[结果]MTT法检测表明,消瘤汤高、中、低剂量组与0.95%氯化钠（对照）组相比,对人肝癌细胞增殖均有抑制作用（P〈0.01）。细胞形态学检测表明消瘤汤各剂量组含药血清均可诱导细胞凋亡。[结论]消瘤汤能明显抑制肝癌Bel-7402细胞的增殖,其抑瘤作用有细胞毒作用和诱导细胞凋亡2种途径。     

去氢骆驼蓬碱通过下调COX-2表达抑制胃癌细胞迁移和侵袭

目的探讨去氢骆驼蓬碱对人胃癌MKN-45细胞环氧化酶-2(cyclooxygenase-2,COX-2)表达、迁移和侵袭的影响。方法 MKN-45细胞接种于含10%胎牛血清的RPMI-1640培养液中,常规培养24h后加去氢骆驼蓬碱(2、4、8、16、32μg/ml),同时设置对照组不加药物,空白组只加培养液不含细胞,分别培养24h、48h、72h,MTT法检测细胞增殖率;western blot法检测COX-2表达;划痕损伤愈合实验及Transwell小室基质侵袭实验检测胃癌细胞体外迁移和侵袭。结果去氢骆驼蓬碱剂量依赖性抑制MKN-45细胞COX-2表达(P0.01);与对照组相比,去氢骆驼蓬碱组MKN-45细胞迁移和侵袭能力明显下降(P0.01)。结论去氢骆驼蓬碱可能通过下调COX-2表达抑制胃癌细胞迁移和侵袭。