摘要
目的:探讨加味左金丸三种组成中药的主要单体成分小檗碱、吴茱萸碱和靛玉红对人胃癌细胞生长抑制、诱导凋亡和细胞周期的影响. 方法:人胃癌细胞株为低分化黏液腺癌MGC803细胞; 小檗碱、吴茱萸碱和靛玉红购自中国药品与生物制品检定所.苔盼蓝染色细胞计数法测定细胞存活率和凋亡率,流式细胞技术进行细胞周期、凋亡百分比分析,甲基绿-派诺宁联合染色法观测凋亡形态,琼脂糖凝胶电泳分析DNA损伤情况. 结果:96 h内小檗碱、吴茱萸碱各组细胞存活率不断下降,其中48 h存活率对照组为100.0±7.9%,小檗碱4 mg/L和8 mg/L组分别为72.9±6.2%(t=4.67, P<0.01)和17.4±4.8%(t=15.48,P<0.001),吴茱萸碱1 mg/L和5 mg/L组分别为37.8±5.7%(t=11.06, P<0.001)和10.7±11.1%(t=11.35,P<0.001);各组均与对照组比较(n=3);对生长的抑制作用呈时间、药物浓度依赖性.甲基绿-派诺宁原位染色,细胞表现出凋亡特征.小檗碱与吴茱萸碱组脱落的悬浮死细胞中有较高比例胞膜完整、抗拒苔盼蓝和酚红染色的凋亡细胞, 其中48 h凋亡率对照组为0.3±0.0%,小檗碱8 mg/L 组为65.2±9.5%(t=11.83,P<0.001);吴茱萸碱1 mg/L组为58.9±11.4%(t=8.90,P<0.001),而阿霉素组脱落死细胞不能抗拒苔盼蓝和酚红染色.流式细胞仪检测表明小檗碱与吴茱萸碱组均出现亚二倍体凋亡峰;细胞周期分别阻滞于G0—G1、G2期;凋亡百分比与对照组11.8±2.5比较,小檗碱4 mg/L组48 h 为18.9±2.7(t=3.34,P<0.05),72 h为23.9±3.3(t=5.06,P<0.01),吴茱萸碱1 mg/L组72 h 为16.6±1.6(t=2.80,P<0.05).琼脂糖凝胶电泳表明,小檗碱组DNA裂成大片段,吴茱萸碱组和阿霉素组DNA断裂呈涂抹状.靛玉红各组对细胞生长、凋亡无影响,DNA无损伤. 结论:小檗碱与吴茱萸碱均能诱导人胃癌细胞凋亡,且作用较阿霉素温和;靛玉红对胃癌细胞的体外作用不明显.
AIM: To explore the effects of berberine, evodiamine and indirubin, the major constituents of Chinese medicinal herbs Coptis, evodia fruit and natural indigo, respectively, on the proliferation and apoptosis of human gastric cancer cells. METHODS: Human gastric cancer cell line MGC803 was derived from low differentiatedmucous adenocarcinoma. Berberine, evodiamine and indirubin were purchased from National Institute for the Control of Pharmaceutical and Biological Products. Cell viability and apoptosis were determined by trypan blue exclusion assay. Cell cycle distribution and the percentage of apoptosis were determined by flow cytometry. The apoptosis morphology was observed through methyl green and pyronin staining. Nucleo-somal DNA fragmentation was assayed by agarose gel electrophoresis. RESULTS: The cell viability was decreasing continuously during 96 h treatment with berberine and evodiamine. At 48 h, the viability was 100% in control group, while the viability in 4 mg/L and 8 mg/L berberine groups were 72.9±6.2% (t = 4.67, P<0.01) and 17.4±4.8% (t = 15.48, P<0.001), respectively. The viability in 1 mg/L and 5 mg/L groups evodiamine were 37.8±5.7% (t = 11.06, P<0.001) and 10.7±11.1% (t = 11.35, P<0.001). The inhibition effects were dose- and time-dependent. MGC-803 cells showed typical apoptosis morphology when stained by methyl green and pyronin. High proportion of trypan blue and phenol red excluding apoptotic cells with integrate membrane were observed in the suspending dead cells in berberine and evodimine groups. At 48 h, the apoptosis cell ratios were 0.3±0.0% in the control group, 65.2±9.5% (t = 11.83, P<0.001) in 8 mg/L berberine group, and 58.9±11.4% (t = 8.90, P<0.001) in 1 mg/L evodiamine group. No such cells were observed in adriamycin group. Flow cytometry analysis indicated that the apoptotic cell death induced by berberine or evodiamine was accompanied with cell cycle arrest in the G0/G1 or G2 phase, respectively. Both exhibited a sub-diploid apoptotic peak. The percentages of apoptosis were 11.8±1.5 % in control group, 18.9±2.7% (t = 3.34, P<0.05) at 48 h, 23.9±3.3 % (t = 5.06, P<0.01) at 72 h in 4 mg/L berberine group, and 16.6±1.6 % (t= 2.80, P<0.05) at 72 h in 1 mg/L evodiamine group. Agarose gel electrophoresis assay showed that the MGC-803 cell DNA was degraded into large fragments when treated with berberine, and smear fragments when treated with evodiamine and adriamycin. At the given concentration, indirubin had no effect on MGC-803. CONCLUSION: Berberine and evodiamine induce apoptotic cell death of MGC-803 gastric cancer cells. The effects are milder than adriamycin. Indirubin has insignificant effect on MGC-803 in vitro.
出处
《世界华人消化杂志》
CAS
北大核心
2005年第4期472-476,共5页
World Chinese Journal of Digestology
基金
国家中医药管理局科学技术研究基金资助项目.No.97Y031~~
