【作者】 付燕燕；

【导师】 段昌柱；

【作者基本信息】 重庆医科大学 ， 遗传学， 2005， 硕士

【摘要】 目的:苦参碱（matrine,Mat）在临床上主要用于肝炎治疗。最近文献报道它还具有抗癌活性,但其作用机理尚不清楚。本文拟研究 Mat对人肝癌细胞 HepG2 凋亡诱导作用;研究细胞周期调节蛋白 P27^kip1在凋亡中变化;探讨细胞凋亡与 p27^kip1 之间的关系。初步探讨 P27^kip1 调控发生的时期。 方法:运用 MTT 法检测细胞增殖情况,形态学观察、流式细胞仪等多种方法检测 HepG2 凋亡及细胞周期分布的改变。免疫细胞法检测细胞内 p27kip1 蛋白表达变化。RT-PCR 技术检测 mRNA 表达水平。 结果:（1）0.5mg/ml～2.0mg/ml Mat 对 HepG2 细胞增殖有明显抑制作用,Mat 与细胞抑制之间具有时间-剂量依赖性。（2）TEM 观察发现,经 1.0mg/ml Mat 处理 24h 的细胞出现了胞膜上微绒毛消失,染色质边集、核浓缩、核碎裂,凋亡小体形成。（3）FCM 发现 1.0 mg/ml﹑2.0 mg/ml Mat 作用 48h,细胞停滞于 G0/G1 期, 细胞发生凋亡,DNA 组方图上出现典型的凋亡峰,随浓度增加,凋亡百分率逐渐增高。（4）免疫细胞化学结果显示苦参碱使人肝癌细胞 HepG2 P27kip1 蛋白表达上调。（5）RT-PCR 结果表明 Mat 作用引起凋亡时 p27kip1 mRNA 水平无变化。 结论:苦参碱抗肿瘤机制是诱导 HepG2 细胞凋亡,与细胞周期蛋更多还原

【Abstract】 Objective: It has been reported that matrine had the anti-tumor activity, while the mechanism（s） by which matrine suppresses tumor growth remains unknown. The study was designed to investigate inhibitory effects and mechanisms of matrine-induced apoptosis. We have designed experiments to explore the expression changes of the cell cyclin inhibitor p27^kip1 during apoptosis and analyzed the correlation between this apoptosis and p27^kip1. And to investigate regulation pattern and time of p27^kip1 protein. Methods: MTT assay was used to examine the changes of the cell proliferation. Microscopic observation, flow cytometry were used to determine the induction of apoptosis and the changes of cell cycle. Immunohistochemistry was performed to determine the protein of p27^kip1, and RT-PCR was used to determine the mRNA levels of p27^kip1. Results: （1） 0.5mg/ml～2.0mg/ml matrine could inhibit the proliferation and induce the apoptosis of HepG2 cells on a time and dose-depending manner. （2） In result of TEM , microvillus disappear, electron density of cytoplasm is high ,large deciduous apoptosis bodies appear after being treated with 1.0mg/ml Mat for 24 houres.（3） In result of FCM,after dealt with 1.0 mg/ml﹑2.0 mg/ml Mat for 48 hours,cell cycle is controlled in G0/G1,and there is a apoptosis peak in DNA tapes.（4） The protein levels of p27^kip1 were gradually upregulated with concentration increasing of matrine. （5） Their mRNA levels remained unchanged. Concludes: The anti-tumor mechanism of matrine is that it can induce apoptiosis in HepG2 cells and there is significant correlation with cell cycle inhibitor p27^kip1. Matrine may influence the protein expression of p27^kip1 in HepG2 cells, while do not influence their mRNA transcription. p27^kip1 protein regulation may happen in post-transcription .更多还原