祁连圆柏提取物诱导人肝癌细胞Bel-7402凋亡的作用及筛选研究

摘要

Objective To investigate the effect of anti - proliferative and induction apoptosis on human hepatocellular carcinoma Bel - 7402 cells of Juniperus przewalskii in vitro and screen the active components. Methods The antitumor active components were extracted by silica gel column chromatography, and contents were measured by spectrophotometer. The effects of proliferation of Bel - 7402 cells were analyzed by clone formation assay, trypan - blue and MTT method. The morphological features of cell apoptosis were observed under light and electrical microscopies. The cell cycle and apoptosis index were detected by flow cytometry ( FCM) and the expression of Bax and Bel - 2 were tested by immunohistochemical S - P method. Results Juniperus przewalskii significantly inhibited the proliferation of Bel -7402 cells, which increased cell numbers at the G0/G1, phase but decreased cell number at the G2/M phase cells are also found clearly in histogram following 10, 20, 50礸/ml C solution's effective component - Group 3 of juniperus przewalskii, which the cell half inhibitory concentration (IC50) was 2. 47礸/ml and immunohistochemical S - P expression of Bax protein was up - regulated and Bel - 2 protein was significantly down - regulated in experimental cell groups. Conclusion The antitumor active compounds of the C - solution - Group 3 of Juniperus przewalskii can significantly inhibit the proliferation of Bel - 7402 cells. The mechanisms include increasing the expression of Bax, decreasing the expression of Bel - 2, the activation of apoptosis gene protein Bax, G0/G1 phase retardant, which plays the important role in anti - tumor effect of Juniperus przewalskii on Bel -7402 cell.%目的 探讨祁连圆柏提取物体外对人肝癌细胞Bel-7402体外增殖抑制、凋亡作用机制并进行有效组分筛选研究.方法 采用硅胶柱层析法提取祁连圆柏有效抗瘤组分,分光光度法测定含量.用克隆形成实验、台盼蓝拒染法及MTT显色法检测祁连圆柏提取物对Bel-7402细胞增殖的影响,流式细胞术检测其诱导细胞凋亡的细胞周期阻滞状况及凋亡率,免疫组化观察对Bcl-2与Bax蛋白的表达水平,光镜、电镜观察凋亡细胞的组织形态和超微结构变化.结果 祁连圆柏提取物对Bel-7402细胞增殖有明显的抑制作用,其中C液-组分-3细胞的半数抑制浓度为2.47μg/ml.流式细胞检测结果G0/G1期细胞增多,G2/M期细胞减少,其中10～50μg/ml的祁连圆柏C液-组分-3在G1期细胞前出现典型“凋亡峰”;免疫组化SP法检测实验组细胞BCL-2基因蛋白表达降低,Bax基因蛋白表达增加.光镜与电镜下均见凋亡细胞明显增多.结论 祁连圆柏C液-组分-3具有显著的抑制Bel-7402细胞增殖以及诱导该细胞系凋亡的作用,其诱导Bel-7402细胞凋亡的作用与其上调Bax、下调Bcl-2蛋白的表达,激活Bax凋亡基因蛋白及Go/G1期阻滞有关,是其重要的抗肿瘤作用机制.