Survivin microRNA对人肝癌细胞间通讯功能影响的实验观察

目的 观察survivin的microRNA对HepG2细胞间通讯功能主要环节的影响作用.方法 人工设计合成特异性靶向survivin的microRNA的序列.肝癌细胞株HepG2接种于细胞培养板内并分为5组.作用后收集各组细胞.流式细胞术检测各组细胞增殖率和凋亡指数.划痕染料示踪技术检测转染前后HepG2的细胞间隙连接通讯功能变化,放射性免疫法定量测定细胞内cAMP含量的变化,钙离子指示剂Fura-2双波长荧光检测技术检测转染前后胞内Ca2＋水平的变化.结果 流式细胞术检测各浓度miRNA转染组细胞凋亡指数明显高于对照组（P＜0.05）,高浓度转染组最明显（P＜0.05）;各浓度microRNA转染组细胞增殖指数明显低于对照组（P＜0.05）,高浓度转染组最明显（P＜0.05）;Survivin microRNA转染组HepG2细胞间隙连接通讯功能较两个对照组有不同程度的恢复和增强;不同浓度Survivin microRNA转染组细胞内cAMP浓度明显高于对照组;Survivin microRNA转染组HepG2细胞内[Ca2＋]与两个对照组相比有升高趋势.结论 Survivin microRNA能显著抑制HepG2细胞的增殖,促进GJIC功能恢复和增强,并能直接影响细胞内的第二信使传递系统.

Effect of microRNA Targeting Surviving on Intercellular Communication of Human Liver Cancer Cells

Objective To investigate the effects communication in human liver cancer cells. Methods of microRNA targeting survivin on intercellular MicroRNA targeting survivin was synthesized and transfected into HepG2 cells by lipofectin. Twenty-four hours before transfection, HepG2 cells were seeded in culture dish overnight, and divided into 5 groups for transfection. The transfected cells were then harvested for the following analyses. Apoptotic index （AI） and proliferative index （PI） were examined by flow cytometry. Effect on GJIC of HepG2 cells was observed with scrape- loading and dye transfer technique. The intracellular cAMP content was detected by 125I-cAMP protein binding assay. The change of cytosolic free calcium concentrations was investigated by Fura-2/ AM. Results As shown by flow cytometry, the apoptotic indexes of the dose-range miRNA groups were significantly higher than those of compare groups （ P 〈 0. 05 ）, with the most obvious apoptotic index for the high dose miRNA group. The proliferative indexes of the dose-range miRNA groups were significantly lower than those of compare groups （P 〈 0.05 ）, with the most obvious proliferation index for high dose miRNA group. Survivin microRNA up-regulated or recovered GJIC of HepG2 cells to a certain extent, compared with two other control groups. The intracellular cAMP content in survivin miRNA groups was notably higher than two control groups. Compared with two control groups, the concentration of cytosolic free calcium increased significantly. Conclusion Survivin microRNA can effectively inhibit the proliferation of HepG2 cells, significantly up-regulating or recovering GJIC of HepG2 cells and directly influencing the second messenger system.