抑癌基因Smad4对HepG2生长的影响及其机制探讨

目的研究抑癌基因Smad4对人肝癌细胞株HepG2．生长的影响，并探讨其作用机制。方法采用脂质体瞬时转染法转染PFTX-5Smad4质粒至人肝癌细胞株HepG2（实验组），以转染空质粒PFTX-5的HepG2细胞为对照组，野生型HepG2细胞为空白组，利用免疫印迹（Westernblot）和逆转录聚合酶链反应（RT—PCR）检测Smad4及血管内皮生长因子（VEGF）表达的变化；用MTT法检测细胞增殖及用FITC—AnnexinV、碘化吡啶（propidiumiodide，PI）双染后流式细胞仪（FCM）检测细胞周期和凋亡情况，Westernblot检测细胞周期素D1（CyclinD1）表达差异。结果PFTX-5Smad4瞬时转染到肝癌细胞后，实验组与对照组和空白组相比，Smad4mRNA和蛋白表达明显上升（P〈0．05），而VEGFmRNA和蛋白的表达显著降低（P〈0．05）。实验组细胞CyclinD1表达明显下降（P〈0．05），细胞周期时相分布出现合成前期（G。／G．期）阻滞。实验组细胞凋亡率明显升高（P〈0．01），增殖受到明显抑制。结论Smad4高表达可能通过降低肝癌细胞中VEGF的表达，抑制eyclinD1转录合成，并诱导强烈的G1期阻滞和细胞凋亡，对细胞的增殖有明显的抑制作用，为进-步研究肝癌生长增殖机制提供了实验基础。

The growth effects of tumor suppressor Smad4 gene on human hepatocellular carcinoma cell line HepG2

Objective To study the growth effects of transfected tumor suppressor Smad4 gene on human hepatocellular carcinoma cell line HepG2. Methods Plasmid of PFTX-5 Smad4 was transfected into human hepatocellular carcinoma cell line HepG2. The HepG2 cells transfected with this plasmid （experimental group） and those treated with PFTX-5 vector （control group） and without any treatment （blank group） were compared. The expression of Smad4 and VEGF were detected by RT-PCR and Western blot. Cell proliferation was detected by MTT and then assessed the cell cycle distribution and apoptosis by flow cytometry with fluoreseein isothiocyanate-conjugated Annexin V （FITC-Annexin V ） and propidium iodide （PI） staining. The expression of CyclinD1 was detected by Western blot. Results After being transfected with PFTX-5 Smad4, with the decrease in VEGF gene expression （P 〈 0.05 ） , the growth rate of HepG2 was also inhibited compared with those of the control and blank groups. Cell cycle blocked at G0/G1 stage and hepatic cell apoptosis was found （P 〈 0.01 ）. CyclinD1 expressed lower （P 〈 0.05）. Conclusion Growth of HepG2 can be controled by increasing Smad4 expression. These provide potent theoretical ground for studying the mechanism of cell proliferation in hepatoma.