Smad4基因抑制人类胃癌细胞株生长的作用机制

目的研究Smad4基因对人类胃癌细胞株细胞周期和细胞凋亡的影响,探讨其对人类胃癌细胞生长抑制的作用机制。方法运用脂质体介导转染方法将Smad4基因导入其表达缺失的胃癌MKN28细胞株中,经G418筛选,获得稳定表达Smad4基因的Smad4 -MKN28细胞和作为对照的Smad4--MKN28细胞。体外培养亲本细胞、Smad4 -MKN28细胞和Smad4--MKN28细胞,各组细胞于第1、3、5、7天取三瓶细胞计数,计算平均值,并绘制生长曲线;细胞培养48h后应用流式细胞仪检测细胞周期和凋亡率的改变;用RT-PCR和Western blotting方法检测细胞Bcl-2、Bax基因的mRNA和蛋白的表达。结果稳定转染Smad4重组质粒的Smad4 -MKN28细胞较未转染的Smad4--MKN28细胞生长速度减慢(P<0.05);流式细胞仪检测结果示Smad4 -MKN28细胞G0-G1期细胞百分数增加(P<0.05),S期细胞百分数减少(P<0.05),细胞凋亡增加(P<0.05);RT-PCR结果示Smad4 -MKN28细胞Bax基因表达增加(P<0.05),Bcl-2基因表达减少(P<0.05),Western blotting结果示两者的编码蛋白也出现相应的变化。结论恢复Smad4表达后细胞增殖减慢,凋亡增加,促凋亡基因表达上调,抑制凋亡基因表达下调,两者比值增大,从而使肿瘤细胞的生长受到抑制。

Mechanism of suppression effect of Smad4 gene on growth of human gastric cancer cell lines

Objective To study the effect of Smad4 gene on the cell cycle and apoptosis in human gastric cancer cell lines,and discuss the mechanism of its suppression effect on the growth of gastric cancer cell lines.Methods Smad4 gene was introduced into human gastric cancer cell lines MKN28 that lacked Smad4 gene by liposome-mediated transfection.After screened by G418,Smad4 -MKN28 cells which steadly expressed Smad4 gene and Smad4--MKN28 cells(a controlled cell line)were obtained.The parent cells,Smad4 -MKN28 cells and Smad4--MKN28 cells were cultured,cell count and average value at 1,3,5 and 7 d after culture were obtained and the growth curve was drawn.The cell cycle distributions and apoptosis were observed by cytometry.The mRNA and protein expressions of bax and Bcl-2 were detected by RT-PCR and Western blotting.Results Smad4 -MKN28 cells steadly transfected with recombinant plasmid gene grew slower than Smad4--MKN28 cells without transfection(P<0.05).Compared with the other two cell lines,G0-G1% of Smad4 -MKN28 cells was higher(P<0.05),while S% was lower(P<0.05),and the apoptosis rate was increased(P<0.05).RT-PCR revealed that the expression level of Bax gene in Smad4 -MKN28 cells was significantly increased(P<0.05),while that of Bcl-2 was much lower(P<0.05).The corresponding changes in encoding proteins were also indicated by Western blotting.Conclusion After transfection of eukaryotic expression vector with Smad4 gene,cell proliferation can be slowed,cell apoptosis can be increased,and the up-regulation of apoptosis-stimulating gene and down-regulation of apoptosis-inhibiting gene may lead to the suppression of tumor cell growth.