DPC4基因转染对结肠癌细胞生物学行为的影响

Effect of transfected DPC4 gene on biological behaviours of human colorectal carcinoma cells

目的:研究DPC4基因转染对结肠癌细胞生物学行为的影响,探讨DPC4在结肠癌的发生和发展中的作用.方法:利用基因重组技术构建PcDNA3.1-DPC4质粒;利用脂质体介导转染技术和G418筛选得到稳定表达Smad4蛋白的DPC4+-SW620(PcDNA3.1-DPC4转染的SW620高转移性结肠癌细胞株)细胞模型;采用免疫组化S-P法和Westernblot检测细胞中Smad4的表达;生长曲线和平板克隆形成实验检测其生物学行为的改变;采用流式细胞术检测其S期细胞百分数(S%)和凋亡率.结果:成功构建了PcDNA3.1-DPC4质粒;DPC4+-SW620细胞的Smad4蛋白表达于细胞质及细胞核,以细胞质为主,且Smad4蛋白表达水平明显高于SW620细胞及PcDNA3.1-SW620细胞(PcDNA3.1转染的SW620细胞);DPC4+-SW620细胞的倍增时间为116h,较SW620细胞(31h,P<0.01)及PcDNA3.1-SW620细胞(29h,P<0.01)明显延长;DPC4+-SW620细胞的克隆形成率为(12%),明显低于SW620细胞(69%,P<0.01)及PcDNA3.1-SW620细胞(67%,P<0.01)的克隆形成率;与SW620细胞及PcDNA3.1-SW620细胞相比较,DPC4+-SW620细胞G0-G1期细胞百分数增多,S期细胞百分数明显减少(P<0.05);与SW620细胞及PcDNA3.1-SW620细胞相比较,DPC4+-SW620细胞的凋亡率较高.结论:成功地构建了质粒PcDNA3.1-DPC4,并转染SW620细胞株,得到稳定且表达Smad4蛋白明显增强的细胞模型;DPC4对结肠癌细胞增生的调控可能是通过DPC4抑制细胞生长和诱导细胞凋亡两方面作用实现的.

AIM:To study the effect of transfected DPC4 gene on bio-logical behaviours of human colorectal carcinoma cells andthe role of DPC4 gene in colorectal carcinogenesis.METHODS: PcDNA3.1-DPC4 plasmid was re-constructed bygene-recombination technology; Sw620 cells, a humancolorectal carcinoma cell line, were transfected with PcDNA3.1-DPC4 plasmid using lipofectamine transfecting technique.Transfected cells were selected with G418; Expression ofSmad4 protein was detected by immunohistochemistry andWestern blot in cells transfected DPC4 gene; Biological char-acters of transfected cells were evaluated by populationdoubling time and cloning efficiency;Alterations of S% andapoptosis rate were determined by flow- cytometry.RESULTS: PcDNA3.1-DPC4 plasmid was constructedsuccessfully; Sw620 cells transfected with PcDNA3.1-DPC4plasmid showed strong intracellular expression of Smad4protein, and the positive signal was localized in cytoplasmand nucleus, mainly in cytoplasm, while the expression ofSmad4 protein in Sw620 cells transfected with PcDNA3.1and SW620 cells without the transfection were weaker thanthat in Sw620 cells transfected with PcDNA3.1-DPC4 plasmid;The population doubling time in Sw620 cells transfectedwith PcDNA3.1-DPC4 plasmid (116h) was much longer thanthose in SW620 cells without transfection (116h versus 31h,P <0.01) and Sw620 cells transfected with PcDNA3.1 plas-mid (116h versus 29h,P < 0.01); The cloning efficienciesof Sw620 cells transfected with PcDNA3.1-DPC4 plasmid(12%) were much lower than those of SW620 cells with-out transfection (12% versus 69%,P <0.01) and Sw620 cells transfected with PcDNA3.1 plasmid (12% versus 67%,P <0.01);Compared with SW620 cells without transfectionand Sw620 cells transfected with PcDNA3.1 plasmid; theG0-G1% of Sw620 cells transfected with PcDNA3.1-DPC4plasmid was much higher while the S% was much lower,P <0.05; Apoptosis rate of Sw620 cells transfected withPcDNA3.1-DPC4 plasmid was much higher than that ofSW620 cells without transfection and Sw620 cells trans-fected with PcDNA3.1 plasmid.CONCLUSIONS: PcDNA3.1-DPC4 plasmid is successfully re-constructed and transfected into SW620 cells with PcDNA3.1-DPC4 plasmid, and cell model with steadily expressedSmad4 is obtained. Proliferation of the cells can be regu-lated by inhibiting growth and inducing apoptosis ofcolorectal carcinoma cells.