DPC_4基因转染结肠癌细胞对VEGF表达的影响

Effect of transfected DPC_4 gene on expression of VEGF in human colorectal carcinoma cells

目的 研究DPC4基因转染人结肠癌细胞株SW 6 2 0对血管内皮生长因子 (VEGF)表达的影响。方法 利用DNA的限制性内切酶双酶切技术鉴定重组质粒PCDNA3 1 DPC4;利用脂质体介导转染技术和G4 18筛选得到稳定表达Smad4蛋白的DPC+ 4 SW 6 2 0 (PCDNA3 .1 DPC4转染的SW 6 2 0高转移性结肠癌细胞株 )细胞模型 ;采用Westernblot检测细胞中Smad4的表达 ;采用ELISA检测细胞上清液中VEGF的蛋白表达量 ;采用半定量逆转录多聚酶链反应RT PCR技术检测细胞中VEGF的mRNA表达量。结果 阳性质粒组DPC+ 4 SW6 2 0细胞的Smad4蛋白表达强于空白质粒组PCDNA3 .1 SW 6 2 0和未转染组SW6 2 0 ;DPC+ 4 SW 6 2 0组细胞上清液VEGF蛋白分泌明显减少 ,与PCDNA3 .1 SW 6 2 0组和SW 6 2 0组比较差异有显著性 (P <0 0 5 ) ,DPC+ 4 SW 6 2 0组细胞与PCDNA3 .1 SW6 2 0组细胞中VEGFmRNA半定量结果和SW6 2 0组比较 ,其表达量分别下降2 8 3%和 4 5 %。结论 DPC4可抑制人结肠癌细胞株SW 6 2 0中VEGF的表达。

Purpose To study the effect of transfected DPC4 gene on expression of VEGF in human colorectal carcinoma cells. Methods The re-constructed plasmid PCDNA 3.1 -DPC 4 was proved by restriction endonuclease technology. SW620 cells, a human colorectal carcinoma cell line, were transfected with PCDNA 3.1 -DPC 4 plasmid using lipofectamine transfecting technique. Transfected cells were selected with G418. Expression of Smad4 protein was detected by Western blot in cells transfected DPC 4 gene. VEGF protein of cell culture supernatants was dectected by ELISA method. The expression of VEGF mRNA was detected by RT-PCR method. Results SW620 cells transfected with PCDNA 3.1 -DPC 4 plasmid showed stronger intracellular expression of Samd4 protein than SW620 cells transfected with PCDNA 3.1 and SW620 cells without the transfection. The secretion of VEGF protein in cell culture supernatants of SW620 cells transfected with PCDNA 3.1 -DPC 4 plasmid was significantly decreased compared with control (P<0.05). The VEGF mRNA expression of SW620 cells transfected with PCDNA 3.1 -DPC 4 plasmid and SW620 cells transfected with PCDNA 3.1 plasmid was lower compared with SW620 cells without transfection. They decreased 28.3% and 4.5% respectively. Conclusion DPC 4 can reduce expression of VEGF in SW620 cells.