小RNA干扰沉默VEGF基因对结肠癌HT29细胞系增殖和凋亡的影响

Role of VEGF gene on the biological behaviour of colorectal cancer HT29 cells

目的探讨血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)基因在调控人大肠癌(colorectal carcinoma,CRC)细胞HT29增殖及凋亡中的作用。方法以脂质体Lip 2000为载体,用针对VEGF特异靶点的siRNA转染人CRC细胞HT29后,应用免疫细胞化学SP法和Western blot法检测VEGF蛋白表达变化;MTT法检测细胞增殖;流式细胞技术检测细胞凋亡。结果免疫细胞化学示:VEGF在正常组、脂质体组及阴性错配组细胞质中均呈强阳性;在VEGF-siRNA组细胞质呈弱阳性。Western blot结果示:VEGF-siRNA组细胞的蛋白条带亮度明显低于正常组、脂质体组和阴性错配组;MTT检测结果发现,与阴性错配组相比,VEGF-siRNA组细胞生长出现明显抑制(P<0.05),且不同时段(24、48、72 h)肿瘤细胞增殖抑制率之间差异有显著性(P<0.05);流式细胞术检测VEGF-siRNA组细胞凋亡比例明显高于正常组及阴性错配组(P<0.05)。结论VEGF基因参与调控制CRC细胞HT29的增殖和凋亡,VEGF有望成为CRC基因治疗的新靶点。

Purpose To explore the influence of VEGF gene on the proliferation and apoptosis of human colon cancer HT29 cells. Methods VEGF-targeted siRNA was transfected into human colorectal cancer HT29 cells with Lipofect 2000 mediation. Expression of VEGF protein was detected by immunocytochemistry and Western blot. Cell proliferation was measured by MTI＂ assay. Cell apoptosis was detected by flow cytometry. Results Immunocytochemical results indicated that VEGF had strongly positive expression in the nor- mal control group, Lip 2000 control group and mismatch control group, and showed weak expression in VEGF siRNA interference group. Western blot indicated that brightness of protein bands of VEGF in VEGF siRNA interference group was significantly lower than those in normal control group, Lip 2000 control group and mismatch control group. Compared with control group and negative mismatch group, the inhibition rate of cell proliferation was significantly increased in VEGF siRNA interference group （ P 〈 0. 05 ）. Cell prolifer- ation rates were significantly different between 24, 48 and 72 h after transfection （ P 〈 0. 05 ）. FCM showed that diploid peak appeared in VEGF siRNA interference group and cell apoptosis rates of VEGF siRNA interference group was obviously higher than those of empty control groups （P 〈 0. 05 ）. Conclusions VEGF gene regulate the proliferation of colorectal cancer cells and induce apoptosis. We speculated that VEGF gene may become a new target in gene therapy for colorectal cancer.