人反义VEGF基因对肾癌细胞VEGF表达的影响

Effect of antisense VEGF gene on VEGF expression in human renal cell carcinoma

目的:构建反义VEGF基因真核表达载体,研究其对肾癌细胞VEGF表达的影响。方法:克隆人 VEGF基因,将反义VEGF基因定向克隆于质粒PCDNA3．1(-)真核表达载体,酶切鉴定;转染入肾癌细胞,G418筛选阳性克隆。RT-PCR方法检测VEGFMRNA的表达,免疫组化法检测VEGF基因的蛋白表达,MTT法测细胞生长,流式细胞术检测细胞周期。结果:成功构建反义VEGF基因真核表达载体;反义VEGF组VEGFMRNA的表达受到抑制, 明显低于空载体组和对照组VEGFMRNA的表达,空载体组VEGFMRNA的表达没有受到影响;反义VEGF组的VEGF 蛋白表达明显低于空载体组和对照组,空载体组和对照组VEGF蛋白的表达无显著差异;反义VEGF组细胞生长减慢,G1期细胞比例增加,S期细胞比例减少。结论:人反义VEGF基因可明显降低肾癌细胞VEGF基因在转录和翻译水平的表达,抑制肾癌细胞生长,为肾癌基因治疗提供一定的实验依据。

AIM： To construct eukaryotic expression vector carrying human antisense VEGF gene and to study its effect on VEGF expression and growth of renal cell carcinoma. METHODS： VEGF gene was cloned. Antisense VEGF gene was inserted into eukaryotic expression vector pcDNA3.1 （ - ）, then using restrict enzyme to confirm the result. The vector was transfected into renal cell carcinoma and positive clone was selected by using G418. The VEGF expression was detected by using RT- PCR and immunohistochemical methed. The growth of cells was measured by MTT and cell cycle by FCM. RESULTS： Antisense VEGF gene eukaryotic expression vector was successfully constructed. Compared with empty vector group and control group, the amount of VEGF mRNA expression and VEGF protein were decreased in the antisense VEGF group, the difference was significant. There was no significant difference between empty vector group and control group. The cell growth in the antisense VEGF group became slower, and the percent of G1 phase increased and the percent of S phase decreased. CONCLUSION： Antisense VEGF gene decreases the VEGF mRNA and protein expression in renal cell carcinoma and inhibits the growth of renal carcinoma. Antisense VEGF gene may be used in gene therapy of renal carcinoma.