人反义血管内皮生长因子基因表达对肾癌细胞的影响

Effect of antisense VEGF gene on VEGF expression and growth of human renal cell carcinoma

目的 探讨人反义血管内皮生长因子 (VEGF)基因对肾癌细胞VEGF表达及生长的影响。 方法 采用RT PCR方法将VEGF基因克隆于 pcDNA3.1反义真核表达载体 ,构建VEGF的pcDNA3.1反义真核表达载体 ,酶切鉴定。转染人肾癌 780 0细胞 ,G4 18筛选阳性克隆。免疫组化法检测VEGF表达 ,MTT法测生长曲线。 结果 pcDNA3.1 (antisense)VEGF组VEGF蛋白表达阳性细胞率 (10 .3% )明显低于 780 0 PC组 (92 .8% )和 780 0组 (96 .6 % ) ,P <0 .0 1;VEGF反义真核表达载体抑制肾癌 780 0细胞生长 ,在培养的第 4、5、6天 ,抑制率分别为 2 6 .38%、4 1.78%和 37.6 4 %。 结论 VEGF的 pcDNA3.1反义真核表达载体可明显降低肾癌细胞VEGF的表达 ,明显减慢肾癌细胞生长。

Objective To study the effect of human antisense VEGF gene on VEGF expression and growth of renal cell carcinoma Methods Reverse transcription-polymerase chain reaction (RT-PCR) for VEGF,VEGF was inserted into eukaryotic expression vector pcDNA3.1 to construct eukaryotic expression vector carrying human VEGF cDNA,then using restrict enzyme to confirm the result.The vector was transfected into renal cell carcinoma 780-0 and positive clone was selected by using G418.VEGF expression was detected by using immunocytochemical technique and the growth curve was detected by using MTT method. Results VEGF gene was gained by RT-PCR and antisense VEGF eukaryotic expression vector pcDNA 3.1 was constructed.The positive cell rate of VEGF expression in pcDNA 3.1-(antisense)VEGF group( 10.3%) is lower than that in 780-0-PC group(92.8%) or 780-0 group(96.6%), P<0.01.The vector can greatly slow the growth of renal cell carcinoma 780-0.The inhibition rate on the fourth,fifth,sixth day of culturing is 26.38%,41.78% and 37.64%,respectively. Conclusions Antisense VEGF eukaryotic expression vector pcDNA3.1 was constructed.The vector can be used for antisense gene therapy of renal cell carcinoma.