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目的 进行抗血管生成治疗人脑胶质瘤实验 ,构建和鉴定携带人反义 VEGF1 6 5 基因的真核表达载体 .方法 将VEGF1 6 5 c DNA反向克隆入 pc DNA3,构建 CMV启动子控制的真核表达载体 pc DNA- AVEGF1 6 5 ,用酶切鉴定结果 ;应用该载体转染人脑胶质瘤细胞 SHG44 ,G418筛选阳性克隆 ;Western blot和免疫组化检测转染前、后瘤细胞的 VEGF表达 .检测转染前、后瘤细胞的生物学性状 .结果 获得反向构建的 pc DNA- AVEGF1 6 5 真核表达载体 ,VEGF1 6 5 反义 RNA阻断了 SHG44细胞的 VEGF表达 ,该细胞生物学性状不受外源基因的表达影响 .结论 成功构建的反义 VEGF1 6 5 真核表达载体 ,为人脑胶质瘤的反义基因治疗提供了必要的条件
Objective To conduct anti-angiogenic treatment of human gliomas and to construct and identify eukaryotic expression vectors carrying human antisense VEGF165 gene. Methods Reversely clone VEGF165 gene into pc DNA3 to construct the CMV promoter. The eukaryotic expression vector pc DNA-AVEGF165 was identified by restriction enzyme digestion. The vector was transfected into human glioma cell line SHG44. Positive clones were screened by G418. Western blot and immunohistochemistry were used to detect the tumor cells before and after transfection. Expression of VEGF. The biological characteristics of the tumor cells before and after transfection were examined. The result was an inverted construction of the pc DNA-AVEGF165 eukaryotic expression vector. VEGF165 antisense RNA blocked the expression of VEGF in SHG44 cells. Biological traits are not affected by the expression of exogenous genes. CONCLUSION: The successfully constructed antisense VEGF165 eukaryotic expression vector provides the necessary conditions for antisense gene therapy of human gliomas.