RI基因真核表达载体的构建及其对C6胶质瘤细胞生长的影响

目的通过构建核糖核酸酶抑制因子(ribonuclease inhibitor, RI)基因的真核表达载体——pLNCX-ri, 并转染C6神经胶质瘤细胞,探讨RI抑制肿瘤生长的作用机制.方法用Nde I / Xho从已构建的pET-ri上切下1.4 kb的RI基因片段,再构建到pLNCX上,获得真核表达载体(pLNCX-ri),采用Lipofect AMINE辅助转染大鼠C6神经胶质瘤细胞,经G418筛选获得稳定转染的细胞克隆,用Western blotting 检测RI基因的表达水平.将转染阳性的C6神经胶质瘤细胞接种于大鼠皮下,观察肿瘤的生长情况.结果在转染的C6神经胶质瘤细胞中,RI基因的表达量明显高于未转染的C6神经胶质瘤细胞,转染阳性的C6神经胶质瘤细胞在大鼠体内的成瘤潜伏期23±5.7天(对照组14±3.5天),瘤组织重量转染组1.35±0.43g比对照组2.40±0.61g(P＜0.01)明显下降,瘤组织的血管密度转染组27.2±4.31比对照组47±6.54(P＜0.01)明显减少.结论RI基因的转染对肿瘤的生长有明显抑制作用,其作用机制可能是抑制肿瘤组织血管的形成.

The construction of eukaryotic expressive vector of RI gene and the effects of the transfected pLNCX-ri on the growth of C6 glioma cells

Purpose:To clone and construct an eukaryotic expressive vector of ribonuclease inhibitor (RI) gene ,as well as to observe the effects of the transfected pLNCX-ri on the growth of C6 glioma cells.Methods:A segment of RI gene of 1.4 kb was obtained by Nde I/Xho digestion and cloned into pLNCX. Transfective agent and selective antibiotic were lipofect AMINE and G418 respectively. The expression of pLNCX-ri in C6 glioma cells was detected by Western blotting. And SD rats were inoculated by the transfected C6 glioma cells.Results:An eukaryotic expressive vector of RI gene was constructed successfully. RI content was remarkably higher in the transfected cells than that of in the untransfected cells. After SD rats were inoculated by the transfected C6 glioma cells,the tumorigenic time was prolonged, the tumor weight was reduced and the density of tumor vessels was notably decreased. Conclusions:These results indicated that RI gene powerfully inhibited the growth of C6 glioma cells via decreasing tumor vessels formation.