摘要
探讨RhoGTPases的 3个主要分子RhoA、Rac1和Cdc4 2对肿瘤血管生成相关分子的作用 .构建负显性RhoA、Rac1和Cdc4 2的真核表达质粒 ,在Lipofectamine2 0 0 0 介导下转染胃癌细胞AGS ,应用ELISA检测细胞培养上清中VEGF的含量 ,应用Western印迹检测肿瘤血管生成相关分子HIF 1α、P5 3和PTEN的表达水平 .成功地构建了负显性RhoA、Rac1和Cdc4 2的真核表达质粒 ,转染胃癌细胞AGS并经G4 18筛选出单克隆 .ELISA表明转染细胞培养上清中VEGF的含量可被明显抑制 ;Western印迹表明 ,负显性RhoGTPases在蛋白水平上可下调HIF 1α表达水平 ,上调P5 3的表达水平 .结果表明 ,Rho家族的 3个主要分子可能通过调节血管生成相关分子的表达来促进肿瘤血管生成 .
The relationship between the three main members of Rho GTPases (RhoA, Rac1 and Cdc42) and tumor angiogenesis was studied. The eucaryotic expression vectors of dominant-negative RhoA, Rac1 and Cdc42 (named pCEFL-GST-N 19 -RhoA, pCEFL-GST-N 17 -Rac1 and pCEFL-GST-N 17 -Cdc42) were constructed and transfected into gastric cancer cell line AGS by Lipofectamine 2000 . The content of VEGF in the conditioned media was detected by ELISA. The expression of HIF-1α, P53 and PTEN was studied by Western blotting. pCEFL-GST-N 19 -RhoA, pCEFL-GST-N 17 -Rac1 and pCEFL-GST-N 17 -Cdc42 were constructed and transfected into gastric cancer cell line AGS. Their single clones were selected by G418 and designated as AGS/DN-RhoA, AGS/DN-Rac1 and AGS/DN-Cdc42. ELISA showed that VEGF was decreased in the conditioned media of all the three kinds of cells. Western blotting showed that the expression of HIF-1α was down-regulated, while the expression of P53 was up-regulated. These results indicated that all the three main members of Rho GTPases could promote tumor angiogenesis, the mechanism might be due to regulation of hypoxia-responsive factors.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2004年第5期664-669,共6页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金资助项目 (No .3 0 3 0 0 12 5
No .3 0 10 0 0 79)~~
