人SH3GL2真核表达载体的构建及其对内皮细胞体外成管能力的影响

Construction of human SH3GL2 recombinant expression plasmid and its effect on endothelial cell tube formation

目的构建人SH3GL2基因的重组真核表达质粒,并转染至人脑微血管内皮细胞,检测对内皮细胞体外成管能力的影响。方法提取人脑微血管内皮细胞的总RNA,逆转录聚合酶链式反应(RT-PCR)扩增人SH3GL2基因的蛋白编码区(CDS),亚克隆至真核表达载体p IRES2-EGFP。测序正确后,将重组质粒转染至人脑微血管内皮细胞,采用matrigel三维血管生成实验检测对内皮细胞成管能力变化。结果人SH3GL2基因克隆至表达载体pIRES2-EGFP,重组质粒双酶切鉴定后,琼脂糖凝胶电泳验证片段大小为1058bp。将重组质粒转染人脑微血管内皮细胞,和对照组相比,pIRES2-EGFP-SH3GL2转染组内皮细胞成管能力显著降低(<0.05)。结论成功构建人SH3GL2真核表达载体,人脑微血管内皮细胞过表达SH3GL2能够抑制内皮细胞的成管能力。

Objective To study the construction and effect of human SH3GL2 expression plasmid on endothelial cell tube formation of human cerebral microvascular endothelial cells. Methods The total RNA was extracted from human cerebral microvascular endothelial cells, the SH3GL2 gene coding sequence was amplified by RTPCR and cloned into p IRES2-EGFP. After the target region was sequenced, the plasmid was transfected into human cerebral microvascular endothelial cells. The effects of SH3GL2 on the endothelial cell tube formation were checked by matrigel tube formation assay. Results SH3GL2 was constructed into expressing vector p IRES2-EGFP succesfully, the length of the fragment was 1058 bp identified by restriction enzymes digestion. The recombinant expression plasmid was transfected into human cerebral microvascular endothelial cells. Compared with p IRES2-EGFP groups, relative tubule length was significantly decreased in p IRES2-EGFP-SH3GL2 group（ 〈0.05）. Conclusion The recombinant expression plasmid SH3GL2 was constructed successfully and over-expression of SH3GL2 suppressed endothelial cell tube formation in vitro.