人内皮高表达脂多糖相关因子1强制表达模型的建立及对ECV304细胞增殖的影响

Establishment of human endothelial-overexpressed lipopolysaccharide-associated factor 1 compelling expression model and its effects on the proliferation of ECV304 cells

目的设计和构建新基因内皮高表达脂多糖相关因子1(EOLA1)诱导表达载体,建立EOLA1强制表达模型,观察其对细胞增殖的影响。方法逆转录聚合酶链反应扩增EOLA1开放阅读框片段,引入NoⅠt和XhoⅠ酶切位点,定向亚克隆入可调控真核表达的载体pOPRSVⅠ,测序验证后共转染pOPRSVⅠ-EOLA1和pCMVLacⅠ质粒于ECV304细胞,经潮霉素和G418筛选,获得稳定转染细胞株。对异丙硫半乳糖苷(IPTG)诱导和非诱导的稳定转染细胞株进行计数,绘制细胞生长曲线。结果成功构建了EOLA1可调控真核表达载体pOPRSVⅠ-EOLA1。诱导后第4天,EOLA1稳定表达的ECV304细胞数量为(44±17)×104个,显著多于未诱导细胞的数量(27±11)×104个(P<0.01)。结论强制高表达EOLA1蛋白具有促进ECV304细胞增殖的作用。

Objective To design and construct the inducible expression vector of endothelial-overex-pressed lipopolysaccharide-assoeiated factor 1 （ EOLA1 ） , in order to establish EOLA 1 compelling expression model, and to observe the effects of EOLA1 compelling expression on the proliferation of ECV304 cells.Methods Inducible nverexpression vector pOPRSV Ⅰ - EOLA1 was constructed by amplifying the open reading fragment of EOLA1 and suhcloning it into the Not Ⅰ site and Xho Ⅰ site of pOPRSV Ⅰ vector. After sequencing, the pOPRSV Ⅰ-EOLA1 recombinant vector and pCMVLac Ⅰ vector were co-transfected into ECV304 cells, The cells resistant to G418 and hygromycin were screened by G418 and hygromycin, so that stable transfected cell strain was obtained. The growth curve of cells with or without isopropy-β-1）-thiogalactoside（IPTG） induction were graphed with cell counting. Results The inducible overexpressed EOLA1 vector was constructed successfully. The proliferation of the cells with EOLA1 compelling expression after induction of IPFG （44 ± 17） × 10^4 was significantly higher than that withuut IPTG induction （27 ± 11 ） × 10^4,（ P 〈 0. 01 ）. Conclusion Compelling expression of EOLA1 protein can enhance the proliferation of ECV304 cell.