人ski基因真核表达载体的构建及促细胞增殖作用

Construction of eukaryotic expression vecter for human ski gene and observation of its role in promoting cell proliferation

背景:c-Ski既可促进组织修复,又能降低瘢痕形成,有望成为一个全新的基因治疗药物。目的:构建人ski基因的真核表达载体,并对其促成纤维细胞增殖效果进行验证。方法:RT-PCR法从人包皮培养原代成纤维细胞总RNA中扩增出人ski基因,连同真核表达启动子CMV克隆到pUC118载体上,酶切和测序验证后,应用脂质体将其转染到培养的大鼠皮肤成纤维细胞中。结果与结论:酶切和测序结果证实表达质粒构建成功,Westernblot显示Ski蛋白在转染细胞中成功表达,且可显著提高转染细胞的增殖效应,说明以pUC118为骨架的重组ski表达质粒具有促进大鼠皮肤成纤维细胞增殖的作用。

BACKGROUND：c-Ski can promote tissue repair and alleviate scar formation,which is expected to be a new gene therapeutic drugs.OBJECTIVE：To construct eukaryotic expression plasmid of human ski gene and to investigate its proliferative role in fibroblasts.METHODS：The ski gene was amplified by polymerase chain reaction（PCR） from human foreskin cultured fibroblast and was sub-cloned into pUC118 with CMV eukaryotic promoter.The recombinant pUC118-Ski plasmids were transfected into rat skin fibroblasts using lipofectamine TM 2000 and then its protein expression and effect of the proliferation were detected.RESULTS AND CONCLUSION：pUC118-Ski vector was constructed successfully which was confirmed by restriction endonuclease digestion and DNA sequencing.It expressed successfully and significantly improved the effect of the proliferation in transfected fibroblasts.Recombinant eukaryotic expression plasmid pUC118-Ski can improve the proliferation of rat skin fibroblasts.