IL-1ra基因修饰角膜内皮细胞及其表达

Expression of interleukin-1 receptor antagonist(IL-1ra) for gene modification of corneal endothelial cells

目的构建真核细胞内表达的重组质粒PEGFPhIL1ra,对角膜内皮细胞进行基因修饰,为构建白细胞介素1受体拮抗剂(interleukin1receptorantagonist,IL1ra)基因化角膜内皮细胞移植膜奠定基础。方法以人cDNA文库为模板进行PCR扩增,获得人IL1racDNA片断,插入PEGFPC2载体,构建真核表达质粒PEGFPhIL1ra。以阳离子聚合物为介导,对饲细胞培养下的角膜内皮细胞(cornealendothelialcells,CECs)进行体外转染。通过绿色荧光蛋白(greenfluorescentprotein,GFP)示踪检测转染效率,蛋白免疫印迹检测外源性IL1ra基因在角膜内皮细胞内表达强度。结果以cDNA文库为模板扩增出hIL1racDNA,通过酶切、DNA测序证实PEGFPhIL1ra构建正确。20%～25%的转染角膜内皮细胞中有绿色荧光。Westernblotting检测可见转染角膜内皮细胞内有相对分子量为44000的hIL1raGFP融合蛋白表达,3d时蛋白表达水平达到峰值,并且持续至5d,7d时表达水平开始回落,9d时表达处于较低水平,对照组在观察期内均未见蛋白表达。结论阳离子聚合物介导下重组质粒PEGFPhIL1ra可对角膜内皮细胞实现有效转染并且持续表达IL1ra蛋白。

Objective To 1ay a foundation for corneal endothelial cells （CECs） transplantation membrane with expression of interleukin-1 receptor antagonist （IL-1ra） gene by constructing eukaryotic recombinant plasmid PEGFP-hIL-1ra with high expression of IL-1ra for gene modification of CECs. Methods IL-1ra cDNA containing the sequences of signal peptide was cloned by RT-PCR,and hmnan IL-1 ra cDNA fragments were obtained and then inserted into eukaryotic expression vector PEGFP-C2 to form eukaryotic expression plasmtd PEGFP-C2-hIL-1ra recombtnants. They were transfected into corneal CECs cultured with feed cells via cation polymer mediation In vltro. Expressions of IL-1ra rnRNA and IL-1ra were detected by green fluorescent protein （GFP） and western blotting. Results Restrictive enzyme digestion analysis of Hind, EcoR ＋ Hind, Pst Ⅰ, BamH Ⅰ and DNA sequence analysis showed that recombinant co-expression plasmid PEGFP-C2 -hIL-1 ra had been constructed successfully. CECs cultured with feed cells grew we11, 20 - 25 percent of which showed green fluorescence after transfectton. The expression of hlL-1ra-GFP fusion protein whose relative molecular weight（RMW）was 44 000 was detected by Western-blotting. The levels of IL-1ra protein peaked at the 3rd day and maintained this level till 5th day. The level began to decrease at 7th day and was very low at 9th day. There was no protein expression in control group. Conclusion Recombinant plasmid PEGFP-hIL-1ra could be transfected into CECs via cation polymer mediation In vitro and IL-1ra protein could be expressed continuously.