阳离子聚合物介导下白细胞介素1受体拮抗剂基因兔角膜原位转染及其表达  

The transfection and expression of IL-1ra gene to the rabbit cornea in situ via cation polymer mediation

作　　者：袁进[1] 陈家祺[1] 周世有[1] 刘祖国[1,2] 王智崇[1] 顾建军[1] 

机构地区：[1]中山大学中山眼科中心,教育部眼科学重点实验室,广州510060 [2]厦门大学医学院

出　　处：《中华眼科杂志》2006年第8期686-693,共8页Chinese Journal of Ophthalmology

基　　金：国家自然科学基金资助项目(30271388);卫生部临床重点基金资助项目(2004-468)

摘　　要：目的探讨阳离子聚合物即线性聚乙烯亚胺(PEI)介导下兔角膜基质注射PEGFP-IL- 1ra质粒进行基因角膜原位转染的有效性和安全性。方法以人cDNA文库为模板进行聚合酶链反应(PCR),获得人IL-1ra cDNA片段,构建重组质粒PEGFP-hIL-1ra。以阳离子聚合物为介导转染角膜内皮细胞,通过绿色荧光蛋白(GFP)示踪、蛋白免疫印迹技术检测转染后IL-1ra基因和蛋白的表达。实验组30只Wistar大鼠角膜基质注射PEGFP-hIL-1ra质粒和PEI-in-vivo的混合溶液20μl(含10μg质粒),对照组15只Wistar大鼠角膜基质注射PEI-in-vivo溶液20μl。注射后1、3、6、14、21 d,收集角膜通过HE染色、透射电镜、锥虫蓝-茜素红染色、免疫组织化学观察1L-1ra基因角膜原位转染后的细胞结构和功能的变化。荧光显微镜下追踪IL-1ra-GFP融合蛋白在角膜的表达部位和表达强度。结果以cDNA文库为模板扩增出hIL-1ra cDNA片段,构建重组质粒PEGFP-hIL-1ra。经PstI和BamHI酶切及DNA测序证实了插入片段方向和大小正确。PEI-in-vitro介导下PEGFP-hIL-1ra转染角膜内皮细胞12 h后,可见10％-15％细胞中有GFP荧光表达。Western-blotting检测可见相对分子质量为44 000的hIL-1ra-GFP蛋白表达。PEGFP-hIL-1ra质粒和PEI-in-vivo角膜基质注射后1 d,角膜上皮基底细胞可见荧光条带,6 d时全角膜荧光强度达到高峰,14 d开始减弱,21d角膜上皮层尚存微弱荧光。对照组观察期内始终未见绿色荧光。实验组角膜HE染色未见病理性改变,角膜上皮基底细胞层p63抗体阳性;锥虫蓝-茜素红联合染色未见角膜内皮细胞损伤;透射电镜显示角膜各层细胞的细胞质内可见IL-1ra-GFP颗粒,未见细胞器的损害。结论阳离子聚合物介导下角膜基质注射PEGFP-hIL-1ra质粒可快速、有效地将IL-1ra基因转入角膜并表达,为临床上使用抗炎细胞因子IL-1ra对角膜免疫炎性反应相关疾病进行基因治疗提供了新的技术平台。Objective To investigate the efficiency and safety of transfection of PEGFP-IL-1ra plasmid via cation polymer mediation （poly-ethylenimine, PEI） by injection into the corneal stroma. Methods Human IL-1ra cDNA fragments were cloned by RT-PCR. Plasmid PEGFP-hlL-1ra recombinants were constructed and transferred into corneal endothelial cells （CEC） via cation polymer mediation. Expression of IL-1ra mRNA and IL-1ra was detected by green fluorescent protein （GFP） and Western-blotting. In the experiment group, 20 μl preparation containing 10 μg plasmid PEGFP-hIL-1ra recombinants and PEI-in-vivo was injected into the corneal stroma of Wistar rats （ n = 30）. Equivalent PEI-in-vivo solution was injected into another 15 corneas as the controls. Corneas were harvested at different time points （day 1, 3, 6, 14 and 21 ） after injection. The changes of tissue structure and function after IL-1ra in situ transfection were studied by HE staining, transmission electron microscopy, trypan blue-alizarin red staining and immunohistochemistry. The location and intensity of IL-1ra-GFP fusion protein expression were monitored by fluorescence microscopy. Results The size of the RT-PCR product of hIL-1ra fragments was approximately 500 bp in agarose gel electrophoresis. Restrictive enzyme digestion analysis of PstⅠ, BamHI and DNA sequence analysis showed that expression of plasmid PEGFP-hIL-1ra recombinants had been constructed successfully. Twelve hours after the transfection of PEGFP-hIL-1 ra, GFP fluorescence was detected in 10% - 15% endothelial cells. IL-1ra protein （RMW： 44 000） was detected by Western-blotting. In PEGFP-hIL- 1ra treated group, fluorescence was appeared at day 1 in cornea basal epithelial cells, peaked at day 6 in whole cornea, began to weaken at day 14, and only weak fluorescence remained in cornea epithelial cells at day 21. No fluorescence appeared in the control group. No significant pathologic changes could be found in HE stained cornea tissues in both transfected group and the cont

关 键 词：受体 白细胞介素1 聚乙烯亚胺 角膜 转染 

分 类 号：R772.2[医药卫生—眼科]