表达人膜辅助因子蛋白基因的猪内皮细胞可抑制人血清补体的细胞毒作用  

Inhibition of the cytotoxicity of human complement by porcine endothelial cells expressing human membrane cofactor protein

作　　者：白成[1] 乔殿华[1] 孙方臻[1] 

机构地区：[1]中国科学院发育生物学研究所分子发育生物学开放实验室

出　　处：《中华微生物学和免疫学杂志》1999年第2期133-136,共4页Chinese Journal of Microbiology and Immunology

基　　金：国家863项目基金

摘　　要：目的研究转染人膜辅助因子蛋白基因的猪内皮细胞抗补体的细胞毒作用。方法从人胎盘组织提取总ＲＮＡ，用ＲＴ－ＰＣＲ技术扩增得到人膜辅助因子蛋白（ｈＭＣＰ）基因全长ｃＤＮＡ，将其克隆到带有巨细胞病毒（ＣＭＶ）ＩＥ启动子的ｐＣＩ－ｎｅｏ哺乳动物表达载体和以ｐＣＩ－ｎｅｏ为基础构建成的带有人ＥＦ－１α启动子的ｐＥＦ－ｎｅｏ哺乳动物表达载体上，得到质粒ｐＣＩＭ和ｐＥＦＭ。利用电穿孔基因转移方法分别转染猪内皮细胞（ＰＥＣ），以Ｇ４１８筛选稳定表达克隆。用正常人血清处理此转基因细胞来检测其抗补体作用。结果Ｎｏｒｔｈｅｒｎ杂交结果表明，在ＥＦ－１α启动子引导下的ｈＭＣＰ基因得到了高效表达。死细胞计数和ＬＤＨ释放实验结果表明，表达ｈＭＣＰ基因的ＰＥＣ可有效抑制人血清补体对其的裂解作用（Ｐ＜０．０１）。结论克隆的ｈＭＣＰ基因在ＥＦ－１α启动子引导下于ＰＥＣ可高效表达，且使ＰＥＣ能够抗人补体的细胞毒作用。Objective To study the resistance of porcine endothelial cells transfected by human membrane cofactor protein gene to cytotoxicity of human complement. Methods The cDNA encoding human membrane cofactor protein(hMCP) was cloned from human placenta total RNA by RT PCR . It was inserted into pCI neo mammalian expression vector and pEF neo vector which was derived from pCI neo vector. Expression plasmids pCIM and pEFM were got. Porcine endothelial cells(PEC) grew to log phase and transfection was performed by electroporation. The stable expression clones were selected with G418. The resistance of transgenic PEC to human complement was tested by treating the cells with normal human serum. Results Northern blot indicated that hMCP gene driven by human EF 1α promoter got expressed efficiently. Dead cell count and LDH release results indicated that the PEC which expressed hMCP gene could effectively inhibit the cytotoxicity of human complement. Conclusions hMCP gene driven by EF 1α promoter in PEC can express effeciently and the transfected PEC can inhibit the cytotoxicity of human complement.

关 键 词：细胞毒作用 内皮细胞 表达 hMCP 基因 补体 

分 类 号：R392.4[医药卫生—免疫学]