表达人源补体调节蛋白hCD55对异种胰岛移植的保护作用

Protective role of expression of human complement regulatory protein hCD55 in islet xenotransplantation

目的验证猪胰岛细胞上表达人类簇分化抗原55(hCD55)蛋白能否抑制人血清中补体成分的激活。方法选取4只基因型分别为WT(野生型)、GTKO[α-1,3-半乳糖基转移酶(GGTA1)基因敲除]、GTKO/hCD55和hCD55的成年猪,分离WT、GTKO、GTKO/hCD55猪胰岛细胞,检测其纯度及胰岛素分泌功能,通过琼脂糖凝胶电泳、逆转录聚合酶链反应(RT-PCR)、流式细胞术从DNA、RNA和蛋白水平检测hCD55的表达。在新鲜人血清孵育条件下,进行补体依赖的细胞毒性实验和补体沉积检测。结果分离得到的3种基因型猪胰岛细胞纯度>75%,糖刺激指数>1。GTKO/hCD55猪胰岛细胞上表达hCD55信使核糖核酸(mRNA)和hCD55蛋白,能够减少人补体C3c和攻膜复合物C5b-9的沉积,降低细胞毒性。结论猪胰岛细胞上表达hCD55蛋白能够抑制人补体激活,降低补体介导的杀伤作用,表明hCD55蛋白在猪胰岛细胞上可发挥补体保护效果,这为hCD55在异种胰岛移植中的应用提供了理论依据。

Objective To validate whether the expression of human cluster of differentiation 55(hCD55)protein in porcine islet cells could inhibit the activation of complement components in human serum.Methods Four adult pigs with WT(wild type),GTKO[α-1,3-galactosyltransferase(GGTA1)knockout],GTKO/hCD55 and hCD55 genotypes were selected.Islet cells were isolated from WT,GTKO and GTKO/hCD55 pigs,and the purity and insulin secretion function were detected.The expression of hCD55 at the DNA,RNA and protein levels was analyzed by agarose gel electrophoresis,reverse transcription polymerase chain reaction(RT-PCR)and flow cytometry,respectively.Complement-dependent cytotoxicity assay and complement deposition assay were performed under the incubation conditions with fresh human serum.Results The purity of isolated porcine islet cells from three genotype pigs was>75%,and the glycemic index was>1.The expression of hCD55 messenger RNA(mRNA)and protein in GTKO/hCD55 porcine islet cells decreased the deposition of human complement component C3c and membrane-attacking complex C5b-9,and reduced the cytotoxicity.Conclusions The expression of hCD55 protein in porcine islet cells could inhibit the activation of human complement and reduce complement-mediated killing effect,indicating that hCD55 protein could exert complement protection effect on porcine islet cells.These findings provide theoretical basis for the application of hCD55 in islet xenotransplantation.