猪供人异种移植超急性排斥反应体外实验模型的初步研究

目的 研究通过转基因方法在猪主动脉内皮细胞上表达人ＣＤ５９蛋白,抑制猪供人移植超急性排斥反应。方法 体我２的猪主动脉和人脐静脉内皮细胞,用免疫荧光和免疫组化的方法得到鉴定。构建含入ＣＤ５９ＣＤＮＡ的真核细胞表达载体ＬＸＳＮ－ＣＤ５９,限制性内切酶酶切,琼脂糖凝胶电泳和多聚酶链反应（ＰＣＲ）法鉴定。脂质体转染法将ＬＸＳＮ－ＣＤ５９转化进猪血管内皮细胞,Ｇ４１８筛选得到稳定转化子流式细胞仪筛选得到表达

In vitro model of hyperacute rejections in swine to human xenotransplantation

OBJECTIVE: To evaluate the efficiency of inhibiting hyperacute rejections in swine to human xenotransplantation through expressing human CD(59) protein on the membrane of cultured pig aortic endothelial cells (PAECs) by transgenic methods. METHODS: The cultured PAECs and human umbilical vein endothelial cells (HUVECs) were identified by fluorescent and immunohistochemical methods. The constructed eukaryorotic expression vectors containing the human CD(59) cDNA, LXSN-CD(59) were identified by the methods of restriction endonucleases cleavage, agarose gel electrophoresis, and polymerase chain reaction. LXSN and LXSN-CD(59) were transfected into the PAECs separately with lipofectinmine mediated method. Stable transfectants were selected using G418. Cells expressing maximum human CD(59) protein were selected by FACS. These clones were incubated with the media containing human serum or monkey serum. The LDH released into the media was measured to assess the function of the hCD(59) protein. RESULT: Comparing with the control cells, we found that the cells expressing the human CD(59) protein released less LDH when they were incubated with human serum or monkey serum (P < 0.01). CONCLUSION: Hyperacute rejections may be inhibited through expressing the human complement regulatory protein CD(59) on the membrane of PAECs by transgenic methods.