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

In vitro model of hyperacute rejections in swine to human xenotransplantation

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 体外培养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。构建含人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人CD59蛋白最强的细胞克隆 ,与含有异种活性抗体和补体的人血清或猴血清共孵育 ,测定乳酸脱氢酶 (LDH)漏出率 ,作为人CD59蛋白的功能活性指标。 结果 与未转基因的对照组细胞比较 ,表达人CD59蛋白组细胞与人血清或猴血清共孵育时 ,LDH漏出率显著降低 (P <0 0 1)。 结论 通过转基因方法在猪血管内皮细胞膜上表达人补体调节蛋白CD59,可抑制异种移植超急性排斥反应。

Objective[WT5”BZ] 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. [WT5”HZ]Methods[WT5”BZ] The cultured PAECs and human umbilical vein endothelial cells (HUVECs) were identified by fluorescent and immunohistochemical methords. 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. [WT5”HZ]Result[WT5”BZ] 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 monky serum ( P <0 01). [WT5”HZ]Conclusion[WT5”BZ] Hyperacute rejections may be inhibited through expressing the human complement regulatory protein CD 59 on the membrane of PAECs by transgenic methods. [WT5”HZ]