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目的 :在培养的鼠主动脉平滑肌细胞膜上表达人CD5 9蛋白 ,研究通过转基因方法抑制异种移植超急性排斥反应。方法 :分别从新鲜的鼠主动脉和人脐静脉分离培养鼠主动脉平滑肌细胞、人脐静脉内皮细胞 ,并用免疫荧光和免疫组化的方法鉴定。构建含人CD5 9cDNA的真核细胞表达载体LXSN CD5 9,用脂质体转染法将LXSN和LXSN CD5 9转化进鼠主动脉平滑肌细胞 ,40 0~ 80 0mg·L-1G418筛选得到稳定转化子。流式细胞仪筛选得到表达人CD5 9蛋白最强细胞株。与含有异种活性抗体和补体的人血清和猴血清共孵育 ,分析乳酸脱氢酶 (lacticde hydrogenase ,LDH)释放率测定人CD5 9蛋白功能活性。结果 :与未转基因的对照组细胞比较 ,表达人CD5 9蛋白组细胞与不同浓度人血清和猴血清共孵育时 ,人血清和猴血清中补体及抗体对这些细胞的杀伤作用被显著抑制 ,统计学检验差异有显著性。结论 :通过转基因方法在鼠血管平滑肌细胞膜上表达人补体调节蛋白 ,能使异种移植超急性排斥反应得到抑制
OBJECTIVE: To express human CD5 9 protein in the cultured mouse aortic smooth muscle cell membrane and investigate the inhibition of hyperacute rejection of xenotransplantation by transgenic method. Methods: Rat aorta smooth muscle cells and human umbilical vein endothelial cells were isolated and cultured from fresh rat aorta and human umbilical vein, respectively, and identified by immunofluorescence and immunohistochemistry. The eukaryotic expression vector LXSN CD5 containing human CD5 9 cDNA was constructed and the LXSN CD5 9 was transformed into mouse aortic smooth muscle cells by lipofection. Stable transformants were screened by 40 0 ~ 80 0 mg · L-1 G418 . The strongest cell line expressing human CD59 protein was obtained by flow cytometry. Human serum and monkey sera containing xenogenic active antibody and complement were co-incubated and the release of lactate dehydrogenase (LDH) was analyzed to determine the functional activity of human CD59 protein. Results: Compared with untransformed control cells, the killing effect of complement and antibody in human serum and monkey serum on these cells was significantly inhibited when human CD59-expressing cells were incubated with different concentrations of human serum and monkey serum Differences in learning test were significant. CONCLUSIONS: The expression of human complement regulatory proteins on the membrane of rat vascular smooth muscle cells by transgenic methods can inhibit the hyperacute rejection of xenotransplantation