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目的探讨HLA-G1基因转染新生猪胰岛细胞的可行性,检测其表达和功能状况。方法体外分离纯化新生猪胰岛细胞,脂质体载体转染HLA-G1基因。免疫荧光法检测HLA-G1基因在胰岛细胞上的表达状况;51Cr释放实验分析其对人NK细胞细胞毒的抑制作用。结果新生猪胰岛细胞HLA-G1基因表达率大约为50%,体外转染24-48 h达高峰;混合淋巴细胞培养(胰岛细胞+ NK细胞)后,空质粒转染组的胰岛细胞溶解率为71.57%,而转染组胰岛细胞溶解率为53.5%。结论利用脂质体Genejammer转染体外培养的新生猪胰岛细胞,可以获得靶基因的瞬时高效表达; HLA-G1基因转染可以有效抑制NK细胞对异种胰岛的细胞毒作用。
Objective To investigate the feasibility of transfecting newborn porcine pancreatic islet cells with HLA-G1 gene and to detect its expression and function. Methods Isolation and purification of neonatal porcine islet cells in vitro, liposome vector transfected HLA-G1 gene. Immunofluorescence method was used to detect the expression of HLA-G1 gene in islet cells; 51Cr release assay was used to analyze the inhibitory effect of HLA-G1 on cytotoxicity of human NK cells. Results The expression rate of HLA-G1 gene in neonatal porcine islet cells was about 50%, reaching the peak in 24-48 h in vitro. The rate of islet cell lysate in empty plasmid transfected group after mixed lymphocyte culture (islet + NK cells) was 71.57%, while the transfection group waslet cell lysate rate was 53.5%. CONCLUSION: Gene transfection with liposome Genejammer can transfect transiently the islet cells of neonatal porcine so as to obtain transient and efficient expression of the target gene. HLA-G1 gene transfection can effectively inhibit the cytotoxic effect of NK cells on the islet cells.