论文部分内容阅读
通过基因克隆及转染,建立分别稳定表达HLA-G5、-G6及HLA-G7抗原的人绒癌JAR细胞株,探讨可溶型HLA-G5~-G7异构体的表达及对NK细胞杀伤功能的影响。采用RT-PCR、流式细胞术、Western blot及免疫细胞化学法分析、鉴定转染细胞中HLA-G的mRNA及蛋白表达。LDH释放法检测HLA-G5、-G6及HLA-G7表达对NK细胞杀伤活性的影响。RT-PCR及免疫细胞化学结果显示,HLA-G5~-G7/pVITRO2-mcs重组质粒成功转染HLA-G表达阴性的人绒癌JAR细胞株。FACS分析显示HLA-G5、-G6抗原在JAR-HLA-G5、-G6细胞胞浆内表达。Western blot分析显示可溶性HLA-G5抗原能成功分泌至细胞外。体外杀伤实验发现可溶型HLA-G5抗原能抑制NK细胞的杀伤活性(P<0.05)。本研究结果提示,HLA-G5具有分泌到细胞外的特性,且能有效抑制NK细胞的杀伤活性,但HLA-G6、-G7主要在细胞内表达,其生物学功能有待进一步鉴定。
The human choriocarcinoma JAR cell line stably expressing HLA-G5, -G6 and HLA-G7 antigens was established by gene cloning and transfection, and the expression of soluble HLA-G5 ~ -G7 isoforms and NK cell killing Effect of function. The expression of HLA-G mRNA and protein in transfected cells was identified by RT-PCR, flow cytometry, Western blot and immunocytochemistry. The effects of HLA-G5, -G6 and HLA-G7 expressions on cytotoxic activity of NK cells were detected by LDH release assay. The results of RT-PCR and immunocytochemistry showed that HLA-G5 ~ -G7 / pVITRO2-mcs recombinant plasmid successfully transfected JAR cell line of human choriocarcinoma with negative HLA-G expression. FACS analysis showed that HLA-G5 and -G6 antigens were expressed cytoplasm in JAR-HLA-G5 and -G6 cells. Western blot analysis showed that soluble HLA-G5 antigen could be successfully secreted to the outside of the cell. In vitro killing experiments showed that soluble HLA-G5 antigen can inhibit NK cell killing activity (P <0.05). The results suggest that HLA-G5 has the characteristics of being secreted extracellularly, and can effectively inhibit the killing activity of NK cells. However, HLA-G6 and -G7 are mainly expressed in the cells, and their biological functions need to be further identified.