可溶型HLA-G5～-G7分子的克隆表达及对NK细胞杀伤功能的影响  

Cloning and expression of soluble HLA-G5～-G7 molecules and their effect on cytotoxic function of NK cells function

作　　者：许惠惠[1] 林爱芬[1] 徐丹萍[1] 颜卫华[1] 

机构地区：[1]温州医学院附属台州医院中心实验室,临海317000

出　　处：《现代免疫学》2010年第6期477-481,共5页Current Immunology

基　　金：浙江省科技计划(2008C33013;2009C33147)

摘　　要：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。To establish the expression of soluble HLA-G5^-G7 isoforms in choriocarcinoma cell line JAR and to investigate their role in NK cytotoxicity in vitro,stable expression of HLA-G5,-G6 and HLA-G7 in JAR cells was established by gene cloning and transfection,and the HLA-G transcripts and protein expression in the transfected JAR cells were tested with RT-PCR,flow cytometry,Western blotting and immunocytochemistry,respectively.Effects of HLA-G5^-G7 isoforms on NK cytotoxicity was performed with lactic dehydrogenase（LDH） releasing method.Results obtained from RT-PCR and immunocytochemical analysis showed that exogenous HLA-G5^-G7 gene were successfully transfected and proteins were stably expressed in the HLA-G negative JAR cells;Flow cytometry data demonstrated that the intracellular HLA-G5 and HLA-G6 isoforms were detectable in the transfected JAR cells.Western blotting data showed that HLA-G5 isoform was secreted by the transfected JAR cells.Among these isoforms,only the HLA-G5 expressing in JAR cells could significantly suppress the cytotoxicity of NK cells（P0.05）.Result of the present study demonstrates the extracellular secretory property of the HLA-G5 molecule and its effective inhibitory effect on the cytotoxicity of NK cells,while the HLA-G6 and HLA-G7 isoforms are mainly intracellular expressed and their biological functions still wait for further investigation.

关 键 词：HLA-G 异构体 真核表达 NK杀伤 

分 类 号：Q343.1[生物学—遗传学]