膜结合型HLA-G1～G4分子的克隆表达及对NK细胞杀伤功能的影响  

Cloning and expression of HLA-G1-G4 molecule in JAR cells and its effects on NK cell function

作　　者：许惠惠[1] 林爱芬[1] 颜卫华[1] 

机构地区：[1]温州医学院附属浙江省台州医院实验中心,临海317000

出　　处：《中华微生物学和免疫学杂志》2010年第11期982-986,共5页Chinese Journal of Microbiology and Immunology

基　　金：基金项日：浙江省科技计划（2008C33013,2009C33147）

摘　　要：目的 探讨膜结合型HLA-G1～G4异构体的表达及对NK细胞杀伤功能的影响.方法 通过基因克隆及转染,分别建立稳定表达HLA-G1～G4抗原的人绒癌JAR细胞株.采用RTPCR、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.通过加载HLA-G高亲和性KIPAQFYIL抗原肽,观察对HLA-G表达的影响.LDH释放法检测HLA-G1～G4表达对NK细胞杀伤活性的影响.结果 RT-PCR、Western blot及免疫细胞化学结果显示,HLA-G1～G4/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-G1抗原能在JAR-HLA-G1细胞株表面表达,HLA-G2～G4抗原不能有效到达细胞表面.体外杀伤试验发现表达HLA-G1～G4抗原的细胞均能抑制NK细胞的杀伤活性（P＜0.05） 加载HLA-G高亲和性KIPAQFYIL抗原肽对HLA-G表达无明显影响,对NK细胞杀伤抑制程度也未见明显改变.结论 HLA-G1～G4能够明显抑制NK细胞的杀伤活性,提示不同膜结合型HLA-G异构体分子均能作为免疫耐受分子,具备免疫调节功能.Objective To establish the expression of membrane-bound HLA-G1-G4 isoforms in choriocarcinoma cell line JAR and to investigate its roles in NK cytotoxicity in vitro. Methods Stable expression of HLA-G1, -G2, -G3 and -G4 in JAR cells was established by gene cloning and transfection.HLA-Gtranscripts and protein expression in the transfected JAR cells was tested by RT-PCR, flow cytometry, Western blot and immunocytochemistry, respectively. High-affinity peptide KIPAQFYIL pulsing was performed to evaluate its effects on HLA-G expression. Effects of HLA-G1-G4 isoforms on NK cytotoxicity was performed with lactic dehydrogenase （LDH） releasing method. Results RT-PCR, Western blot and immunocytochemistry results showed that exogenous HLA-G1-G4 gene were successfully transfected and proteins were stably expressed in the HLA-G negative JAR cells Flow cytometry data showed that only HLAG1, but not HLA-G2-G4 isoform was detectable in those transfected JAR cells and the peptide pulsing did not affect their expression status. However, all HLA-G1-G4 isoform expressed JAR cells could significantly decreased the NK cell cytotoxicity （P〈0.05）. Conclusion HLA-G1-G4 isoform expression could dramatically inhibit NK-92 cell lysis, indicating that membrane-bound HLA-G isoforms are importantly immunotolerant and may play immune regulation roles in various physio-pathological situations.

关 键 词：HLA-G 异构体 转染 细胞毒性 

分 类 号：R392.1[医药卫生—免疫学]