人类白细胞抗原-GRNA干扰载体的构建及效应检测  

Construction of Recombinant Expression Vector Carrying Human Leukocyte Antigen-G-shRNA and Detection of Its Effect

作　　者：曾宪成[1] 陈伟[2] 张彤[3] 李华[3] 聂常富 张琪 陈规划[3] 

机构地区：[1]增城市人民医院(中山大学附属博济医院)普通外科,广东增城511300 [2]浙江大学医学院附属第二医院普通外科,浙江杭州310009 [3]中山大学器官移植研究所,中山大学附属第三医院肝移植中心,广东广州510630 [4]郑州大学附属肿瘤医院肝胆外科,河南郑州450003

出　　处：《中国普外基础与临床杂志》2012年第6期604-609,共6页Chinese Journal of Bases and Clinics In General Surgery

基　　金：国家自然科学基金资助项目(项目编号:30571769);广东省科技计划项目重大专项(项目编号:2007A032000001);广东省自然科学基金(项目编号:10451130001004472);增城市创新扶持基金(项目编号:ZC201004)~~

摘　　要：目的构建表达人类白细胞抗原-G(HLA-G)基因shRNA载体,探讨其对NK细胞杀伤效应的影响。方法构建4个HLA-G-shRNA真核表达质粒,瞬时转染至人肝癌Bel-7402细胞,检测HLA-G mRNA和蛋白水平的表达。将NK-92MI细胞(效应细胞)与转染后的Bel-7402细胞(靶细胞)共同培养,以LDH释放法观察不同效靶比时NK-92MI细胞对靶细胞的杀伤效应。结果经酶切及测序鉴定证实,插入序列与设计的序列相符。RT-PCR和Western blot结果均表明重组载体抑制了Bel-7402细胞HLA-G基因的表达。NK-92MI细胞对转染HLA-GshRNA的Bel-7402细胞杀伤作用明显增强(P<0.01)。封闭NK-92MI细胞KIR2DL4受体后,杀伤作用减弱(P<0.01)。结论成功构建了HLA-G shRNA载体,下调HLA-G可以增强NK细胞对肝癌细胞的杀伤效应,HLA-G与NK细胞表面KIR2DL4受体结合后,向NK细胞传递抑制效应。Objective To construct the expression vector of HLAGshRNA and investigate the effect of HLAG shRNA from NK cell lysis. Methods Four HLAG shRNA plasmids were constructed and transiently transfected to Bel7402 cell lines, the levels of mRNA and protein of HLAG were detected by RealTime PCR and Western blot. The cytotoxicity of NK92MI cells against the transfected cells was analyzed by LDH releasing assay. Results The gel electrophoresis and sequencing showed that the inserted sequence was identical to the one which we designed, and no aberrations such as mutation, deletion or insertion occurred. The expressions of HLAG confirmed by Real TimePCR and Western blot were significantly downregulated. Bel7402 cell lines transfected HLAG shRNA showed higher lytic activity （P〈0. 01）. After KIR2DL4 receptor blocked, lytic activity ofNK92 MI cell were decreased G〈0. 01）. Con clusions HLAG shRNA plasmids are successfully constructed and HLAG downregulated can increase NK cytolysis against Bel7402 cell. After HLAG combines with KIR2DL4 receptor at the surface of NK cells, the inhibition effect is transferred.

关 键 词：人类白细胞抗原-G RNA干扰 肝癌 

分 类 号：R735.7[医药卫生—肿瘤]