髓源性抑制细胞在鸟苷酸结合蛋白1促进胶质瘤U87细胞增殖中的作用及其机制  

Effect of myeloid-derived suppressor cells on guanylate binding protein 1 in promoting the proliferation of glioma U87 cells and its mechanism

作　　者：陈丽莉 高策 郑彦文 李明[1] 王爱东[1] Chen Lili;Gao Ce;Zheng Yanwen;Li Ming;Wang Aidong(Central Laboratory,the Second Affiliated Hospital of Soochow University,Suzhou 215004,China;Clinical Laboratory,Suzhou Municipal Hospital,Suzhou 215008,China)

机构地区：[1]苏州大学附属第二医院实验中心,215004 [2]苏州市立医院北区检验科,215008

出　　处：《肿瘤研究与临床》2020年第11期745-752,共8页Cancer Research and Clinic

基　　金：苏州市科技计划(SYS201627);苏州大学附属第二医院青年预研项目(SDFEYQN1716)。

摘　　要：目的:探讨髓源性抑制细胞(MDSC)在鸟苷酸结合蛋白1(GBP1)促进胶质瘤U87细胞增殖中的作用及其相关机制。方法:选择过表达GBP1的胶质瘤U87细胞(U87-GBP1细胞)和转染空载体的对照U87-Lacz细胞进行实验。采用实时荧光定量聚合酶链反应(RT-qPCR)、蛋白质印迹法、酶联免疫吸附试验(ELISA)检测两种U87细胞GBP1和CC趋化因子配体2(CCL2)mRNA相对表达量或蛋白表达水平,采用CCK-8法检测两种细胞增殖变化。利用免疫磁珠从健康人外周血中分选出CD14 +单核细胞和CD3 + T淋巴细胞。用U87-Lacz或U87-GBP1细胞培养液处理CD14 +单核细胞,分别为U87-Lacz培养液组和U87-GBP1培养液组;采用流式细胞术检测CD14 +单核细胞中MDSC比例,用两培养液组作用的CD14 +单核细胞与活化的CD3 + T淋巴细胞共培养,流式细胞术检测活化的CD3 + T细胞增殖情况,分别以未经U87细胞培养液处理的单核细胞或活化CD3 + T淋巴细胞作为对照组。用加入抗人CCL2抗体U87-Lacz或U87-GBP1细胞培养液处理CD14 +单核细胞,分别为U87-Lacz+抗CCL2培养液组和U87-GBP1+抗CCL2培养液组,采用流式细胞术分析CD14 +单核细胞中MDSC比例。分别将U87-GBP1细胞和U87-Lacz细胞原位接种至BALB/c裸鼠颅内致瘤,1周后各分为CC趋化因子受体2(CCR2)抑制剂RS504393治疗组(U87-Lacz+RS裸鼠组和U87-GBP1+RS裸鼠组)和未经治疗的对照组(U87-Lacz裸鼠组和U87-GBP1裸鼠组);30 d后处死裸鼠,分离全脑、脾脏和骨髓组织,采用苏木精-伊红(HE)染色观察裸鼠脑中移植瘤,计算移植瘤体积,采用流式细胞术检测各组织MDSC比例。 结果:U87-GBP1细胞中GBP1蛋白表达水平高于U87-Lacz细胞,但两组细胞体外增殖水平差异无统计学意义( P>0.05)。U87-GBP1培养液组MDSC比例高于U87-Lacz培养液组[(7.75±0.80)%比(4.50±0.08)%, P=0.003],两组均高于对照组[(2.55±0.31)%)]( F=18.27, P=0.002);U87-GBP1培养液组作用的活化CD3 + T淋巴细胞增殖率低于U87-Lacz�Objective To investigate the effect of myeloid-derived suppressor cells(MDSC)on guanylate binding protein 1(GBP1)in promoting the proliferation of glioma U87 cells and its mechanism.Methods Glioma cells U87 with GBP1 overexpression(U87-GBP1)and control cells U87-Lacz transfected with empty vector were used as experimental cells.The mRNA and protein expressions of GBP1 and chemokine(C-C motif)ligand 2(CCL2)in two groups of cells were detected by reverse transcription-quantitative polymerase chain reaction(RT-qPCR),Western blot and enzyme linked immunosorbent assay(ELISA),and the proliferation of U87 cells were detected by CCK-8.CD14+monocytes and CD3+T lymphocytes were isolated from peripheral blood of healthy people by immunomagnetic beads.The CD14+monocytes were treated with culture medium of U87-Lacz cells or U87-GBP1 cells,and then the cells were divided into U87-Lacz culture medium group and U87-GBP1 culture medium group.The proportion of MDSC in CD14+monocytes was analyzed by flow cytometry.CD14+monocytes treated by two culture medium groups were cocultured with activated CD3+T lymphocytes,and flow cytometry was used to detect the proliferation of activated CD3+T lymphocytes.Monocytes untreated by U87 cells culture medium or activated CD3+T lymphocytes were used as the control group.CD14+monocytes were treated with U87-Lacz or U87-GBP1 cell culture medium anti-human CCL2 antibody,which were U87-Lacz+anti-CCL2 culture medium group and U87-GBP1+anti-CCL2 culture medium group,and the proportion of MDSC in CD14+monocytes was analyzed by flow cytometry.U87-GBP1 and U87-Lacz cells were inoculated into BALB/c nude mice to cause tumors in the brain.One week later,they were divided into chemokine(C-C motif)receptor 2(CCR2)inhibitor RS504393 treatment group(U87-Lacz+RS nude mice group and U87-GBP1+RS nude mice group)and untreated control group(U87-Lacz nude mice group and U87-GBP1 nude mice group).After 30 days,the mice were sacrificed and the brain,spleen and bone marrow were isolated.Hematoxylin-eosin(HE)staining wa

关 键 词：胶质瘤 鸟苷酸结合蛋白1 髓源性抑制细胞 CC趋化因子配体2 

分 类 号：R739.41[医药卫生—肿瘤]