胶质母细胞瘤脂肪酸合酶调控巨噬细胞M1极化影响细胞迁移和侵袭能力的实验研究  

Research of the regulation of macrophage M1 polarization and influence of cell migration and invasion by FASN expression in glioblastomas

作　　者：张文欣 季佳宇 周贤喆 王佳琳 袁桂强 张梦梦 张俊文 刘福生[1,2] 金贵善[1] Zhang Wenxin;Ji Jiayu;Zhou Xianzhe;Wang Jialin;Yuan Guiqiang;Zhang Mengmeng;Zhang Junwen;Liu Fusheng;Jin Guishan(Beijing Neurosurgical Institute,Capital Medical University,Beijing 100070,China;Neurosurgery Center,Beijing Tiantan Hospital,Capital Medical University,Beijing 100070,China)

机构地区：[1]首都医科大学,北京市神经外科研究所,北京100070 [2]首都医科大学附属北京天坛医院神经外科学中心,北京100070

出　　处：《中华神经外科杂志》2024年第3期283-290,共8页Chinese Journal of Neurosurgery

基　　金：国家自然科学基金(81972344);北京市自然科学基金(7222019)。

摘　　要：目的 探究胶质母细胞瘤(GBM)细胞中脂肪酸合酶(FASN)表达对巨噬细胞U937极化状态的影响.方法 采用慢病毒转染GBM细胞(U87和LN229)构建稳定干扰FASN的细胞株,获得干扰RNA对照组(sh-con,即U87sh-con组、LN229sh-con组)和敲低FASN实验组(sh-FASN,即U87sh-FASN组、LN229sh-FASN组),收集以上4组的细胞上清液(GBM细胞条件培养基)后分别与经佛波酯(PMA)诱导分化后的U937巨噬细胞共培养(共培养后的细胞上清为巨噬细胞条件培养基),再分别将4组巨噬细胞条件培养基与GBM细胞(U251)进行共培养.通过蛋白质免疫印迹法(WB)和(或)细胞免疫荧光(IF)实验检测 U87sh-con、U87sh-FASN、LN229sh-con、LN229sh-FASN 4 组细胞中FASN蛋白的表达情况及GBM条件培养基与U937细胞共培养后环氧合酶2(COX-2)、精氨酸酶1(ARG1)蛋白的表达情况;通过CCK-8实验检测敲低FASN后U87和LN229细胞的增殖情况;通过实时荧光定量聚合酶链反应(qRT-PCR)实验检测4组GBM条件培养基与U937细胞共培养后成熟巨噬细胞标志物CD68,巨噬细胞M1表型标志物CD11c、COX-2及M2表型标志物白细胞介素10(IL-10)、ARG1的表达情况;通过Transwell小室实验检测4组巨噬细胞条件培养基对U251细胞迁移、侵袭能力的影响.统计学以P<0.05为差异有统计学意义.结果 成功构建了FASN蛋白稳定敲低的U87、LN229细胞株,WB和IF结果均显示,与对应细胞的sh-con组相比,U87、LN229细胞sh-FASN组的FASN蛋白表达均降低(均P<0.05);CCK-8结果显示,48 h时U87sh-con组与U87sh-FASN组细胞活力的差异具有统计学意义[(588.391±10.032)%对比(547.009±1.860)%,P=0.002];24、48 h 时LN229sh-con组与LN229sh-FASN组细胞活力的差异均具有统计学意义[24 h:(235.033±1.598)%对比(218.026±3.593)%;48 h:(441.190±5.196)%对比(388.306±9.887)%;均P<0.05),而 8、12 h时U87、LN229细胞sh-con组与sh-FASN组细胞活力的差异均无统计学意义(均P>0.05).qRT-PCR结果显示,与U937细胞相比,经诱导分化�Objective To investigate the effect of the expression of fatty acid synthase(FASN)in glioblastoma(GBM)cells on polarization of human monocyte macrophage U937.Methods Stable interference RNA control group(U87sh-con group and LN229sh-con group)and FASN knockout experimental group(U87sh-FASN group and LN229sh-FASN group)were obtained by lentivirus transfection of U87 and LN229 cell lines.The cell supernatant(GBM conditioned medium)of the above 4 groups was collected and co-cultured with the induced by PMA differentiated U937 macrophages(the cell supernatant after co-culture used as the macrophage conditioned medium),and then the macrophage conditioned medium of the 4 groups were co-cultured with U251 cells.Protein expressions of FASN in U87sh-con,U87sh-FASN,LN229sh-con,LN229sh-FASN groups and COX-2 and ARG1 after co-culture of GBM conditioned medium with U937 cells were detected by Western blot(WB)and(or)immunofluorescence(IF).The proliferation after FASN knockout in U87 and LN229 of cells was detected by CCK-8 assay.The expressions of mature macrophage marker CD68,M1 marker CD11c,COX-2 and M2 marker IL-10 and ARG1 in macrophages were detected by quantitative real-time fluorescent polymerase chain reaction(qRT-PCR)after co-culture of GBM conditioned medium with U937 cells in 4 groups.The effect of macrophage conditioned medium on the migration and invasion ability of U251 cells was detected by Transwell assay.Results We successfully constructed U87 and LN229 cell lines with stable knockdown of FASN protein.WB and IF results showed that compared with sh-con group of corresponding cells,FASN protein expression in sh-FASN group of U87 and LN229 cells was decreased,and the differences were statistically significant(both P<0.05).The CCK-8 results showed that at 48 hours,the difference of cell viability between U87sh-con and U87sh-FASN groups was statistically significant(588.391±10.032%vs.547.009±1.860%,P=0.002).At 24 hours and 48 hours,the differences of the cell viability between LN229sh-con and LN229sh-FASN groups w

关 键 词：胶质母细胞瘤 脂肪酸合酶 巨噬细胞 M1极化 细胞迁移 细胞侵袭 

分 类 号：R739.4[医药卫生—肿瘤]