SiRNA干扰BTN3A3对脑胶质瘤细胞凋亡作用的研究  

Effect of siRNA interfering with BTN3A3 on the apoptosis of glioma cells

作　　者：张梦梦 张俊文 米蕊芳[1] 金贵善[1] 刘福生[1,2] Zhang Mengmeng;Zhang Junwen;Mi Ruifang;Jin Guishan;Liu Fusheng(Beijing Neurosurgical Institute,Capital Medical University,Beijing 100070,China;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,100070 [2]首都医科大学附属北京天坛医院神经外科,100070

出　　处：《中华神经外科杂志》2020年第9期951-956,共6页Chinese Journal of Neurosurgery

基　　金：生物医用材料北京实验室基金(京教函[2013]133号);国家自然科学基金(81772671)。

摘　　要：目的探讨采用siRNA干扰嗜乳脂蛋白亚家族成员A3(BTN3A3)对脑胶质瘤细胞凋亡的影响。方法实验采用胶质瘤细胞株(HS683、U251)以及原代胶质瘤细胞,均设立阴性对照组(NC组)以及转染不同序列siRNA的Si-1组(转染1序列)和Si-2组(转染2序列)。应用蛋白质免疫印迹方法分析不同胶质瘤细胞株、小胶质细胞株(HMC3)中BTN3A3的表达情况。通过转染siRNA敲减BTN3A3后,采用流术细胞术分析各细胞株中细胞凋亡的变化情况。分离培养手术切除的脑胶质瘤组织,获得原代胶质瘤细胞后,采用蛋白质免疫印迹方法分析原代细胞中BTN3A3的表达水平;通过转染siRNA敲减BTN3A3后,采用流式细胞术分析原代细胞的细胞凋亡变化情况。结果 BTN3A3在HS683(1.12±0.03)和U87-MG细胞(1.08±0.05)中的表达水平显著高于HMC3细胞(0.66±0.02)(均P<0.01)。U251细胞Si-1组、Si-2组BTN3A3的表达水平分别为0.11±0.01、0.12±0.01,均低于NC组(0.21±0.01)(均P<0.01);HS683细胞Si-1组、Si-2组BTN3A3的表达水平分别为0.77±0.03、0.78±0.04,均低于NC组(1.01±0.03)(均P<0.01)。敲减BTN3A3后,与NC组相比,HS683细胞的早期凋亡比率(NC组:31.6%,Si-1组:53.7%,Si-2组:63.3%)、U251细胞的晚期凋亡比率(NC组:10.4%,Si-1组:18.9%,Si-2组:26.5%)升高。BTN3A3在原代胶质瘤细胞中的表达水平高于HMC3细胞(分别为0.75±0.07和0.55±0.06,P<0.05)。采用siRNA敲减后,原代胶质瘤细胞中BTN3A3的表达水平降低(NC组:0.90±0.01,Si-1组:0.44±0.01,Si-2组:0.49±0.02,Si-1组和Si-2组与NC组相比,均P<0.01),晚期细胞凋亡比率升高(NC组:12.3%,Si-1组:22.6%,Si-2组:18.2%)。结论采用siRNA干扰BTN3A3可促进胶质瘤细胞株、原代胶质瘤细胞的凋亡。Objective To investigate the effect of siRNA interfering butyrophilin family member BTN3A3 on the apoptosis of glioma cells.Methods Glioma cell lines(HS683 and U251)and primary glioma cells were cultured and divided into negative control(NC)group(blank cells),Si-1 group and Si-2 group(transfected with plasmids with 1 and 2 sequences,respectively).Western blot was used to analyze the expression levels of BTN3A3 in glioma cell lines and microglia cell line(HMC3).Flow cytometry was used to analyze the changes of cell apoptosis following knockdown of BTN3A3.Glioma tissue obtained during surgical resection was cultured to obtain primary glioma cells.The expression levels of BTN3A3 in the primary cells were examined by Western blot,and changes of cell apoptosis following knockdown of BTN3A3 were detected by flow cytometry.Results BTN3A3 was expressed at higher levels in HS683 and U87-MG cell lines than in HMC3 cell line(HS683:1.12±0.03,U87-MG:1.08±0.05,HMC3:0.66±0.02,both P<0.01).The expression levels of BTN3A3 of Si-1(0.11±0.01)and Si-2(0.12±0.01)groups in U251 cell line were lower than that of NC group(0.21±0.01)(both P<0.01).The expression levels of BTN3A3 of Si-1 and Si-2 groups in HS683 cell line were 0.77±0.03 and 0.78±0.04 respectively,which were lower than that of NC group(1.01±0.03)(both P<0.01).When BTN3A3 was knocked down,the proportions of early apoptotic cells in HS683 cell line(NC:31.6%,Si-1:53.7%,Si-2:63.3%)and late apoptotic cells in U251 cell line(NC:10.4%,Si-1:18.9%,Si-2:26.5%)were increased.BTN3A3 expression level was higher in primary glioma cells(0.75±0.07)than in HMC3 microglia cells(0.55±0.06)(P<0.05).After transfection of siRNA,expression levels in primary glioma cells decreased(Si-1:0.44±0.01,Si-2:0.49±0.02,NC:0.90±0.01,both P<0.01),whereas the proportion of late apoptotic cells in primary glioma cells was increased(NC:12.3%,Si-1:22.6%,Si-2:18.2%).Conclusion Interference of siRNA with butyrophilin family member BTN3A3 promotes cell apoptosis of glioma cell lines and primary glioma

关 键 词：神经胶质瘤 细胞凋亡 嗜乳脂蛋白 嗜乳脂蛋白亚家族成员A3 

分 类 号：R739.41[医药卫生—肿瘤]