线粒体钙离子单向转运体对脑胶质瘤干细胞增殖和自我更新的影响  

Effect of mitochondrial calcium uniporter on proliferation and self-renewal of glioma stem cells

作　　者：李琳 陈佳意 韩秋影[1] 李爱玲[1] 满江红[1] 潘欣[1] LI Lin;CHEN Jia-yi;HAN Qiu-ying;LI Ai-ling;MAN Jiang-hong;PAN Xin(National Center of Biomedical Analysis,Beijing 100850,China)

机构地区：[1]国家生物医学分析中心,北京100850

出　　处：《中国药理学与毒理学杂志》2022年第9期665-672,共8页Chinese Journal of Pharmacology and Toxicology

基　　金：国家杰出青年科学基金(82025028)。

摘　　要：目的研究线粒体钙离子单向转运体(MCU)对脑胶质瘤干细胞增殖和自我更新的影响,探讨MCU作为治疗胶质瘤新靶点的可能性。方法Western印迹法和实时荧光定量PCR(RT-qPCR)检测脑胶质瘤干细胞(T456,T4121,T387和T3832)和胶质瘤细胞(U87和U251)中MCU蛋白和mRNA表达水平。构建短发夹RNA干涉阴性对照(shNT)、shMCU#1和shMCU#2质粒,利用磷酸钙转染法包装病毒,转染上述4种胶质瘤干细胞,分别获得该4种细胞的shNT、shMCU#1和shMCU#2细胞,并用RT-qPCR检测MCU敲低效果。将上述转染的细胞接种到96孔板中,于培养第0,2,4和6天用CellTiter-Glo?Luminescent细胞活力检测试剂盒检测细胞活力;第6天用成像显微镜观察每孔形成肿瘤球的大小并计数。用MCU抑制剂DS1657051125和50μmol·L^(-1)处理脑胶质瘤干细胞(T456和T4121)、人神经干细胞(16157)和人星形胶质细胞(NHA),同时设溶剂对照(0.1%DMSO)组,于培养第0,2,4和6天检测细胞活力,第6天观察形成肿瘤球的大小和细胞状态。构建LUC-shNT,LUC-shMCU#1和LUC-shMCU#2质粒,磷酸钙转染法包装病毒,并转染T387和T4121细胞,随后将转染的细胞注射到BALB/c裸小鼠颅内,25 d后用IVIS活体成像系统对小鼠脑内形成移植瘤体积进行生物发光检测,并记录小鼠存活时间。结果除T456细胞,4121,T387和T3832细胞MCU mRNA和蛋白表达水平均高于胶质瘤细胞U87和U251。与各自shNT对照组相比,4种脑胶质瘤干细胞shMCU#1和shMCU#2组MCU蛋白表达均显著下降(P<0.01),细胞活力亦明显降低(P<0.01),肿瘤球大小和数量显著减少(P<0.01)。与细胞对照组相比,DS1657051125μmol·L^(-1)可显著抑制T387,T4121和16157细胞活力(P<0.01),但对NHA细胞活力抑制作用相对较弱(P<0.05);DS1657051150μmol·L^(-1)可抑制T387,T4121,16157和NHA细胞活力(P<0.01)。小鼠活体成像检测显示,与接种LUCshNT细胞组相比,接种敲低MCU的T387和T4121细胞小鼠颅内移植瘤区域发光显著减弱,移植瘤小鼠�OBJECTIVE To investigate the effects of mitochondrial calcium uniporter(MCU)on proliferation and self-renewal of brain glioma stem cells(GSGs),and to explore the possibility of MCU being used as a new therapeutic target for glioblastoma multiforme.METHODS The protein and mRNA expressions of MCU in GSGs(T456,T4121,T387 and T3832)and glioma cells(U87 and U251)were detected by Western blotting and real-time fluorescence quantitative PCR(RT-q PCR).Short hairpin RNA ngeative control(sh NT),sh MCU#1 and sh MCU#2 plasmids were constructed,and the virus was packaged with the calcium phosphate transfection method before GSCs were infected,which were named corresponding shNT,shMCU#1 and shMCU#2 cells,respectively.The knockdown effect was detected by RT-q PCR.Cell viability was detected on the 0,2nd,4th,and 6thday of culture using the Cell Titer-Glo?luminescent cell viability assay.On the 6thday,the formation size of tumor spheres in each hole was recorded with an imaging microscope,and the number of tumor spheres in each group was counted.T456 and T4121(GSCs),16157(glioma cells)and NHA(human astrocytes)were treated with MCU inhibitor DS1657051125 and 50μmol·L^(-1).Cell viability was detected on the 0,2nd,4th,and 6thday using the CellTiter-Glo?luminescent cell viability assay,and on the 6thday the formation of tumor ball size and cell state was detected using an imaging microscope.LUC-shNT,LUC-shMCU#1 and LUCshMCU#2 plasmids were constructed,and the virus was packaged using the calcium phosphate transfection method and transfected to T387 and T4121 cells.Then,the transfected cels were injected into the intracranial of BALB/c nude mice.After some time,in situ glioma grafts were formed in the brain of the mice.Bioluminescence imaging was performed on the brains of BALB/c nude mice after 25 d,and the mouse survival time was recorded.RESULTS The protein and mRNA expressions of MCU in T387,T3832and T4121 cells were higher than in U87 and U251 cells.RT-q PCR results showed that the MCU mRNA expressions of T387,T4121,T3832 and T

关 键 词：线粒体钙离子单向转运体 神经胶质瘤 脑胶质瘤干细胞 细胞增殖 

分 类 号：R963[医药卫生—微生物与生化药学]