王举波,权瑜,吕健,董丹凤
WANG Jubo,QUAN Yu,LV Jian,DONG Danfeng

• 1:西安交通大学第二附属医院神经外科
• 2:西安交通大学第一附属医院肿瘤内科

摘要(Abstract)：

目的:探讨人异柠檬酸脱氢酶1(mIDH1)基因突变对替莫唑胺(TMZ)干预下脑胶质瘤U87细胞凋亡的影响。方法:用基因重组技术构建真核表达载体mIDH1/wIDH1,转染技术构建目的基因的U87细胞的稳转细胞系,采用流式细胞术检测TMZ干预24 h后的细胞凋亡率;构建裸鼠移植瘤模型,采用免疫组织化学法(Western blot)检测mIDH1对TMZ干预U87细胞24 h时及TMZ连续灌胃5 d时裸鼠移植瘤相关凋亡蛋白Caspase-3、Caspase-9、Bax及Bcl-2的表达水平。结果:成功构建胶质瘤稳转细胞系,流式细胞术结果显示,mIDH1能提高TMZ作用后U87细胞的凋亡率,也能上调TMZ干预24 h时U87细胞及TMZ连续灌胃5 d时裸鼠移植瘤中Caspase-3、Caspase-9、Bax蛋白表达水平,下调Bcl-2蛋白表达水平(P<0.05)。结论:mIDH1通过上调TMZ作用的U87细胞、裸鼠移植瘤组织的Caspase-3、Caspase-9、Bax表达、下调Bcl-2表达促进胶质瘤细胞凋亡。
Objective: To investigate the effect of human isocitrate dehydrogenase 1(IDH1) gene mutation(mutIDH1) on the apoptosis of glioma U87 cells induced by temozolomide(TMZ).Methods:Human wild type IDH1 cDNA was amplified by PCR and cloned into pCMV expression vector.Mutated IDH1(mutIDH1)was generated using a mutagenesis kit.The recombinant vectors expressing IDH1 or mutIDH1 gene were transfected into U87 cells to establish stable cell lines.Flow cytometry was used to detect the TMZ-induced U87 cell apoptosis.Glioma-bearing nude mice were treated with TMZ for 5 days.Immunohistochemistry and Western blot were used to detect the expression levels of caspase-3,Caspase-9,Bax and Bcl-2 in glioma.Results:IDH1 or mutIDH1 were stably expressed in U87 cells.The mutation of IDH1 enhanced TMZ-induced apoptosis compared to wild type IDH1.Moreover,the mutation of IDH1 upregulated expression levels of proapoptotic protein such as Caspase-3,Caspase-9,Bax,while downregulated Bcl-2 expression level(P<0.05).Conclusion:mutIDH1 can syngerize TMZ-induced apoptosis by up-regulating Caspase-3,Caspase-9 and Bax expression in vivo,and down-regulating Bcl-2 expression.

关键词(KeyWords)： 胶质瘤细胞;异柠檬酸脱氢酶1;凋亡,细胞;凋亡相关蛋白;替莫唑胺
glioma cells;mutated isocitrate dehydrogenase 1;apoptosis;apoptosis-related protein;temozolomide

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金青年科学基金项目(81801121);; 西安交通大学基本科研业务自由探索项目(xjj2018136)

作者(Author): 王举波,权瑜,吕健,董丹凤
WANG Jubo,QUAN Yu,LV Jian,DONG Danfeng

DOI: 10.19367/j.cnki.1000-2707.2019.08.005

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