GPx4在体细胞重编程为干细胞中的作用研究  

Role of GPx4 in somatic cells reprogramming into stem cells

作　　者：杨洋[1] 林厦华 杨君怡 郑茵 李鹏东 欧阳资章[1] 赵国军 廖宝剑 YANG Yang;LIN Xiahua;YANG Junyi;ZHENG Yin;LI Pengdong;OUYANG Zizhang;ZHAO Guojun;LIAO Baojian(Affiliated Qingyuan Hospital,Guangzhou Medical University,Qingyuan People’s Hospital,Qingyuan,Guangdong 511518,China;Guangzhou Institutes of Biomedicine and Health,Chinese Academy of Sciences,Guangzhou,Guangdong 510530,China)

机构地区：[1]广州医科大学附属清远医院/清远市人民医院,广东清远511518 [2]中国科学院广州生物医药与健康研究院,广州510530

出　　处：《重庆医学》2024年第19期2898-2906,共9页Chongqing Medical Journal

基　　金：广东省自然科学基金项目(2024A1515013168)。

摘　　要：目的探究谷胱甘肽过氧化酶4(GPx4)对小鼠体细胞重编程的影响。方法为了比较OG2小鼠胚胎成纤维(MEFs)细胞(MEFs组)与小鼠胚胎干细胞(mESCs,mESCs组)中GPx4的表达,利用转录组测序技术和Western blot确定细胞内GPx4的表达水平;为了验证GPx4对体细胞重编程效率的影响,将GPx4基因完整的开放阅读框序列以及其硒代半胱氨酸插入序列(SECIS)连接到反转录病毒载体pMXs上,构建过表达质粒pMXs-GPx4。合成靶向GPx4的短发夹RNA(shRNA)并连接到pSUPER载体上,构建GPx4 shRNA1和GPx4 shRNA2来敲低GPx4的表达。将上述质粒及对应的对照质粒与pMXs-Sox2、pMXs-Klf4、pMXs-Oct4共转染到MEFs细胞中进行重编程诱导,得到pMXs GPx4组、pMXs空载对照(pMXs NC)组、GPx4 shRNA1组、GPx4 shRNA2组、pSUPER空载对照(pSUPER NC)组。实时荧光定量PCR检测GPx4基因及多功能性标记基因Rex1、Sox2、Dappa3、Sall4、Oct4、Nanog表达;免疫荧光染色检测诱导性多能干细胞(iPSC),多能干细胞碱性磷酸酶染色检测iPSC克隆生成数量;Western blot检测GPx 4蛋白表达。结果mESCs组中GPx4 mRNA及蛋白表达水平高于MEFs组;与pMXs NC组比较,pMXs GPx4组细胞内GPx4 mRNA表达水平明显升高;与pSUPER NC组比较,GPx4 shRNA1组和GPx4 shRNA2组中GPx4 mRNA和蛋白表达水平下降(P<0.05);pMXs GPx4组iPSC克隆数高于pMXs NC组,但差异无统计学意义(P>0.05);GPx4 shRNA1组和GPx4 shRNA2组的iPSC克隆数均明显低于pSUPER NC组,差异有统计学意义(P<0.05)。完成重编程后,各组生成的iPSC与原始MEFs细胞比较,各多能性标记基因Rex1、Sox2、Dappa3、Sall4、Oct4、Nanog表达水平均升高。结论敲低GPx4可抑制体细胞重编程效率,其生成的诱导性多能干细胞具有正常的多能基因表达能力。Objective To investigate the effect of the glutathione peroxidase 4(GPx4)on mouse somatic cell reprogramming.Methods To compare the expressions of GPx4 in OG2 mouse embryonic fibroblast(OG2-MEF)cells(MEFs group)and mouse embryonic stem cells(mESC,mESCs group),the expression level of intracellular GPx4 was determined by transcriptome sequencing technique and Western blot.To verify the effect of GPx4 on the efficiency of the somatic cells reprogramming,the complete open reading frame sequence of GPx4 gene and its selenocysteine insertion sequence(SECIS)were connected to the retroviral vector pMXs for constructing the overexpressed plasmid pMXs-GPx4.Gpx4-targeting short hairpin RNA(shRNA)was synthesized and connected to pSUPER vector,GPx4 shRNA1 and GPx4 shRNA2 were constructed to knockdown GPx4 expression.The above plasmids were co-transfected with pMXs-Sox2,pMXs-Klf4 and pMXs-Oct4 into MEF cells for reprogramming induction to obtain the pMXs no-load control group(pMXs NC),pMXs GPx4 group,pSUPER no-load control group(pSUPER NC),GPx4 shRNA1 group and GPx4 shRNA2 group.The expressions of GPx4 gene and multifunctional marker genes Rex1,Sox2,Dappa3,Sall4,Oct4 and Nanog were detected by real-time fluorescence quantitative PCR.The induced pluripotent stem cells(iPSC)were detected by immunofluorescence staining;the number of iPSC clones generation was detected by alkaline phosphatase staining of pluripotent stem cells;the GPx4 protein expression was detected by Western blot.Results The mRNA and protein expression of GPx4 in the mESCs group was higher than that in the MEFs group;compared with the pMXs NC group,the expression level of GPx4 mRNA in the pMXs GPx4 group was significantly increased;compared with the pSUPER NC group,the GPx4 mRNA and protein levels in the GPx4 shRNA1 group and GPx4 shRNA2 group were decreased(P<0.05);the iPSC clone number in the pMXs GPx4 group was higher than that in the pMXs NC group,but the difference was not statistically significant(P>0.05).The number of iPSC clones in the GPx4 shRNA1 group

关 键 词：体细胞重编程 谷胱甘肽过氧化酶4 干细胞多能性 诱导性多能干细胞 细胞命运调控 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]