精氨酸代琥珀酸合成酶-1对胰岛β细胞增殖与凋亡的影响  

Effects of argininosuccinate synthetase 1 on cell proliferation and apoptosis of pancreatic isletβcells

作　　者：夏芷晴 张紫晨 付麒[1] 何云强 杨涛[1] 孙敏[1] XIA Zhiqing;ZHANG Zichen;FU Qi;HE Yunqiang;YANG Tao;SUN Min(Department of Endocrinology,the First Affiliated Hospital of Nanjing Medical University,Nanjing 210029;Department of Endocrinology,Yancheng Third People’s Hospital,Yancheng 224000,China)

机构地区：[1]南京医科大学第一附属医院内分泌科,江苏南京210029 [2]盐城市第三人民医院内分泌科,江苏盐城224000

出　　处：《南京医科大学学报（自然科学版）》2023年第6期764-771,共8页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金(82070803)。

摘　　要：目的:探讨精氨酸代琥珀酸合成酶⁃1(argininosuccinate synthetase 1,ASS1)对胰岛β细胞增殖与凋亡的影响及机制。方法:利用siRNA和慢病毒载体在胰岛β⁃TC6细胞中分别敲低和过表达ASS1;5⁃乙炔基⁃2′⁃脱氧尿嘧啶核苷(5⁃ethynyl⁃2′⁃deoxyuridine,EdU)和CCK⁃8检测细胞增殖能力;Annexin V⁃PI流式细胞术和原位末端转移酶标记技术(terminal dUTP nick⁃end labeling assay,TUNEL)检测细胞凋亡水平;Western blot检测B细胞淋巴瘤⁃2(B⁃cell leukaemia⁃2,Bcl⁃2)蛋白、Bcl⁃2相关X蛋白(Bcl⁃2 associated X protein,Bax)、半胱氨酸天冬氨酸酶3(Caspase3)、切割型半胱氨酸天冬氨酸蛋白水解酶⁃3(cleaved Caspase⁃3)、凋亡诱导因子(apoptosis inducing factor,AIF)、细胞核增殖抗原(Ki67)和哺乳动物雷帕霉素靶蛋白(mammalian rapamycin target protein,mTOR)的表达水平;RT⁃qPCR检测AIF、Ki67和mTOR mRNA水平。结果:①与对照相比,敲低ASS1后,胰岛β细胞EdU阳性率和CCK⁃8细胞增殖活力降低,TUNEL阳性细胞数和AV/PI检测的细胞凋亡率升高。胰岛β细胞内AIF表达量明显升高,Bax/Bcl⁃2比值下降,Caspase⁃3活性降低。②过表达ASS1后,TUNEL阳性细胞数和AV/PI检测的细胞凋亡率均较对照组降低,伴随胰岛β细胞内Ki67和mTOR表达量升高。但胰岛β细胞EdU阳性率和CCK⁃8细胞增殖活力无明显变化。结论:ASS1过表达可能激活mTOR信号通路促进胰岛β细胞增殖;ASS1表达降低时,胰岛β细胞可通过AIF途径启动细胞凋亡。ASS1可能在胰岛β细胞的增殖和凋亡中发挥一定调控作用。Objective:The current study aims to investigate the effect of argininosuccinate synthetase 1(ASS1)on cell proliferation and apoptosis of pancreatic isletβcells.Methods:Small interference RNA was used to knockdown ASS1,and lentiviral vector to overexpress ASS1 in pancreatic isletβcells.The 5⁃ethynyl⁃2′⁃deoxyuridine(EdU)and CCK⁃8 methods were used to detect proliferation ability,and Annexin V⁃PI flow cytometry and terminal dUTP nick⁃end labeling(TUNEL)were used to detect apoptosis.Western blot was used to examine protein expression of B⁃cell leukaemia⁃2(Bcl⁃2),Bcl⁃2 associated X protein(Bax),Caspase⁃3,cleaved Caspase⁃3,apoptosis inducing factor(AIF),nuclear proliferation antigen Ki67 and mammalian rapamycin target protein(mTOR).RT⁃qPCR was applied to detect mRNA expression of AIF,Ki67 and mTOR.Results:①Proliferation activity of pancreaticβcells detected by EdU and CCK⁃8 was lower when ASS1 was knockdown,and apoptosis of isletβcells examined by TUNEL method and flow cytometry was higher than that in the control group.Meanwhile,the increased AIF expression and the decreased ratio of Bax/Bcl⁃2 and the activity of Caspase⁃3 were observed.②After overexpression of ASS1,the number of TUNEL positive cells and the apoptosis rate detected by AV/PI decreased compared to the control group,accompanied by expression levels of Ki67 and mTOR increased in pancreatic isletsβcells.But there was no significant change in the positive rate of EdU and proliferative activity of cells by CCK⁃8 detected.Conclusion:Overexpressing ASS1 in pancreatic isletβcells may activate mTOR signaling pathway to promote cell proliferation.Pancreatic isletβcells with decreased ASS1 expression may initiate apoptosis through AIF pathway.In summary,ASS1 may play a role in proliferation and apoptosis of pancreatic isletβ⁃cells.

关 键 词：精氨酸代琥珀酸合成酶 胰岛Β细胞 增殖 凋亡 

分 类 号：R587.1[医药卫生—内分泌]