PKC通过调控TRPC/Ca^(2+)通道影响iPS-mpMSC-fbs分化为功能性胰岛β细胞的机制研究  

Mechanism study of PKC effect on differentiation of iPS-mpMSC-fbs into functional slet β cells by regulating TRPC/Ca^(2+) channels

作　　者：郭鹏 谭烨 李文婧 丁钧 GUO Peng;TAN Ye;LI Wenjing;DING Jun(Department of Hepaticbiliary Pancreatic Surgery;Department of Gastroenterology,The Third Affiliated Hospital of Chongqing Medical University,Chongqing 401120,China)

机构地区：[1]重庆医科大学附属第三医院肝胆胰外科,重庆401120 [2]重庆医科大学附属第三医院消化内科,重庆401120

出　　处：《重庆医学》2023年第9期1298-1302,1308,共6页Chongqing medicine

基　　金：重庆市渝中区基础研究与前沿探索项目(20180113)。

摘　　要：目的探讨影响诱导性多能干细胞-间充质样-成纤维细胞(iPS-mpMSC-fbs)分化为功能性胰岛β细胞的分子机制。方法从健康成年小鼠胰腺中分离纯化间充质样-成纤维细胞(mpMSC-fbs),通过慢病毒将Oct4、Sox2、c-Myc和Klf44种转录因子转染至mpMSC-fbs,诱导mpMSC-fbs转变为诱导性多能干性球体细胞(iPS-mpMSC-fbs),并通过免疫荧光染色实验和Western blot检测其是否表达瞬时受体离子电位通道蛋白(TRPCs);分别将蛋白激酶C(PKC)激动剂佛波醇酯(PMA)和拮抗剂(Bis-1)与iPS-mpMSC-fbs共孵育,通过荧光影像系统测定细胞内游离钙离子(Ca^(2+))水平,观察PKC对iPS-mpMSC-fbs TRPC/Ca^(2+)通道的调节关系。结果从成年小鼠胰腺中成功分离纯化mpMSC-fbs,该细胞群与骨髓来源的间充质干细胞(MSCs)有相似表面标志物(CD90、CD105和CD146),并成功将mpMSC-fbs诱导为iPS-mpMSC-fbs,且诱导后细胞表达TRPCs(TRPC1、TRPC3和TRPC4)。iPS-mpMSC-fbs与PKC激动剂PMA共孵育后,使用磷脂酶C(PLC)/Diac途径刺激可见细胞内Ca^(2+)未出现第2高峰;而与PKC抑制剂(Bis-1)共孵育后,使用PLC/Diac途径刺激细胞内Ca^(2+)出现第2高峰。结论PKC可负性调控TRPC/Ca^(2+)通道,从而影响iPS-mpMSC-fbs分化为功能性胰岛β细胞。Objective To explore the molecular mechanism affecting the differentiation of induced pluripotent stem cell-mesenchymal-fibroblasts(iPS-mpMSC-fbs)into functional isletβcells.Methods Mesenchymal-fibroblast(mpMSC-fbs)were isolated and purified from the pancreas of healthy adult mice.The four transcription factors Oct4,Sox2,c-Myc and Klf4 were transfected into mpMSC-fbs by lentivirus to induce the transformation of mpMSC-fbs into iPS-mpMSC-fbs.Immunofluorescence staining and Western blot were used to detect the expression of transient receptor potential channel proteins(TRPCs).The protein kinase C(PKC)agonist phorbol myristate acetate(PMA)and antagonist(Bis-1)were co-incubated with iPS-mpMSCfbs,and the intracellular free Ca^(2+)concentration was measured by fluorescence imaging system to observe the relationship between PKC and iPS-mpMSC-fbs TRPC/Ca^(2+)channels.Results The mpMSC-fbs were successfully isolated and purified from pancreas of the adult mouse,which had similar surface markers(CD90,CD105 and CD146)to bone marrow derived mesenchymal stem cells(MSCs).MpMSC-fbs were successfully induced into iPS-mpMSC-fbs,and the induced cells expressed TRPC proteins including TRPC 1,3 and 4.After co-incubation of iPS-mpMSC-fbs with PKC agonist phorbol myristate acetate(PMA),intracellular Ca^(2+)did not show a second peak after stimulation with PLC/Diac pathway.After co-incubation with PKC inhibitor(Bis-1),the second peak of intracellular Ca^(2+)appeared after PLC/Diac pathway stimulation.Conclusion PKC can negatively regulate TRPC/Ca^(2+)channels,which affects the differentiation of iPS-mpMSC-fbs into functional isletβcells.

关 键 词：1型糖尿病 多能干细胞 转录因子 间充质干细胞 成纤维细胞 蛋白激酶 胰岛Β细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]