阻断Notch信号通路促骨髓间质干细胞体外分化为胰岛样细胞
조단Notch신호통로촉골수간질간세포체외분화위이도양세포
Inhibition of Notch signaling pathway promotes the differentiation of bone marrow mesenchymal stem cells into islet
  • 万方数据
    中华肾脏病杂志 중화신장병잡지

    2014年 8期 619-626 ,共8页

    张一婷%尹爱平%李利利%孙吉平

    장일정%윤애평%리리리%손길평

    间质干细胞,骨髓%胰岛%Notch信号 間質榦細胞,骨髓%胰島%Notch信號
    간질간세포,골수%이도%Notch신호
    Mesenchymal stem cells%bone marrow%Islet of langerhans%Notch signaling
    目的 通过Notch信号途径抑制剂DAPT构建Notch信号敲除模型,探讨Notch信号通路在骨髓间质干细胞(BMSCs)体外分化为胰岛样细胞中的作用.方法 体外分离培养BMSCs并鉴定,体外诱导其分化.用γ-分泌酶特异性抑制剂DAPT阻断Notch信号通路.采用双硫腙(DTZ)染色法鉴定胰岛细胞;间接免疫荧光染色,RT-PCR和Western印迹等方法检测诱导后细胞的胰岛素、胰高血糖素、胰十二指肠同源框1基因(Pdx-1)和神经元素3(Ngn3)的表达.ELISA法检测细胞葡萄糖刺激后分泌胰岛素水平.结果 (1)BMSCs鉴定:间接免疫荧光染色结果显示:BMSCs表达CD59和CD90抗原,体外可表达神经元特异性烯醇化酶(NSE)、神经胶质纤维酸性蛋白(GFAP)等神经细胞标记物,证实其具多向分化潜能.(2)MTT结果显示:γ-分泌酶抑制剂(DAPT)可抑制BMSCs增殖,其效应有时间和剂量依赖性.并以剂量和时间依赖性方式抑制Notch信号通路靶基因Hes1的表达.用1、5、20 μmol DAPT处理96 h后细胞的Hes1表达分别为对照组的92.06％、71.40％、46.89％,提示其对Notch信号通路阻断率分别达到7.94％、28.6％和53.11％(均P＜0.05).(3)间接免疫荧光结果显示:DAPT阻断组的胰岛素和胰高血糖素阳性细胞百分比较对照组明显增多[分别为(74.03±3.96)％比(36.49±3.24)％;(64.81±4.37)％比(37.50±3.69)％,均P＜0.05].提示阻断Notch信号通路后BMSCs体外分化效率增加.(4)RT-PCR和Western印迹结果提示:BSMCs诱导14 d后可以检测到胰岛素、胰高血糖素表达,DAPT阻断后上述蛋白表达上调;BMSCs诱导早期(7 d)即表达Pdx-1和Ngn3,14d后达到高峰,之后逐渐下降.DAPT阻断后Pdx-1和Ngn3的表达增加.(5)ELISA结果显示:诱导后细胞对葡萄糖刺激有分泌胰岛素反应,DAPT阻断后诱导细胞的反应更好.结论 大鼠BMSCs体外可分化为胰岛样细胞;阻断Notch信号通路可通过改变胰腺关键转录因子的表达而增强BMSCs体外分化能力.
    목적 통과Notch신호도경억제제DAPT구건Notch신호고제모형,탐토Notch신호통로재골수간질간세포(BMSCs)체외분화위이도양세포중적작용.방법 체외분리배양BMSCs병감정,체외유도기분화.용γ-분비매특이성억제제DAPT조단Notch신호통로.채용쌍류종(DTZ)염색법감정이도세포;간접면역형광염색,RT-PCR화Western인적등방법검측유도후세포적이도소、이고혈당소、이십이지장동원광1기인(Pdx-1)화신경원소3(Ngn3)적표체.ELISA법검측세포포도당자격후분비이도소수평.결과 (1)BMSCs감정:간접면역형광염색결과현시:BMSCs표체CD59화CD90항원,체외가표체신경원특이성희순화매(NSE)、신경효질섬유산성단백(GFAP)등신경세포표기물,증실기구다향분화잠능.(2)MTT결과현시:γ-분비매억제제(DAPT)가억제BMSCs증식,기효응유시간화제량의뢰성.병이제량화시간의뢰성방식억제Notch신호통로파기인Hes1적표체.용1、5、20 μmol DAPT처리96 h후세포적Hes1표체분별위대조조적92.06％、71.40％、46.89％,제시기대Notch신호통로조단솔분별체도7.94％、28.6％화53.11％(균P＜0.05).(3)간접면역형광결과현시:DAPT조단조적이도소화이고혈당소양성세포백분비교대조조명현증다[분별위(74.03±3.96)％비(36.49±3.24)％;(64.81±4.37)％비(37.50±3.69)％,균P＜0.05].제시조단Notch신호통로후BMSCs체외분화효솔증가.(4)RT-PCR화Western인적결과제시:BSMCs유도14 d후가이검측도이도소、이고혈당소표체,DAPT조단후상술단백표체상조;BMSCs유도조기(7 d)즉표체Pdx-1화Ngn3,14d후체도고봉,지후축점하강.DAPT조단후Pdx-1화Ngn3적표체증가.(5)ELISA결과현시:유도후세포대포도당자격유분비이도소반응,DAPT조단후유도세포적반응경호.결론 대서BMSCs체외가분화위이도양세포;조단Notch신호통로가통과개변이선관건전록인자적표체이증강BMSCs체외분화능력.
    Objective To investigate the effect of Notch signaling during bone marrow mesenchymal stem cells (BMSC) differentiating into islet in vitro.Methods The specific inhibitor of γ-secretase DAPT was used to inhibit the Notch signaling pathway.Mter induction,DTZ staining,indirect immunofluorescence staining,RT-PCR and Westenn blotting were used to detect the expression of insulin,glucagon,Pdx-1 and Ngn3.Results (1) Identification of BMSCs:Indirect immunofluorescence staining showed that BMSCs could express CD59 and CD90,which both were makrers of mesenchymal stem cells.Besides,BMSCs could express nerve culluar markers such as NSE,GFAP,suggesting multi-directional differentiation.(2) The result of MTT showed DAPT could inhibit the cell proliferation in a time-dependent manner and a dose-dependent mannar.Besides,DAPT could inhibit the expression of target gene of Notch signal pathway in a time-dependent manner and a dosedependent mannar.After treated by 1,5,20 μmol DAPT,the expression of Hes1 had reached to 92.06％,71.40％ and 46.89％ of controls respectively,suggesting efficiency of inhibition on Notch reached 7.94％,28.6％ and 53.11％ respectively (all P＜ 0.05).(3) Indirect immunofluorescence staining showed the expression of pancreas-specific markers such as insulin and glucagon were much higher in DAPT treated BMSCs than that in controls,which was confirmed by RT-PCR and Western blotting analyses.The proportion of insulin-producing cells differentiated from DAPT treated BMSCs was (74.03 ± 3.96)％,which was higher than that from controls[(36.49 ± 3.24)％,P＜ 0.05].(4)Furthermore,RT-PCR and Western blotting analysis showed that the expressions of Pdx-1 and Ngn3 were earlier than that of insulin and glucagon,and the expressions of Pdx-1 and Ngn3 were higher in DAPT treated BMSCs than that in controls.Conclusions Notch signaling pathway plays a role in the differentiation of BMSCs into islet in vitro.Pharmacological interference with Notch signaling pathway may provide a novel method to obtain islet for therapeutic use.
    원문보기 원문다운로드 사이트로이동
저자의 최근 논문