摘要
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信号通路在骨髓问质干细胞(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ν-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)MTr结果显示:ν-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Hcsl的表达。用1、5、20μmol DAPT处理96h后细胞的Hesl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<0.051。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14d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(7d)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。
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 ν- secretase DAPT was used to inhibit the Notch signaling pathway. After induction, DTZ staining, indirect immunofluorescence staining, RT-PCR and Western blotting were used to detect the expressionof 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 MTI' 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 dose- dependent mannar. After treated by 1, 5, 20 μmol DAPT, the expression of Hesl 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.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2014年第8期619-626,共8页
Chinese Journal of Nephrology
基金
国家自然科学基金(81200528)
陕西省自然科学基金(2009JQ4012)
“十二五”国家科技支撑计划课题(2011BAll0804)
关键词
间质干细胞
骨髓
胰岛
NOTCH信号
Mesenchymal stem cells
bone marrow
Islet of langerhans
Notch signaling