贵州医科大学学报

2020, v.45;No.238(07) 773-780

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hUMSCs向胰岛前体细胞诱导分化过程中Notch1和神经元素3的表达
Expression of Notch1 and Ngn3 during the Differentiation of h UMSCs into Islet Progenitor Cells

韩晶;洪艳;王琪;檀梦天;
HAN Jing;HONG Yan;WANG Qi;TAN Mengtian;Department of Histology and Embryology,School of Basic Medical Sciences,Guizhou Medical University;Department of Science and Education,the Affiliated Hospital of Hebei Engineering University;

摘要(Abstract):

目的:探讨人间充质干细胞(h UMSCs)向胰岛前体细胞诱导分化过程中Notch信号通路受体Notch1与神经元素3(Ngn3)变化。方法:分离培养并鉴定h UMSCs,采用分阶段联合诱导法(前10 d用含EGF低糖培养基诱导,后11 d用含激活素A和尼克酰胺高糖培养基诱导)诱导h UMSCs向胰岛前体细胞分化,设单独培养的h UMSCs作为对照;观察诱导分化前后细胞的形态变化,免疫细胞化学法检测诱导后细胞Ngn3、胰高血糖素(Glucagon)表达鉴定胰岛前体细胞,电镜下观察诱导后胰岛前体细胞改变;采用免疫细胞化学法、real-time PCR法及Western blot法检测诱导第7天、第14天及第21天时细胞中Notch1及Ngn3的表达; Western blot法检测诱导并加入γ分泌酶抑制剂(DAPT)后第21天时细胞中Notch1及Ngn3的表达水平。结果:诱导后Glucagon免疫组化染色呈阳性,出现胰岛前体细胞集落,电镜下可见胞体内有分泌颗粒; real-time PCR和Western blot结果显示,Notch1水平在诱导后第7天时最高,Ngn3水平至第14天时达到峰值;添加Notch信号通路抑制剂后Ngn3表达水平升高。结论:Notch信号通路可能通过激活Notch1受体与下游基因Ngn3共同调控体外诱导h UMSCs向胰岛前体细胞分化过程。
Objective: To investigate the expression of Notch signaling receptor Notch1 and nerve element 3( Ngn3) during the differentiation of human umbilical mesenchymal stem cells( h UMSCs)into islet precursor cells. Methods: hUMSCs were isolated,cultured and verified. h UMSCs without the treatment was used as a control. The morphological changes of cells were observed before and after the differentiation. Immunohistochemical( IHC) staining was used to detect the expression of Ngn3 and glucagon for identifying islet precursor cells. The changes of islet precursor cells were observed under electron microscope. IHC staining,real-time PCR and Western blot were used to detect the expression of Notch1 and Ngn3 in the cells on days 7,14 and 21. The effect of gamma secretase inhibitor on Notch1 and Ngn3 expression was determined by Western blot. Results: After induction,IHC staining showed that cells were Glucagon positive. Islet precursor cells formed colonies. Secretory particles were observed in cells under electron microscopy. Real-time PCR and Western blot analyses showed that the levels of Notch1 was the highest on the 7 thday after induction,and Ngn3 reached a peak on the 14 thday. The inhibitor of Notch signaling pathway upregulated Ngn3 expression.Conclusion: The Notch signaling pathway may regulate the differentiation of hUMSCs into islet precursor cells in vitro by activating Notch1 receptor and its downstream gene Ngn3.

关键词(KeyWords): 间质干细胞;胰岛前体细胞;Notch1;Ngn3;人脐带间充质干细胞;γ分泌酶抑制剂;诱导
mesenchymal stem cells;islet precursor cells;Notch1;neurogenin-3(Ngn3);human umbilical cord mesenchymal stem cells(hUMSCs);gamma secretase inhibitor;induction

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金地区科学基金项目(81160099)

作者(Author): 韩晶;洪艳;王琪;檀梦天;
HAN Jing;HONG Yan;WANG Qi;TAN Mengtian;Department of Histology and Embryology,School of Basic Medical Sciences,Guizhou Medical University;Department of Science and Education,the Affiliated Hospital of Hebei Engineering University;

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DOI: 10.19367/j.cnki.2096-8388.2020.07.005

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