中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (13): 2088-2093.doi: 10.3969/j.issn.2095-4344.1696

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

胰腺组织裂解液诱导骨髓间充质干细胞分化为胰岛样结构的鉴定

刘红静1，王慧丰1，韦雅淑2，赵文婧1，陈维平1   

  1. 1广西医科大学组织学与胚胎学教研室，广西壮族自治区南宁市 530021；2广西壮族自治区计划生育研究中心，广西壮族自治区南宁市 530021
  • 修回日期:2019-02-11 出版日期:2019-05-08 发布日期:2019-05-08
  • 通讯作者: 陈维平，教授，广西医科大学组织学与胚胎学教研室，广西壮族自治区南宁市 530021； 并列通讯作者：赵文婧，讲师，广西医科大学组织学与胚胎学教研室，广西壮族自治区南宁市 530021
  • 作者简介:刘红静，女，1991年生，黑龙江省同江市人，汉族，广西医科大学在读硕士，主要从事组织工程化胰岛模型建立的研究。
  • 基金资助:

    广西自然科学基金(2012GXNSFAA05-3126)，项目负责人：陈维平；广西科技厅资助项目(桂科自0832157)，项目负责人：陈维平；广西科技厅资助项目(桂科攻0592007-1G)，项目负责人：陈维平；广西医科大学青年科学基金(GXMUYSF201528)，项目负责人：赵文婧；2017年度广西高校中青年教师基础能力提升项目(2017KY0096)，项目负责人：赵文婧；广西再生医学重点实验室开放课题资助项目(桂再重开201703)，项目负责人：赵文婧

Identification of islet-like structures differentiated from bone marrow mesenchymal stem cells induced by pancreatic tissue lysate

Liu Hongjing1, Wang Huifeng1, Wei Yashu2, Zhao Wenjing1, Chen Weiping1   

  1. 1Department of Histology and Embryology, Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China; 2Guangxi Zhuang Autonomous Region Family Planning Research Center, Nanning 530021, Guangxi Zhuang Autonomous Region, China
  • Revised:2019-02-11 Online:2019-05-08 Published:2019-05-08
  • Contact: Chen Weiping, Professor, Department of Histology and Embryology, Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China; Zhao Wenjing, lecturer, Department of Histology and Embryology, Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China
  • About author:Liu Hongjing, Master candidate, Department of Histology and Embryology, Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China
  • Supported by:

    Guangxi Natural Science Foundation, No. 2012GXNSFAA05-3126 (to CWP); grants from Guangxi Science and Technology Department, No. 0832157 and 0592007-1G (both to CWP); the Young Scientific Fund of Guangxi University of Medical Sciences, No. GXMUYSF201528 (to ZWJ) ; the Basic Ability Promotion Project for Young and Middle-aged Teachers of Guangxi Universities in 2017, No. 2017KY0096 (to ZWJ); the Key Laboratory Opening Project of Guangxi Key Laboratory of Regenerative Medicine, No. 201703 (to ZWJ)

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摘要：

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文题释义：
体外诱导干细胞分化为胰岛样细胞的主要方法：目前，体外诱导干细胞分化为胰岛样细胞的主要方法包括化学试剂诱导(二甲基亚砜、烟酰胺等)、导入外源性转录因子(如PDX1、NeuroD1和Mafa等)和微环境诱导法。组织裂解液保留了组织结构的小分子物质和生物活性物质，模拟特定的微环境，提供促进细胞分化或保持细胞特性的因子和激素，通过相互作用的信号分子实现对细胞行为的调控。
胰岛样细胞鉴定：定向诱导骨髓间充质干细胞分化为胰岛内分泌细胞，诱导后需要通过组织化学染色检测细胞内的富集离子(如锌离子、氨基)，初步判断是否向胰岛内分泌细胞方向分化；结合免疫荧光细胞化学染色检测胰岛内分泌细胞的表达产物(如胰岛素、C肽、胰高血糖素及生长抑素等)进行综合评价。

 

摘要
背景：目前，体外已成功将骨髓间充质干细胞定向诱导分化为能够分泌胰岛素的胰岛样细胞团，但是对细胞的形态变化规律、胰岛样细胞团中内分泌细胞的类型及其相互关系的研究报道较少。
目的：观察骨髓间充质干细胞定向分化为胰岛样结构过程中细胞的形态变化、内分泌细胞的组成及分布规律。
方法：取生长良好的第3代骨髓间充质干细胞，扩增培养至细胞集落汇合占据瓶底面积80%，加入胰腺组织裂解液连续诱导培养。以双硫腙染色方法对骨髓间充质干细胞定向分化形成的细胞团进行初步筛选，Mallory染色鉴别胰岛内分泌细胞的类型及其分布；采用免疫荧光细胞化学染色法检测胰岛内分泌细胞中胰岛素、C肽、胰高血糖素和生长抑素蛋白表达。
结果与结论：①双硫腙染色显示，随着诱导时间延长，阳性细胞数量逐渐增多；②Mallory染色显示，胰岛样细胞团内α样细胞位于周边，β样细胞分布在中央及周边，成纤维样细胞分布在周围；③免疫荧光染色显示，胰岛样细胞团内有胰岛素、C肽、胰高血糖素和生长抑素阳性细胞；④结果表明，在特定的微环境条件下，骨髓间充质干细胞能分化为胰岛样结构，内含α、β、δ样细胞，周围有类成纤维细胞包绕。


中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
ORCID: 0000-0003-0223-8736(陈维平)

关键词: 骨髓间充质干细胞, 胰腺组织裂解液, 诱导分化, 胰岛样结构, 内分泌细胞, 广西自然科学基金

Abstract:

BACKGROUND: Bone marrow mesenchymal stem cells have been induced into islet-like cell mass in vitro. However,little researches reported on the morphological changes of cells, the types of endocrine cells in the islet-like cell mass and their relationships.
OBJECTIVE: To investigate the morphological changes of cells in the differentiation of bone marrow mesenchymal stem cells into islet-like structure and to explore the composition and distribution of endocrine cells.
METHODS: Passage 3 bone marrow mesenchymal stem cells growing well were cultured and expanded until the cell colonies occupies 80% of the bottom of the culture bottle, and the pancreatic tissue lysate was added for continuous induction. Dithizone staining was used to screen the islet-like cell mass directly differentiated from bone marrow mesenchymal stem cells. The types and distribution of endocrine cells were identified by Mallory staining. Expressions of insulin, C-peptide, glucagon and somatostatin protein were detected by immunofluorescence cytochemical staining.
RESULTS AND CONCLUSION: (1) Dithizone staining showed that the number of positive cells was increased over the induction time. (2) Mallory staining showed the red α-like cells were located in the periphery of the islet-like cell mass, the yellow β-like cells located in the center and periphery, and the light blue fibroblasts were distributed around the cell mass. (3) Immunofluorescence staining showed insulin, C-peptide, glucagon and somatostatin positive cells in the islet-like cell mass. To conclude, under certain microenvironment, bone marrow mesenchymal stem cells can differentiate into islet-like structures which containing α, β, δ-like cells, and are surrounded by fibroblast-like cells.

Key words: Bone Marrow, Mesenchymal Stem Cells, Cell Differentiation, Islets of Langerhans, Endocrine Cells, Tissue Engineering

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