中国组织工程研究

中国组织工程研究 ›› 2017, Vol. 21 ›› Issue (13): 1974-1979.doi: 10.3969/j.issn.2095-4344.2017.13.002

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

骨髓间充质干细胞分化为心肌样细胞相关基因表达与形态结构发生的关联

徐亦辰，刘玲珑，赵文婧，王慧丰，韦雅淑，陈维平   

  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
  • 修回日期:2016-12-22 出版日期:2017-05-08 发布日期:2017-06-09
  • 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
  • 作者简介:徐亦辰，男，1988年生，黑龙江省哈尔滨市人，汉族，广西医科大学在读硕士，主要从事组织工程化心肌模型的建立研究。
  • 基金资助:

    广西急诊与医学救援人才小高地•广西高校急诊医学重点实验室开放课题(GXJZ201505)；广西医科大学青年科学基金资助项目 (GXMUYSF201528)；广西自然科学基金(2012GXNSFAA053126，桂科自 0832157，桂科攻0592007-1G，桂科青0229009)；广西高校人体发育与疾病研究重点实验(0102402214009C)

Cardiomyocyte-like differentiation of bone marrow mesenchymal stem cells:an association between related gene expression and cell morphological changes

Xu Yi-chen, Liu Ling-long, Zhao Wen-jing, Wang Hui-feng, Wei Ya-shu, Chen Wei-ping   

  1. Department of Histology and Embryology, Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China
  • Revised:2016-12-22 Online:2017-05-08 Published:2017-06-09
  • Contact: Chen Wei-ping, Professor, Department of Histology and Embryology, Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China
  • About author:Xu Yi-chen, Studying for master’s degree, Department of Histology and Embryology, Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China
  • Supported by:

    Guangxi Emergency and Medical Rescue Talent Small Highland • Guangxi University Emergency Laboratory Key Laboratory Open Topics, No. GXJZ201505; the Science Foundation for the Youth in the Guangxi Medical University, No. GXMUYSF201528; the Natural Science Foundation of Guangxi Zhuang Autonomous Region, No. 2012GXNSFAA053126, guikezi0832157, guikegong0592007-1G, guikeqing0229009; Key Experiments of Human Development and Disease Research in Guangxi Universities, No. 0102402214009C

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摘要：

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文题释义：
骨髓间充质干细胞分化为心肌细胞：心肌细胞的发生过程中，细胞的形态变化表现出典型的规律：发育早期，心内皮管外周的间充质细胞以突起连接成网；胞体增长、突起缩短成为成肌细胞；成肌细胞不断发育，相互衔接形成闰盘，细胞内肌原纤维由较少、排列散乱，逐步趋于整齐并插向闰盘；当胞内肌原纤维的肌微丝和Z线出现时，心肌纤维将开始出现收缩活动。将骨髓间充质干细胞诱导分化为心肌细胞，细胞的形态变化也呈现相似的规律：由“长梭形”的骨髓间充质干细胞，逐渐呈现“短杆状-突起形成-突起交错相连呈网状-细胞端端相连、胞膜增厚呈竹节状-相邻细胞融合形成肌管样结构”的变化过程。
心脏的发育过程中相关基因的表达：具有准确的时序性和空间性，在定向诱导骨髓间充质干细胞分化为心肌细胞过程中，本实验对细胞形态、相关基因表达和相应结构蛋白产生的关联性进行研究，认为相关基因的表达、细胞的形态变化、细胞内相关结构的发生密切相关，且具有规律性。

 

摘要
背景：前期已完成体外定向诱导骨髓间充质干细胞分化为心肌样细胞的实验。
目的：观察骨髓间充质干细胞分化为心肌样细胞过程中，细胞形态变化、结构发生规律与GATA-4、Nkx-2.5和a-MHC表达的关联性。
方法：采用心肌组织裂解液诱导SD乳鼠骨髓间充质干细胞分化为心肌细胞，以免疫组织化学染色检测心肌肌钙蛋白T和间隙连接蛋白43表达情况，鉴定心肌细胞；在定向诱导分化过程中，RT-PCR检测GATA-4、Nkx2.5与α-MHC的表达情况。
结果与结论：①细胞形态：在定向诱导过程中，细胞形态由长梭形经历短杆状、形成短小突起、网状结构、呈竹节状及肌管样结构；②心肌细胞鉴定：竹节样结构开始出现心肌肌钙蛋白T和间隙连接蛋白43阳性细胞，肌管样结构心肌肌钙蛋白T和间隙连接蛋白43阳性细胞增多；③RT-PCR检测：在定向分化过程中，细胞GATA-4、Nkx2.5与α-MHC基因表达持续增加；细胞交错连接成网状时，GATA-4表达明显增加，之后持续高表达；相邻细胞融合形成肌管样结构时，α-MHC表达达到峰值；Nkx2.5表达随诱导时间的延长逐渐增加；④结果表明：骨髓间充质干细胞分化为心肌样细胞过程中，心肌细胞特异基因的表达与心肌样细胞的形态特征、特殊结构发生密切相关，具有典型的时序性和空间性。

 

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: 干细胞, 骨髓干细胞, 骨髓间充质干细胞, 心肌细胞, NKx2.5, GATA-4, α-MHC, 细胞形态, 广西壮族自治区自然科学基金

Abstract:

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) have been induced to differentiate into cardiomyocyte-like cells in vitro.
OBJECTIVE: To explore the association between GATA-4, Nkx-2.5 and α-myosin heavy chain (α-MHC) expression and cell morphological changes and structure formation in the process of BMSCs differentiation into cardiomyocyte-like cells.
METHODS: By using myocardial lysate, BMSCs were induced to differentiate into cardiomyocytes. Immunocytochemistry staining was used to detect cardiac troponin T (cTnT) and connexin43, for the identification of cardiomyocytes. In the process of directional differentiation, RT-PCR was used to detect the expression of GATA-4, Nkx2.5 and α-MHC.
RESULTS AND CONCLUSION: During the directional differentiation of BMSCs, the cells were changed from long fusiform to short rod, forming protrusions that were interconnected to form mesh-like, bamboo-like or myotube-like structure. When the cells were interconnected like a bamboo, cTNT and connexin43 positive cells were visible, and then the number of positive cells increased with the presence of myotube-like structure. RT-PCR results showed that during the induced directional differentiation of BMSCs, GATA-4, Nkx2.5 and α-MHC mRNA levels increased continuously. When interconnected cells formed a mesh-like structure, GATA-4 expression reached the peak and then kept a high level. When adjacent cells were fused into a myotube-like structure, α-MHC reached the peak. Additionally, the expression of Nkx2.5 presented a time-dependent increase trend. Overall, during the induced differentiation of BMSCs into cardiomyocyte-like cells, the expression of cardiomyocyte specific genes, characterized by temporality and spatiality, is related to the changes of cell morphology and special structure formation.

 

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

Key words: Mesenchymal Stem Cells, Flow Cytometry, Immunohistochemistry, Myocytes, Cardiac, Tissue Engineering

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