中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (31): 4946-4953.doi: 10.12307/2022.736

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

内皮前体细胞促骨髓间充质干细胞向肝样细胞转化中的VEGF-Notch信号通路

于云宝1,2,陈  琳1,2,伍希雅3,闫乐蓉3,苗  章3,满  洋3,荆仁一3,雷  振1,褚志强1,张宏伟1,4   

  1. 1石河子大学医学院第一附属医院肝胆外科,新疆维吾尔自治区石河子市   832008;2成都京东方医院,四川省成都市   610000;3石河子大学医学院,新疆维吾尔自治区石河子市   832008;4国家卫生健康委中亚高发病防治重点实验室,新疆维吾尔自治区石河子市   832008
  • 收稿日期:2021-07-06 接受日期:2021-10-25 出版日期:2022-11-08 发布日期:2022-04-24
  • 通讯作者: 张宏伟,博士,副主任医师,石河子大学医学院第一附属医院肝胆外科,新疆维吾尔自治区石河子市 832008;国家卫生健康委中亚高发病防治重点实验室,新疆维吾尔自治区石河子市 832008
  • 作者简介:于云宝,男,1984年生,山东省潍坊市人,硕士,主要从事普通外科疾病的相关研究。
  • 基金资助:
    中国医学科学院中央级公益性科研院所基本科研业务费专项资金资助(2020-PT330-003),子项目负责人:张宏伟;兵团基础与应用研究项目(2016AG019),项目负责人:张宏伟

Vascular endothelial growth factor-Notch signaling pathways in endothelial precursor cells in promoting the transformation of bone marrow mesenchymal stem cells into hepatocyte-like cells

Yu Yunbao1, 2, Chen Lin1, 2, Wu Xiya3, Yan Lerong3, Miao Zhang3, Man Yang3, Jing Renyi3, Lei Zhen1, Chu Zhiqiang1, Zhang Hongwei1, 4   

  1. 1Department of Hepatobiliary Surgery, First Affiliated Hospital, Shihezi University School of Medicine, Shihezi 832008, Xinjiang Uygur Autonomous Region, China; 2Chengdu BOE Hospital, Chengdu 610000, Sichuan Province, China; 3Shihezi University School of Medicine, Shihezi 832008, Xinjiang Uygur Autonomous Region, China; 4NHC Key Laboratory of Prevention and Treatment of Central Asia High Incidence Diseases, Shihezi 832008, Xinjiang Uygur Autonomous Region, China
  • Received:2021-07-06 Accepted:2021-10-25 Online:2022-11-08 Published:2022-04-24
  • Contact: Zhang Hongwei, MD, Associate chief physician, Department of Hepatobiliary Surgery, First Affiliated Hospital, Shihezi University School of Medicine, Shihezi 832008, Xinjiang Uygur Autonomous Region, China; NHC Key Laboratory of Prevention and Treatment of Central Asia High Incidence Diseases, Shihezi 832008, Xinjiang Uygur Autonomous Region, China
  • About author:Yu Yunbao, Master, Department of Hepatobiliary Surgery, First Affiliated Hospital, Shihezi University School of Medicine, Shihezi 832008, Xinjiang Uygur Autonomous Region, China; Chengdu BOE Hospital, Chengdu 610000, Sichuan Province, China
  • Supported by:
    the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences, No. 2020-PT330-003 (to ZHW); Corps Foundation and Applied Research Project, No. 2016AG019 (to ZHW)

摘要:

文题释义:
Notch信号通路:在进化中高度保守,影响细胞正常形态发生的多个过程,包括多能祖细胞的分化、细胞凋亡、细胞增殖及细胞边界的形成。在特定条件下,内皮祖细胞可以促进骨髓间充质干细胞向肝样细胞转化,Notch信号通路参与了此过程。
肝再生:是指肝脏受损而肝细胞数量急剧减少时,各种反馈信号刺激处于G0期的肝细胞进行增殖,残肝细胞通过细胞增殖由基本不生长状态转变为快速生长状态,以补偿丢失、损伤的肝细胞和恢复肝脏的生理功能。

背景:在前期研究中发现骨髓血管内皮前体细胞在体外培养过程中通过旁分泌的方式产生了能够促进骨髓间充质干细胞分化的因子,血管内皮生长因子是其中之一。
目的:探讨骨髓血管内皮前体细胞促进骨髓间充质干细胞向肝样细胞转化中是否有VEGF-Notch信号通路参与。
方法:①分离培养SD大鼠骨髓间充质干细胞和骨髓血管内皮前体细胞,将第3代骨髓间充质干细胞分为3组:实验组使用培养骨髓血管内皮前体细胞的不含血清及因子的上清液,按照体积1∶1的比例与肝细胞转化培养基混合;模型对照组使用无血清的α-MEM 培养基,按照体积1∶1比例与肝细胞转化培养基混合;空白对照组使用正常α-MEM 培养基。在培养3,5,7,9 d,应用免疫荧光检测白蛋白、甲胎蛋白的表达。②取第3代骨髓间充质干细胞向肝样细胞转化分组培养,在肝细胞转化培养基中按体积1∶1比例分别加入培养骨髓血管内皮前体细胞的无血清及因子的上清液、血管内皮生长因子、培养血管内皮生长因子干扰质粒转染骨髓血管内皮前体细胞的无血清及因子的上清液;对照组使用肝细胞转化培养基培养。培养48 h后,使用实时定量RT-PCR和Western blot方法检测骨髓间充质干细胞中Notch下游基因Dll4、Hes1及Hey1的表达变化。
结果与结论:①空白对照组骨髓间充质干细胞中未见白蛋白、甲胎蛋白的表达,实验组和模型对照组骨髓间充质干细胞中甲胎蛋白、白蛋白表达水平均增高(P < 0.05),其中实验组甲胎蛋白、白蛋白表达水平高于模型对照组(P < 0.05);②肝细胞转化培养基中加入培养过骨髓血管内皮前体细胞的无血清及因子的上清液或血管内皮生长因子诱导骨髓间充质干细胞,Hey1、Dll4蛋白和mRNA表达均增高(P < 0.05),Hes1蛋白和mRNA表达均降低(P < 0.05);而在肝细胞转化培养基中加入血管内皮生长因子干扰质粒转染骨髓血管内皮前体细胞的无血清及因子的上清液诱导骨髓间充质干细胞,Dll4、Hey1蛋白和mRNA的表达均降低,Hes1蛋白和mRNA表达均增高(P < 0.05);③结果表明,骨髓血管内皮前体细胞在骨髓间充质干细胞向肝样细胞转化中可能起一定促进作用,在此过程中可能有VEGF-Notch信号通路参与。
缩略语:骨髓血管内皮前体细胞:bone marrow endothelial progenitor cells,BM-EPCs; 血管内皮生长因子:vascular endothelial growth factor,VEGF

https://orcid.org/0000-0002-3505-5824 (张宏伟) 

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

关键词: 骨髓间充质干细胞, 内皮前体细胞, 血管内皮生长因子, Notch信号通路, 肝样细胞

Abstract: BACKGROUND: Previous studies have shown that endothelial precursor cells produced factors that could promote bone marrow mesenchymal stem cells differentiation by paracrine in vitro, and vascular endothelial growth factor was one of them.  
OBJECTIVE: To investigate whether vascular endothelial growth factor-Notch signaling pathway participates in the way that endothelial precursor cells promote the transformation of bone marrow mesenchymal stem cells to hepatocyte-like cells.
METHODS:  (1) Bone marrow mesenchymal stem cells and endothelial precursor cells of Sprague-Dawley rats were isolated and cultured. The bone marrow mesenchymal stem cells at passage 3 were divided into three groups. In the experimental group, the serum-free and factor endothelial precursor cells-conditioned medium was mixed with hepatocyte transformation medium in a volume ratio of 1:1. In the model control group, serum-free alpha-minimal essential medium was mixed with the hepatocyte transformation medium in a volume ratio of 1:1. In the blank control group, normal alpha-minimal essential medium was used. The expression levels of alpha-fetoprotein and albumin were detected by immunofluorescence at 3, 5, 7, and 9 days of culture. (2) The third generation of bone marrow mesenchymal stem cells was taken to transform into hepatocyte-like cells and grouped. The serum-free and factor endothelial precursor cells-conditioned medium, vascular endothelial growth factor, and serum-free and factor vascular endothelial growth factor-siRNA-endothelial precursor cells-conditioned medium were added in hepatocyte transformation medium in a volume ratio of 1:1. In the control group, the samples were cultured with the hepatocyte transformation medium. At 48 hours after culture, the expression changes of Dll4, Hes1, and Hey1 (downstream gene of Notch) in bone marrow mesenchymal stem cells were detected by RT-PCR and western-blot assay.  
RESULTS AND CONCLUSION: (1) Alpha-fetoprotein and albumin were not found in the blank control group. Alpha-fetoprotein and albumin levels were increased in the model control group and the experimental group (P < 0.05), and alpha-fetoprotein and albumin levels were significantly increased in the experimental group compared with the model control group (P < 0.05). (2) After adding serum-free and factor endothelial precursor cells-conditioned medium or vascular endothelial growth factor, the expression of Dll4 and Hey1 protein and mRNA increased (P < 0.05), but the expression of Hes1 protein and mRNA decreased (P < 0.05). After adding serum-free and factor vascular endothelial growth factor-siRNA-endothelial precursor cells-conditioned medium, the expression of Dll4 and Hey1 protein and mRNA decreased, and the expression of Hes1 protein and mRNA increased (P < 0.05). (3) The results show that the bone marrow endothelial precursor cells may play a certain role in the transformation of bone marrow mesenchymal stem cells into hepatocyte-like cells, and the vascular endothelial growth factor-Notch signaling pathway may be involved in this process.

Key words: bone marrow mesenchymal stem cell, endothelial precursor cell, vascular endothelial growth factor, Notch signaling pathway, hepatocyte-like cell

中图分类号: