天津医药

天津医药 ›› 2019, Vol. 47 ›› Issue (1): 1-5.doi: 10.11958/20181206

• 细胞与分子生物学 •    下一篇

骨髓间充质干细胞旁分泌HGF体外调控肝星状细胞

李静，郑雪，丁新，向辉，陈卫刚△   

  1. 基金项目：兵团农社处社会发展与科技攻关计划项目（2015AD015） 作者单位：石河子大学医学院第一附属医院消化内科（邮编832000） 作者简介：李静（1991），女，在读硕士，主要研究领域为慢性肝病与消化道肿瘤的临床与基础研究 △通讯作者 E-mail: 13579456959@126.com
  • 收稿日期:2018-08-13 修回日期:2018-11-17 出版日期:2019-01-15 发布日期:2019-01-15
  • 通讯作者: 李静 E-mail:drsyy2lj@163.com
  • 基金资助:
    骨髓/脐血干细胞肝内移植治疗肝硬化

Hepatic stellate cells are regulated by paracrine HGF from bone marrow mesenchymal stem cells in vitro

LI Jing, ZHENG Xue, DING Xin, XIANG Hui, CHEN Wei-gang△   

  1. Department of Gastroenterology, the First Affiliated Hospital of the Medical College, Shihezi University, Shihezi 832000，China △Corresponding Author E-mail:13579456959@126.com
  • Received:2018-08-13 Revised:2018-11-17 Published:2019-01-15 Online:2019-01-15

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摘要： ：目的 探讨大鼠骨髓间充质干细胞（BMSCs）与肝星状细胞（HSCs）体外共培养体系中，BMSCs旁分泌肝细 胞生长因子（HGF）对 HSCs 增殖、凋亡、活化的影响。方法 全骨髓贴壁法分离、培养、纯化大鼠 BMSCs，另培养 HSCs。6 孔板半透膜建立上下两层细胞非直接接触共培养体系，实验设 H 组（HSCs 单独培养）、H-H 组（HSCs 与 HSCs共培养）、M-H组（BMSCs与HSCs共培养）、M-H-C组（BMSCs与HSCs共培养并加c-met抑制剂），各组细胞培 养48 h后，流式细胞仪鉴定BMSCs，检测HSCs凋亡率，MTT法检测HSCs的增殖，免疫荧光共聚焦定量检测HSCs中 α-肌动蛋白（α-SMA）的表达量，ELISA法检测共培养体系上清液中HGF的浓度。结果 MSCs高表达阳性表面分子 CD29、CD90，低表达造血细胞表面标记CD45；BMSCs能明显抑制HSCs的增殖、活化并促进其凋亡，且M-H组上清液 中HGF的浓度明显高于其他组。结论 BMSCs与HSCs共培养过程中，BMSCs通过旁分泌HGF促进HSCs的凋亡，抑 制HSCs的增殖、活化。

关键词: 间质干细胞, 骨髓, 肝细胞生长因子, 共同培养技术, 旁分泌, 肝星状细胞

Abstract: Objective To investigate the effects of paracrine hepatocyte growth factor (HGF) secreted by rat bone marrow mesenchymal stem cells on proliferation, apoptosis and activation of hepatic stellate cells after rat bone marrow mesenchymal stem cells (BMSCs) and hepatic stellate cells (HSCs) were co-cultured in vitro. Methods The whole bone marrow adherent method was used to isolate, culture and purify rat BMSCs, and HSCs were cultured. The semi-permeable membrane of six-well plate was used to establish a two-layer cell co-culture system with indirect contact. The study consisted of group H (HSCs were cultured alone), H-H group (HSCs and HSCs were co-cultured), M-H group (BMSCs and HSCs were co-cultured), M-H-C group (BMSCs and HSCs were co-cultured with c-met inhibitor). Cells in each group were cultured for 48 hours. BMSCs were identified and the apoptosis rate of HSCs was detected by flow cytometry. The proliferation of HSCs was detected by MTT assay, and the expression level of alpha smooth muscle actin (α-SMA) in HSCs was detected quantitatively by immunofluorescence confocal method. The concentration of HGF in the supernate of the coculture system was determined by ELISA. Results MSCs overexpressed positive surface molecules CD29 and CD90, and low expression of hematopoietic cell surface marker CD45. BMSCs could significantly inhibit the proliferation and activation of HSCs, and promote apoptosis of HSCs. The concentration of HGF in the supernate was significantly higher in M-H group than that in the other groups. Conclusion BMSCs can promote the apoptosis of HSCs, inhibit the proliferation and activation of HSCs through paracrine HGF in the co-culture of BMSCs and HSCs.

Key words: mesenchymal stem cells, bone marrow, hepatocyte growth factor, coculture techniques, paracrine, hepatic stellate cells