中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (14): 2540-2544.doi: 10.3969/j.issn.1673-8225.2011.14.016

• 干细胞因子及调控因子 stem cell factors and regulatory factors • 上一篇    下一篇

肿瘤坏死因子α刺激骨髓间充质干细胞表达及分泌肝细胞生长因子

陆正峰，姜海行，覃山羽，肖  健，张君红，孟云超   

  1. 广西医科大学第一附属医院消化内科，广西壮族自治区南宁市530021
  • 收稿日期:2010-12-15 修回日期:2011-02-09 出版日期:2011-04-02 发布日期:2013-11-02
  • 通讯作者: 姜海行，教授，博士生导师，广西医科大学第一附属医院消化内科，广西壮族自治区南宁市 530021 jihaxi@263.net
  • 作者简介:陆正峰★，男，1985年生，广西壮族自治区百色市人，壮族，广西医科大学在读硕士，主要从事干细胞及肝损伤修复机制研究。 nnaassaa117@yahoo.com.cn
  • 基金资助:

    广西自然科学基金资助项目(0897008)，广西“新世纪十百千人才工程”专项基金资助项目(2006206)。

Expression and secretion of hepatocyte growth factor on bone marrow mesenchymal stem cells stimulated by tumor necrosis factor alpha

Lu Zheng-feng, Jiang Hai-xing, Qin Shan-yu, Xiao Jian, Zhang Jun-hong, Meng Yun-chao   

  1. Department of Gastroenterology Medicine, the First Affiliated Hospital of Guangxi Medical University, Nanning  530021, Guangxi Zhuang Autonomous Region, China
  • Received:2010-12-15 Revised:2011-02-09 Online:2011-04-02 Published:2013-11-02
  • Contact: Jiang Hai-xing, Professor, Doctoral supervisor, Department of Gastroenterology Medicine, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China jihaxi@263.net
  • About author:Lu Zheng-feng★, Studying for master’s degree, Department of Gastroenterology Medicine, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China nnaassaa117@yahoo.com.cn
  • Supported by:

    the Natural Science Foundation of Guangxi, No. 0897008*; the “New Century Talent Project” of Guangxi, No. 2006206*

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摘要：

背景：有研究表明，肿瘤坏死因子α可以刺激骨髓间充质干细胞发挥免疫抑制功能，并促进骨髓间充质干细胞表达肝细胞生长因子。
目的：观察肿瘤坏死因子α刺激大鼠骨髓间充质干细胞是否可以促进肝细胞生长因子的表达及分泌。
方法：全骨髓贴壁法分离、纯化SD大鼠骨髓间充质干细胞，传代至第三四代使用。以100 µg/L肿瘤坏死因子α预刺激骨髓间充质干细胞 5 h后弃去培养基，换上新鲜培养基作为实验组，以正常培养的骨髓间充质干细胞为空白组。采用倒置相差显微镜观察细胞形态；流式细胞仪鉴定骨髓间充质干细胞表面标记物CD29、CD34、CD44、CD45的表达；RT-PCR 、Western blot检测细胞肝细胞生长因子mRNA及蛋白表达；ELISA测定细胞上清液中肝细胞生长因子水平。
结果与结论：获得的骨髓间充质干细胞形态均一，呈典型的旋涡样生长。骨髓间充质干细胞表达CD29+99.45%，CD34-97.91%、CD44+99.52%、CD45-98.42%；骨髓间充质干细胞中肝细胞生长因子 mRNA及蛋白与上清液中肝细胞生长因子表达量呈时间依赖性增加，实验组肝细胞生长因子 mRNA及蛋白与上清液中肝细胞生长因子的表达量明显高于空白组(P < 0.01)。说明肿瘤坏死因子α刺激骨髓间充质干细胞可以有效促进肝细胞生长因子的表达及分泌。

关键词: 骨髓间充质干细胞, 肿瘤坏死因子α, 肝细胞生长因子, 干细胞, 干细胞培养与分化

Abstract:

BACKGROUND: Previous studies demonstrated that tumor necrosis factor-α (TNF-α) can stimulate the immunosuppression function of bone marrow mesenchymal stem cells (BMSCs), and promote hepatocyte growth factor (HGF) expression of BMSCs.
OBJECTIVE: To investigate whether BMSCs stimulated by TNF-α is an effective way to promote the expression and secretion of HGF in rat BMSCs.
METHODS: BMSCs from SD rats were isolated, cultured and purified by the whole bone marrow adherence method, generated to passage3-4 to use. TNF-α pre-stimulated BMSCs, the medium was discarded after 5 hours, and then cultured with fresh medium as experimental group, normal cultured BMSCs as blank group. The morphology of BMSCs was observed by the inverted phase contrast microscope. The expression of surface makers CD 29, CD 34, CD 44 and CD 45 of BMSCs were identified by flow cytometer. HGF mRNA and protein expression were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. HGF protein level in cell supernatants was determined by ELISA.
RESULTS AND CONCLUSION: BMSCs were isolated by the whole bone marrow adherence method with uniform and showed spindle shaped, typical swirl morphology. BMSCs expressed CD29+ (99.45%), CD34- (97.91%), CD44+ (99.52%), CD45- (98.42%); the expression level of HGF mRNA and protein and HGF in cell supernatants increased with time-dependent. The expression levels of HGF mRNA and protein and HGF in cell supernatants in experimental group were significantly higher than that in blank group (P < 0.01). BMSCs stimulated by TNF-α can effectively improve the expression and secretion of HGF.

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