上海交通大学学报（医学版）

›› 2011, Vol. 31 ›› Issue (7): 913-.doi: 10.3969/j.issn.1674-8115.2011.07.010

• 论著（基础研究） • 上一篇    下一篇

低氧通路对骨髓间充质干细胞多向分化能力的影响

曾 文1, 张 伟1, 王 君1, 邓若晛2, 周 琦1, 魏 立1, 齐 进1, 邓廉夫1   

  1. 1.上海交通大学 |医学院附属瑞金医院上海市伤骨科研究所 上海市中西医结合防治骨与关节病损重点实验室, 上海 200025； 2．上海理工大学 医疗器械与食品学院, 上海 200093
  • 出版日期:2011-07-28 发布日期:2011-07-27
  • 通讯作者: 邓廉夫, 电子信箱: lianfudeng@126.com。
  • 作者简介:曾 文（1982—）, 男, 硕士生；电子信箱: safeng1234@163.com。
  • 基金资助:

    国家自然科学基金（30872641）；上海市科委研发基地（09DZ2200500, 10DZ2211500）；上海交通大学医学院科技基金项目（YZ1029）

Effects of hypoxia pathway on multipotential differentiation of bone marrow stromal stem cells

ZENG Wen1, ZHANG Wei1, WANG Jun1, DENG Ruo-xian2, ZHOU Qi1, WEI Li1, QI Jin1, DENG Lian-fu1   

  1. 1.Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai Institute of Traumatology and Orthopedics, Shanghai Key Laboratory of Combination of Traditional Chinese and Western Medicine in Prevention and Therapy of Osteo-arthropathy, Shanghai 200025, China；2.School of Medical Devices and Food Sciences, University of Shanghai for Science and Technology, Shanghai 200093, China
  • Online:2011-07-28 Published:2011-07-27
  • Supported by:

    National Natural Science Foundation of China, 30872641；Shanghai Science and Technology Committee Foundation, 09DZ2200500,10DZ2211500；Technology Fund of Shanghai Jiaotong University School of Medicine, YZ1029

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摘要：

目的 探讨低氧通路中低氧诱导因子1α(HIF-1α)对骨髓间充质干细胞(MSCs)多向分化能力的影响。方法 以腺病毒载体AdCre对转基因鼠MSCs 的VHL基因进行基因敲除（基因敲除组），设立对照组（感染腺病毒载体Ad-GPF的转基因鼠MSCs），采用Real-Time PCR技术检测HIF-1α mRNA表达。两组MSCs分别经成脂肪和成软骨诱导分化培养14 d，成骨诱导分化培养21 d；其中基因敲除组MSCs于5% O2条件下培养，对照组MSCs于20% O2条件下培养。采用Real-Time PCR技术检测软骨细胞标志物Ⅱ型胶原(ColⅡ)、脂肪细胞标志物过氧化物酶体增殖物激活受体γ（PPARγ）及成骨细胞标志物骨钙素（OC）和碱性磷酸酶（ALP）的mRNA表达；ColⅡ染色和ALP染色光学显微镜观察两组ColⅡ和ALP染色阳性细胞的分布情况。结果 基因敲除组HIF-1α mRNA表达显著高于对照组（P<0.05）。5% O2条件下培养的基因敲除组ColⅡ、PPARγ、OC、ALP mRNA表达均显著高于20% O2条件下培养的对照组（P<0.05）。与对照组比较，基因敲除组ColⅡ染色和ALP染色阳性细胞数量较多且染色较深。结论 在5% O2条件下，HIF-1α具有促进MSCs向软骨细胞、脂肪细胞和成骨细胞分化的作用。

关键词: 低氧诱导因子1α, 骨髓间充质干细胞, 基因敲除, 诱导分化

Abstract:

Objective To investigate the effects of hypoxia-inducible factor 1α(HIF-1α) on the multipotential differentiation of bone marrow stromal stem cells (MSCs) in the hypoxia pathway. Methods Conditional gene knockout of VHL gene of MSCs from transgenic mice was performed with Ad-Cre (gene knockout group), and control group was established (MSCs from transgenic mice infected with Ad-GPF). The expression of HIF-1α mRNA was detected by Real-Time PCR. Chondrogenic culture and osteogenic culture of MSCs were conducted for 14 d in two groups, and osteogenic culture of MSCs was conducted for 21 d in two groups, with culture of MSCs under 5% O2 in gene knockout group and culture of MSCs under 20% O2 in control group. The expression of chondrocyte marker of typeⅡcollagen (ColⅡ), adipocyte marker of peroxisome proliferator-activated receptors gamma (PPARγ) and osteoblast markers of osteocalcin (OC) and alkaline phosphatase (ALP) mRNA was detected by Real-Time PCR. The distributions of positive cells with ColⅡ staining and ALP staining were observed by light microscopy in two groups. Results The expression of HIF-1α mRNA in gene knockout group was significantly higher than that in control group (P<0.05). The expression of ColⅡ, PPARγ, OC and ALP mRNA in gene knockout group cultured under 5% O2 was significantly higher than that in control group cultured under 20% O2 (P<0.05). Compared with control group, the numbers of positive cells with ColⅡ staining and ALP staining were larger. Conclusion HIF-1α can promote the differentiation of MSCs into chondrocytes, adipocytes and osteoblasts under 5% O2.

Key words: hypoxia-inducible factor 1α, bone marrow stromal stem cells, gene knockout, differentiation