中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (23): 4217-4221.doi: 10.3969/j.issn.1673-8225.2010.23.009

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

转化生长因子β1/胰岛素样生长因子1与髓核细胞诱导脂肪间充质干细胞向髓核样软骨细胞分化的比较

刘连江，李 放，文天用   

  1. 1解放军第四军医大学，陕西省西安市  710032，  
    2解放军北京军区总医院全军骨科中心，北京市   100700
  • 出版日期:2010-06-04 发布日期:2010-06-04
  • 通讯作者: 李 放，教授，硕士生导师，解放军北京军区总医院全军骨科中心，北京市 100700 FANGL6722@ sina.com
  • 作者简介:刘连江，男，1980年生，新疆维吾尔自治区阿克苏地区人，汉族，解放军第四军医大学在读硕士，医师，主要从事椎间盘退行性变及椎间盘组织工程的研究。 woodl@126.com
  • 基金资助:

    北京市自然科学基金资助项目（5062039）

Transforming growth factor-beta 1/ insulin-like growth factor-1 versus nucleus pulposus cells in inducing the differentiation of adipose-derived mesenchymal stem cells into nucleus pulposus-like cells

Liu Lian-jiang1,2, Li Fang2, Wen Tian-yong1   

  1. 1Fourth Military Medical University of Chinese PLA, Xi’an  710032, Shaanxi Province, China;
    2Orthopaedic Research Institute, General Hospital of Beijing Military Area Command of Chinese PLA, Beijing  100700, China
  • Online:2010-06-04 Published:2010-06-04
  • Contact: Li Fang, Professor, Master’s supervisor, Orthopaedic Research Institute, General Hospital of Beijing Military Area Command of Chinese PLA, Beijing 100700, China FANGL6722@sina. com
  • About author:Liu Lian-jiang, Studying for master’s degree, Physician, Fourth Military Medical University of Chinese PLA, Xi’an 710032, Shaanxi Province, China; Orthopaedic Research Institute, General Hospital of Beijing Military Area Command of Chinese PLA, Beijing 100700, China woodl@126.com
  • Supported by:

    the Natural Science Foundation of Beijing City, No. 5062039*

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摘要：

背景：以往研究脂肪间充质干细胞培养方式多为单层培养，诱导方式主要集中于细胞因子的方法，而椎间盘体内微环境，除细胞因子对细胞影响外，在其三维环境中细胞间的相互作用还值得进一步研究。

目的：在体外分别通过髓核细胞和转化生长因子β1/胰岛素样生长因子1两种条件诱导，观察脂肪间充质干细胞向髓核细胞分化的差异。

方法：分别单层培养兔的脂肪间充质干细胞与髓核细胞细胞，脂肪间充质干细胞培养至3代经鉴定后，按5×106个细胞制成微球，置于Transwell培养板中培养，分别通过髓核细胞微球及转化生长因子β1/胰岛素样生长因子1诱导；于诱导前、诱导7，14 d，观察细胞形态变化，并通过RT-PCR对Ⅱ型胶原、蛋白多糖水平进行测定。

结果与结论：在体外诱导7，14 d，两组脂肪间充质干细胞微团体积、形态无明显区别。RT-PCR检测结果显示7 d两组与髓核诱导组均有Ⅱ型胶原及蛋白多糖mRNA表达，但转化生长因子β1/胰岛素样生长因子1组表达更强；诱导14 d髓核诱导组Ⅱ型胶原及蛋白多糖mRNA表达明显增高，优于转化生长因子 β1/胰岛素样生长因子1组。结果表明，在体外髓核细胞和转化生长因子β1/胰岛素样生长因子1对脂肪间充质干细胞进行诱导均有促进其向髓核细胞分化作用,而转化生长因子β1、胰岛素样生长因子1为正常髓核细胞分泌重要因子，说明髓核细胞对脂肪间充质干细胞的诱导之间除细胞因子作用外，尚存在相互促进增殖分化作用。

关键词: 髓核细胞, 脂肪间充质干细胞, 转化生长因子&beta, 1, 胰岛素样生长因子1, 兔

Abstract:

BACKGROUND: Previous studies have addressed adipose-derived mesenchymal stem cells (ADSCs) culture way for monolayer culture; induction way was mainly cytokine method, and under intervertebral discs micro-environment, cell interactions also deserved further study in its three-dimensional environment, in addition to the effects on cell cytokines.

OBJECTIVE: To observe differences in differentiation of ADSCs into NPCs under two induced conditions: nucleus pulposus cells (NPCs) and transforming growth factor β1 (TGF-β1)/ insulin-like growth factor (IGF-1) in vitro.

METHODS: Rabbit ADSCs monolayer culture and NPCs were cultured respectively, 3 generations of ADSCs were qualified; 5×106 cells were made into microspheres and placed in Transwell culture plates for culture under TGF-β 1/IGF-1 induction. Cell shape changes were observed before, 7, 14 days after induction. Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine levels of type II collagen and proteoglycan.

RESULTS AND CONCLUSION: At 7 and 14 days, no significant difference was detected in size and shape of ADSCs microspheres in both groups. RT-PCR detection results displayed that type II collagen and proteoglycan mRNA expression was determined in 7-day TGF-β1/IGF-1 group and NPCs-induced group, but expression was stronger in the TGF-β1/IGF-1 group. Type II collagen and proteoglycan mRNA expression was obviously increased in 14-day NPCs-induced group, superior to TGF-β1/IGF-1 group. Results have suggested that in vitro NPCs and TGF-β1/IGF-1 lead to the ADSCs, which had promoting effects on NPCs differentiation. TGF-β 1 and IGF-1 are normal NPCs-secreted important factors. The promoting effects on proliferation and differentiation exist, with the exception of cytokine induction effect, in differentiation of ADSCs into NPCs.

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