中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (36): 6681-6684.doi: 10.3969/j.issn.1673-8225.2010.36.007

• 脂肪干细胞 adipose-derived stem cells • 上一篇    下一篇

脂肪基质细胞体外分离培养及成软骨定向诱导

赵大庆1，2，李  青2，马  钰2，邓志宏1，乔  莉1，卢连军1，邱建华1   

  1. 解放军第四军医大学西京医院，  1耳鼻咽喉头颈外科，2病理科，陕西省西安市  710032
  • 出版日期:2010-09-03 发布日期:2010-09-03
  • 通讯作者: 邱建华，教授，博士生导师，解放军第四军医大学西京医院耳鼻咽喉头颈外科，陕西省西安市 710032 qiujh@fmmu.edu.cn
  • 作者简介:赵大庆☆，男，1969年生，陕西省咸阳市人，汉族，1993年解放军第四军医大学毕业，博士后，副教授，主要从事软骨组织工程及其修复应用的研究。 zhaodq@fmmu.edu.cn
  • 基金资助:

    国家自然科学基金资助项目(30772261，30671087)；全军医药卫生科研基金资助项目(06MA233)。

Isolation, cultivation and chondrogenic differentiation of adipose-derived stromal cells in vitro

Zhao Da-qing 1,2, Li Qing2, Ma Yu2, Deng Zhi-hong1, Qiao Li1, Lu Lian-jun1, Qiu Jian-hua1   

  1. 1 Department of Otorhinolaryngology-Head and Neck Surgery, 2 Department of Pathology, Xijing Hospital, Fourth Military Medical University of Chinese PLA, Xi’an  710032, Shaanxi Province, China
  • Online:2010-09-03 Published:2010-09-03
  • Contact: Qiu Jian-hua, Professor, Doctoral supervisor, Department of Otorhinolaryngology-Head and Neck Surgery, Xijing Hospital, Fourth Military Medical University of Chinese PLA, Xi’an 710032, Shaanxi Province, China qiujh @fmmu.edu.cn.
  • About author:Zhao Da-qing☆, Doctor, Associate professor, Department of Otorhinolaryngology-Head and Neck Surgery; Department of Pathology, Xijing Hospital, Fourth Military Medical University of Chinese PLA, Xi’an 710032, Shaanxi Province, China zhaodq @fmmu.edu.cn
  • Supported by:

    the National Natural Science Foundation of China, No. 30772261*, 30671087*; Chinese Army Medical Research Council, No. 06MA233*

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被引次数

1

摘要：

背景：组织工程技术是解决软骨等组织缺损的理想途径，但其种子细胞的来源问题仍未很好地解决。
目的：观察脂肪基质细胞体外分离培养及其在体外向成软骨细胞诱导分化的能力。
方法：取新西兰白兔皮下脂肪组织，以胶原酶等消化分离获得脂肪基质细胞，以成软骨诱导培养液对其进行成软骨诱导分化，倒置显微镜下观察脂肪基质细胞体外培养传代情况；免疫组织化学、阿尔辛蓝染色、天狼猩红染色等方法观察脂肪基质细胞成软骨诱导可行性；MTT法观察脂肪基质细胞与诱导培养的脂肪基质细胞体外增殖情况。
结果与结论：脂肪基质细胞可于体外分离培养，在成软骨诱导后，可定向分化为软骨样细胞，其可分泌软骨细胞特有的Ⅱ型胶原及酸性黏多糖。原代培养的脂肪基质细胞呈长梭形，胞核椭圆，第3天进入指数增长期，群体倍增时间为55 h左右，细胞在第4天进入平台期，体外传代培养9代后，细胞开始出现衰老征象；成软骨诱导细胞增殖速度显著增快，随诱导培养时间的延长，细胞体积明显增大，形态由长梭形变为宽大、多伪足的多角形，胞核亦变大，核仁明显，培养第2天进入指数增长期，群体倍增时间为30 h左右，在第8天进入平台期，经体外传代培养15代后，细胞增殖速度减慢，开始出现衰老征象。提示脂肪基质细胞在一定条件下可定向分化为软骨细胞，可作为软骨组织工程种子细胞来源。

关键词: 脂肪基质细胞, 软骨, 诱导, 体外, 种子细胞, 软骨组织工程

Abstract:

BACKGROUND: Tissue engineering has been investigated as a potential means for generation of replacement cartilage. But the ideal seed cell which is still a problem has to be further solved.
OBJECTIVE: To observe the solation and cultivation of adipose-derived stromal cells (ADSCs), and to investigate its chondrogenic differentiation ability in vitro.
METHODS: Subcutaneous fatty tissue from New Zealand rabbits were digested with collagenase. The obtained ADSCs were cultured in the chondrogenic induced medium. Incubation and passage of ADSCs were observed under inverted microscope. The feasibility of ADSCs cartilage induction was detected by immunohistochemistry, Alcian Blue stain, and picrosirius red stain. Proliferation of ADSCs was observed by MTT methods.
RESULTS AND CONCLUSION: ADSCs could be isolated and cultured in vitro. After cultured in chondrogenic medium, the cells differentiated toward cartilage cells and secreted the specific cartilaginous matrices acid mucopolysaccharide and type Ⅱ collagen. Primary cultured ADSCs were spindle-shaped, with oval nucleus. At day 3, ADSCs were in the exponential growth phase, and the population doubling time was about 55 hours. At day 4, cell growth was stable. At day 9, the cells were observed aging symptoms. Cartilage-induced cell proliferation rate increased significantly, while with the incubation time prolonging, cell volume increased significantly, from long fusiform shape into large, multi-polygonal pseudopodia, furthermore nuclei were also larger, and nucleolus was clear. At day 2 after incubation, ADSCs were in the exponential growth phase, and the population doubling time was about 30 hours. At days 8, the cell growth was stable. After the 15th generation, cell proliferation slowed down and cells were observed signs of aging. This suggested that ADSCs differentiated into chondrocytes under a certain degree; therefore, ADSCs could be served as seed cell for cartilaginous tissue engineering.

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