Chinese Journal of Tissue Engineering Research ›› 2013, Vol. 17 ›› Issue (36): 6489-6494.doi: 10.3969/j.issn.2095-4344.2013.36.017

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Quantitative analysis of SOX9 and type Ⅱ collagen mRNA in the three-lineage differentiation of rat mesenchymal stem cells

Liang Da-chuan, Bai Jie-yu, Du Shao-hua, Cheng Peng, Kang Ning, Wang Zhen, Huang Qiang-kai, Yang Zi-quan   

  1. Department of Orthopedics, the Second Hospital of Shanxi Medical Univeristy, Taiyuan  030001, Shanxi Province, China
  • Received:2012-11-28 Revised:2013-01-10 Online:2013-09-03 Published:2013-09-03
  • Contact: Yang Zi-quan, M.D., Professor, Department of Orthopedics, the Second Hospital of Shanxi Medical Univeristy, Taiyuan 030001, Shanxi Province, China yzqonline@126.com
  • About author:Liang Da-chuan★, Master, Physician, Department of Orthopedics, the Second Hospital of Shanxi Medical Univeristy, Taiyuan 030001, Shanxi Province, China lmhey1983@126.com
  • Supported by:

    International Scientific and Technological Cooperation Program, No. 2010DFA32450*; the National Natural Science Foundation of China, No. 30973048*; Scientific Research Foundation of the National Personnel Department and Personnel Department of Shanxi Province for Returned Chinese Scholars; Foundation of Shanxi Province for Returned Chinese Scholars, No. 107*; the Natural Science Foundation of Shanxi Province, No. 2010011050-6*

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Abstract:

BACKGROUND: The main component of cartilage, type Ⅱ collagen gene expression in chondrocyte is positively correlated with SOX9 concentration in a dose-dependent manner.
OBJECTIVE: To observe the variation of SOX9 and type Ⅱ collagen mRNA content at different periods in the differentiation process (osteogenic, chondrogenic, adipogenic induction) of mesenchymal stem cells, and to explore the correlation of SOX9 expression and type Ⅱ collagen.
METHODS: Bone marrow mesenchymal stem cells were isolated from 4-week-old Kunming mice, and cultured in vitro to passage 3. The cell phenotype was identified with flow cytometry. Cells were divided into three groups and subjected to three kinds of induction conditions favorable for adipogenic, chondrogenic and osteogenic differentiation, and each group was observed at three time points. In addition, the non-induced cells were used as a control group. The total RNA of cells was extracted at 3, 7, 14 days after induction, and SOX9 and type Ⅱ collagen mRNA was quantified with reverse transcription-polymerase chain reaction. The induced cells were stained by immunofluorescence to observe the differentiation and perform statistical analysis.     
RESULTS AND CONCLUSION: Passage 3 bone marrow mesenchymal stem cells grew well, and cell phenotype was confirmed as stem cells by flow cytometry. The staining results showed that, the cells differentiated into chondrocytes, adipocytes and osteoblasts. The SOX9 mRNA levels in the induced cells were the highest in chondrogenic differentiation group, then in osteogenic differentiation group, and the lowest in adipogenic differentiation group. Type Ⅱ collagen mRNA levels in the induced cells were the highest in chondrogenic differentiation group, then in adipogenic differentiation group, and the lowest in osteogenic differentiation group. SOX9 expression in chondrogenic differentiation group increased at  3 and 7 days, and then decreased at 14 days. While type Ⅱ collagen expression increased at 3, 7, 14 days. SOX9 mRNA levels increased as the osteogenic differentiation, while type Ⅱ collagen expression gradually decreased. There was no significant difference in the SOX9 mRNA expression between adipogenic differentiation group and control group (P > 0.05), while type Ⅱ collagen expression was not regularly changed. Experimental findings suggest that, critical effect of SOX9 in chondrogenic differentiation is better than that in osteogenic and adipogenic differentiation. SOX9 is associated with type Ⅱ collagen, which may alter along with the SOX9 in the early chondrogenic differentiation; SOX9 may play a fine-tuning role in the process of chondrogenic and osteogenic differentiation.

Key words: stem cells, mesenchymal stem cells, collagen type Ⅱ, SOX9 transcription factor

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