Chinese Journal of Tissue Engineering Research ›› 2011, Vol. 15 ›› Issue (19): 3463-3466.doi: 10.3969/j.issn.1673-8225.2011.19.010

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Retroviral-mediated tissue inhibitor of metalloproteinase-1 gene transfected human mandible bone marrow mesenchymal stem cells and their osteogenic potential

Gao Wen-ling1, Liu Fa-keng2, Hu Kai-shun3, Zhu Shuang-lin1   

  1. 1The Affiliated Stomatology Hospital, Sun Yat-sen University, Guangzhou  510055, Guangdong Province, China
    2First Affiliated Hospital of Sun Yat-sen University, Guangzhou  510080, Guangdong Province, China
    3Affiliated Tumor Hospital of Sun Yat-sen University, Guangzhou  510080, Guangdong Province, China
  • Received:2010-02-12 Revised:2011-03-04 Online:2011-05-07 Published:2011-05-07
  • Contact: Zhu Shuang-lin, Associate professor, The Affiliated Stomatology Hospital, Sun Yat-sen University, Guangzhou 510055, Guangdong Province, China
  • About author:Gao Wen-ling★, Studying for master’s degree, Physician, The Affiliated Stomatology Hospital, Sun Yat-sen University, Guangzhou 510055, Guangdong Province, China gaowenling8775@163.com

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Abstract:

BACKGROUND: Extracellular matrix (ECM) accumulation of mesenchymal stem cells (MSCs) occurs at the early phase of osteogenesis. And the main components of ECM deposited in that process are specific substrates of matrix metalloproteinases (MMPs) suppressed by tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). Whether MSCs can inhibit the activity of MMPs and improve the ability of osteogenic differentiation by increasing the expression of TIMP-1?
OBJECTIVE: To investigate the osteogenic potential of human mandible bone marrow mesenchymal stem cells (hMBM-MSCs) modified by TIMP-1 gene mediated with retroviral transfection.
METHODS: In-Fusion primers for TIMP-1 gene was designed, and reacted with digested linearized pBABE-Puro retroviral expression vector by homologous recombination sites and verified TIMP-1 expression with reverse transcription-polymerase chain reaction (RT-PCR) and sequencing methods, and then packaged with GP293 cell line. Bone marrow mesenchymal stem cells in mandibular surgery patients were separated by density gradient method, and their surface markers were verified by flow cytometry. TIMP-1 packaging liquid after retrovirus transfection infected and selected hMBM-MSCs cell lines which stably expressed TIMP-1 gene, and RT-PCR and protein electrophoresis validate its expression. HMBM-MSCs were induced using osteogenic induction medium for 10 days, and their osteogenic potential was observed. The expression of osteogenic related genes was verified by RT-PCR.
RESULTS AND CONCLUSION: Confirmed by DNA sequencing, RT-PCR obtained cDNA sequences of TIMP-1 gene totally matched with NM_003254.2 (624 bp) identical sequences. HMBM-MSCs were successfully separated according to surface makers identification, and manifested a good osteogenic and adipogenic ability. RT -PCR and Western Blot were used to verify the successful expression of hMBM-MSCs by retroviral vector-mediated transfection. RT-PCR showed that when hMBM-MSCs were induced with osteogenic induction media for 10 days, TIMP-1 increased markedly the mRNA expression of RUNX-2 and COL1. TIMP-1 gene can be successfully transfected into hMBM-MSCs by retroviral transfection, and promote the effect of osteogenesis.

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