Chinese Journal of Tissue Engineering Research ›› 2011, Vol. 15 ›› Issue (7): 1186-1190.doi: 10.3969/j.issn.1673-8225.2011.07.010

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Establishment of gene expression system of transforming growth factor in intermediate split thickness skin wound by real-time RT-PCR in Banna mini-pig inbred line

Li Ying1, Huo Jin-long2, Pan Wei-rong2, Zeng Yang-zhi2, Wang Ji-hua1   

  1. 1Department of Plastic Surgery, the Second Affiliated Hospital of Kunming Medical University, Kunming  650101, Yunan Province, China
    2Key Laboratory of Banna Mini-pig Inbred Line of Yunnan Province, Yunnan Agricultural University, Kunming  650201, Yunnan Province, China
  • Received:2010-09-29 Revised:2010-11-07 Online:2011-02-12 Published:2011-02-12
  • Contact: Wang Ji-Hua, Doctor, Professor, Department of Plastic Surgery, the Second Affiliated Hospital of Kunming Medical University, Kunming 650101, Yunan Province, China wangjihua1966@ 163.com
  • About author:Li Ying★, Master, Department of Plastic Surgery, the Second Affiliated Hospital of Kunming Medical University, Kunming 650101, Yunan Province, China ly4617@163.com Huo Jin-long, Doctor, Experimentalist, Key Laboratory of Banna Mini-pig Inbred Line of Yunnan Province, Yunnan Agricultural University, Kunming 650201, Yunnan Province, China jinlonghuo@ynau. edu.cn Li Ying and Huo Jin-long contributed equally to this paper.
  • Supported by:

    the Graduate’s Innovation Foundation of Kunming Medical University, No. 2009S030*

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Abstract:

BACKGROUND: Banna mini inbred pigs were used to construct animal models of skin wound healing, which is similar with patients. This method can detect the expression of transforming growth factor beta 1 (TGF-β1), a gene closely associated with wound healing and scar. 
OBJECTIVE: To detect the expression of TGF-β1 during wound healing.
METHODS: Banna mini inbred pigs, aged 4-6 months, were used to construct animal models of skin wound healing. The total RNA from skin wounds was extracted, designated with specific primers, and then amplified through RT-PCR to isolate TGF-β1. The purified PCR product was linked with a pMD18-T vector and transferred into the bacterium DH5α for replication. The recombinant plasmid picked out from positive clones was amplified by PCR, digested with EcoR Ⅰ and Hind Ⅲ, and then sequenced. This process was used to calculate the standard concentration of recombinant plasmids from real-time quantitative PCR.
RESULTS AND CONCLUSION: The sensitivity of this method for creating TGF-β1 by expressing mRNA genes through PCR was good. Specifically, the fewest number of copies was 103, with a range of 103-109 copies. A clear linear relationship was found between the threshold cycle number and the PCR system (R2=0.988), and amplification efficiency was determined to be 107.433%. This detection system was used in 45 test samples and worked well. This method can serve as a biological foundation for the study the role of TGF-β1 in wound healing.

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