中国组织工程研究与临床康复2011，Vol.15Issue(7)：1186-1190,5.DOI:10.3969/j.issn.1673-8225.2011.07.010
实时荧光定量RT-PCR法建立版纳微型猪近交系中厚皮创面转化生长因子表达体系
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
摘要
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 DH5a 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.关键词
版纳微型猪近交系/实时荧光定量/转化生长因子/基因克隆/质粒标准品/基因表达分类
医药卫生引用本文复制引用
李颖,霍金龙,潘伟荣,曾养志,王继华..实时荧光定量RT-PCR法建立版纳微型猪近交系中厚皮创面转化生长因子表达体系[J].中国组织工程研究与临床康复,2011,15(7):1186-1190,5.基金项目
昆明医学院研究生创新基金"藻酸盐敷料对创面中VEGF、TGF-β1及bcl-2/bax因子表达的影响"(No.2009S030). （No.2009S030）
