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

背景:版纳微型猪近交系能较好的模拟人取皮区创面,构建动物模型,检测与创面愈合及瘢痕形成密切相关的转化生长因子的表达。目的:观察创面愈合过程中转化生长因子β1基因的表达情况。方法:利用版纳微型猪近交系4～6月龄猪构建了皮肤创面愈合动物模型,通过提取皮肤创面总RNA,设计特异引物,对与创面愈合相关密切的转化生长因子β1基因进行了RT-PCR扩增。纯化目的片段并与pMD18-T载体连接,转化宿主菌DH5α,提取重组质粒DNA,并经酶切、PCR和测序鉴定,计算重组质粒原液拷贝数浓度并制备梯度浓度标准品,进行实时荧光定量PCR。结果与结论:建立的转化生长因子β1基因mRNA表达实时荧光定量PCR检测方法特异性较好,检测灵敏度可达103拷贝/μL,线性范围达103～109拷贝/μL,阈值循环数与PCR体系中起始模板量的对数值之间存在良好的线性关系(R2=0.988),扩增效率高(E=107.433%)。利用该检测体系检测了45份样品,效果良好,该方法可为研究TGF-β1基因在创面愈合过程中的作用机理奠定分子生物学基础。

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

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.