中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (7): 1186-1190.doi: 10.3969/j.issn.1673-8225.2011.07.010

• 组织构建与生物活性因子 tissue construction and bioactive factors • 上一篇    下一篇

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

李  颖1,霍金龙2,潘伟荣2,曾养志2,王继华1   

  1. 1昆明医学院第二附属医院整形外科,云南省昆明市 650101
    2云南农业大学云南省版纳微型猪近交系重点实验室,云南省昆明市650201
  • 收稿日期:2010-09-29 修回日期:2010-11-07 出版日期:2011-02-12 发布日期:2011-02-12
  • 通讯作者: 王继华,博士,教授,昆明医学院第二附属医院整形外科,云南省昆明市 650101 wangjihua1966@163.com
  • 作者简介:李颖★,女,1984年生,新疆维吾尔自治区塔城市人,硕士,主要从事创面愈合及瘢痕防治方面的研究。ly4617@163. com 并列第一作者:霍金龙,男,1975年生,山西省五寨县人,博士,实验师,主要从事动物分子遗传学方面的研究。 jinlonghuo@ ynau.edu.cn
  • 基金资助:

    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)。

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*

摘要:

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。

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

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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