Analysis of the specific function of EGF family in epidermal keratinocytes using siRNA transgenic mouse

本研究的目的是阐明每个因子在表皮中的特定功能，利用siRNA（小干扰RNA）转基因小鼠，该小鼠缺陷四种EGF家族生长因子（tgf - α, HB-EGF, amphiregulin, epiregulin）在表皮中发挥最重要的作用。利用siRNA选择软件确定了几种候选siRNA排列（19个序列），通过体外转录合成siRNA，并使用siRNA构建试剂盒在柱上提纯。下一步将候选siRNA转染到角质细胞，回顾mRNA的表现，确定最佳的siRNA排列方式。通过实时荧光定量PCR、RNase保护实验，回顾了各mRNA在EGF刺激中的表现。我们尝试了每一只siRNA转基因小鼠，但是在研究阶段我们没有成功。因此，我们利用角化细胞特异性敲除HB-EGF小鼠，分析了HB-EGF在皮肤伤口愈合过程中的作用。在体外创面愈合模型中加入HB-EGF中和抗体可抑制角质形成细胞的迁移，而添加HB-EGF可促进创面愈合。此外，刮痧法在EGF家族中可立即显著诱导HB-EGF mRNA表达。最后，我们使用角化细胞特异性HB-EGF敲除小鼠进行伤口愈合试验。HB-EGF敲除小鼠伤口愈合延迟。我们认为HB-EGF在皮肤创面愈合中起重要作用。

The purpose of this study is to clarify a specific function of each factor in epidermis, using a siRNA(small interfering RNA) transgenic mouse which defects four kinds of EGF family growth factor (TGF-alpha, HB-EGF, amphiregulin, epiregulin) having the most important work in the epidermis. Several kinds determined a candidate siRNA arrangement (19 sequence) using software for siRNA choice and we composed siRNA by in vitro transcription and, using siRNA construction kit, refined it in a column. Transfection did candidate siRNA to keratinocyte to be next and reviewed mRNA manifestation and determined a best siRNA arrangement. We reviewed manifestation of each mRNA in EGF stimulation by real time PCR, RNase protection assay. We tried to generate each siRNA transgenic mice, however we could not get in the research periods. Therefore, using keratinocyte specific knockout mouse of HB-EGF, we analyzed a function of HB-EGF in a skin healing of wound process. Migration of keratinocyte was inhibited when we used neutralizing antibody of HB-EGF with the in vitro wound healing model whereas HB-EGF addition promoted wound healing. In addition, the mRNA of HB-EGF was induced immediately and conspicuously among EGF families by scraping. Finally we performed wound healing assay using keratinocyte-specific HB-EGF knockout mice. A delay of healing of wound was accepted in HB-EGF knockout mouse. We conclude that HB-EGF plays an important role in skin wound healing.