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

本研究的目的是利用siRNA(小干扰RNA)转基因小鼠，该转基因小鼠缺失了表皮中最重要的四种EGF家族生长因子(转化生长因子-α、HB-EGF、两相调节蛋白、表型调节蛋白)，以阐明每种因子在表皮中的特定功能。几种类型的siRNA利用siRNA选择软件确定了一个候选的siRNA排列(19个序列)，我们通过体外转录合成了siRNA，并使用siRNA构建试剂盒在柱子中进行了提纯。将siRNA转入角质形成细胞，观察其表达情况，确定最佳的siRNA排列方式。通过实时荧光定量聚合酶链式反应、核糖核酸酶保护实验，对EGF刺激过程中各基因的表达情况进行综述。我们试图培育出每一只siRNA转基因小鼠，但未能进入研究期。因此，利用角质形成细胞特异的HB-EGF基因敲除小鼠，我们分析了HB-EGF在皮肤创伤愈合过程中的作用。将HB-EGF中和抗体应用于体外创面愈合模型，可抑制角质形成细胞的迁移，而加入HB-EGF则促进创面愈合。此外，刮刮法可在EGF家系中即刻、明显地诱导HB-EGF的表达。最后，我们利用角质形成细胞特异性的HB-EGF基因敲除小鼠进行了伤口愈合实验。HB-EGF基因敲除小鼠可接受伤口延迟愈合。结论：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.