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

siRNA转基因小鼠分析EGF家族在表皮角质形成细胞中的特异性功能

• 批准号: 15390343
• 负责人: SAYAMA Koji
• 金额: $ 9.6万
• 依托单位: Ehime University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15390343/
• 关键词: siRNA; HB-EGF; TGF-alpha; amphiregulin; epiregulin; knockout mouse; keratinocyte; cre recombinase; knockout mouse; cre recombinase; TGF-α

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.

本研究的目的是使用具有四种EGF家族生长因子（TGF-α、HB-EGF、双调蛋白、上皮调节蛋白）缺陷的siRNA（小干扰RNA）转基因小鼠，阐明表皮中各因子的特定功能。表皮中最重要的工作。使用siRNA选择软件确定了几种候选siRNA排列（19个序列），我们通过体外转录合成了siRNA，并使用siRNA构建试剂盒将其在柱中精制。将候选 siRNA 转染至下一个角质形成细胞，并检查 mRNA 表现并确定最佳 siRNA 排列。我们通过实时PCR、RNase 保护测定回顾了每种mRNA 在EGF 刺激中的表现。我们试图产生每种siRNA转基因小鼠，但我们无法进入研究阶段。因此，使用HB-EGF的角质形成细胞特异性敲除小鼠，我们分析了HB-EGF在皮肤伤口愈合过程中的功能。当我们在体外伤口愈合模型中使用HB-EGF中和抗体时，角质形成细胞的迁移受到抑制，而添加HB-EGF则促进伤口愈合。此外，通过刮擦，HB-EGF的mRNA在EGF家族中被立即且显着地诱导。最后，我们使用角质形成细胞特异性 HB-EGF 敲除小鼠进行伤口愈合测定。 HB-EGF 敲除小鼠的伤口愈合延迟被接受。我们得出结论，HB-EGF 在皮肤伤口愈合中发挥着重要作用。

项目成果

All-trans-retinoic acid induces interleukin-8 via the nuclear factor-kappaB and p38 mitogen-activated protein kinase pathways in normal human keratinocytes.

• 发表时间: 2004
• 期刊: The Journal of investigative dermatology
• 作者: X. Dai;K. Yamasaki;Y. Shirakata;K. Sayama;K. Hashimoto

Yamasaki K, Hanakawa Y, Tokumaru S, Shirakata Y, Sayama K, et al.: "SOCS1/JAB and SOCS3/CIS3 negatively regulate the STATs signaling pathway in normal human epidermal keratinocytes"The Journal of Investigative Dermatology. 120. 571-580 (2003)

Yamasaki K、Hanakawa Y、Tokumaru S、Shirakata Y、Sayama K 等人：“SOCS1/JAB 和 SOCS3/CIS3 负向调节正常人表皮角质形成细胞中的 STATs 信号通路”《皮肤病学研究杂志》。

Yamasaki K, Toriu N, Hanakawa Y, Shirakata Y, Sayama K, et al.: "Keratinocyte growth inhibition by high-dose epidermal growth factor is mediated by transforming growth factor β autoinduction : A negative feedback mechanism for keratinocyte growth"The Jour

Yamasaki K、Toriu N、Hanakawa Y、Shirakata Y、Sayama K 等人：“高剂量表皮生长因子对角质形成细胞生长的抑制是通过转化生长因子 β 自诱导介导的：角质形成细胞生长的负反馈机制”The Jour

Cre/loxP mediated gene transfer into living skin equivalent model

Cre/loxP 介导的基因转移到活体皮肤等效模型中

• 期刊: Br.J Dermatol (In press)
• 作者: Hanakawa Y;Shirakata Y;Sayama K;et al.
• 通讯作者: et al.

Tsuda T, Thoyama M, Yamasaki K, Shirakata Y, Yahata Y, Sayama K et al.: "Lack of evidence for TARC/CCL17 production by normal human keratinocytes in vitro"The Journal of Dermatological Science. 31. 37-42 (2003)

Tsuda T、Thoyama M、Yamasaki K、Shirakata Y、Yahata Y、Sayama K 等人：“缺乏正常人角质形成细胞在体外产生 TARC/CCL17 的证据”皮肤病学杂志。