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siRNA（小干扰RNA）转基因小鼠来阐明表皮中每种因子的特定功能，所述siRNA转基因小鼠缺失在表皮中具有最重要作用的四种EGF家族生长因子（TGF-α、HB-EGF、双调蛋白、表皮调节蛋白）。几种利用siRNA选择软件确定候选siRNA排列（19个序列），并通过体外转录合成siRNA，使用siRNA构建试剂盒，在柱中对其进行纯化。将候选siRNA转染到角质形成细胞中，观察mRNA表达情况，确定最佳siRNA排列。本文通过真实的时间PCR、RNase保护实验等方法对EGF刺激后各mRNA的表达进行了综述。我们尝试产生每种siRNA转基因小鼠，但在研究期间我们无法获得。因此，使用角质形成细胞特异性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功能。在体外伤口愈合模型中，应用HB-EGF中和抗体可抑制角质形成细胞的迁移，而加入HB-EGF则可促进伤口愈合。此外，HB-EGF mRNA的诱导立即和明显的EGF家族刮。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。在HB-EGF基因敲除小鼠中，伤口愈合延迟是可以接受的。我们认为HB-EGF在皮肤创伤愈合中起重要作用。

项目成果

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

TGF-beta is not involved in early phase growth inhibition of keratinocytes by 1alpha, 25(OH)(2)vitamin D(3).

TGF-β 不参与 1α、25(OH)(2) 维生素 D(3) 对角质形成细胞的早期生长抑制。

• 发表时间: 2004
• 期刊: J Dermatol Sci. 36
• 作者: Shirakata;Ueno H;Hanakawa Y;Kameda K;Yamasaki K;Tokumaru S;Yahata Y;Tohyama M;Sayama K;Hashimoto K
• 通讯作者: Hashimoto K

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》。

Keratinocyte G2/M growth arrest by 1,25-dihydroxyvitamin D3 is caused by Cdc2 phosphorylation through Wee1 and Myt1 regulation

• DOI: 10.1111/j.0022-202x.2004.22522.x
• 发表时间: 2004-06-01
• 期刊: JOURNAL OF INVESTIGATIVE DERMATOLOGY
• 影响因子: 6.5
• 作者: Dai, X;Yamasaki, K;Hashimoto, K
• 通讯作者: Hashimoto, K

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