Construction of keratin disease keratinocyte model

角蛋白病角质形成细胞模型的构建

• 批准号: 12670805
• 负责人: YONEDA Kozo
• 金额: $ 2.05万
• 依托单位: AKITA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670805/
• 关键词: Keratin; Filaggrin; Keratin Disease; Ubiqutin; 角化細胞; ユビチキン; 26Sプロテアソーム

Recently, mutations in keratin genes have been identified in many hereditary skin diseases. These skin disorders are now termed as keratin diseases. However, there are lots of unsolved problems in the pathophysiology of these keratin diseases. For example, we do not know why acanthosis of epidermis is induced following the keratin gene mutation yet. The reasonable rationale to the hyperkeratosis of some keratin disorders has not been obatined. In order to acquire the clue to solve these unanswered questions, we try to make keratin disease keratincyte model. At first, we introduced the mutation into keratin 14 cDNA (Arg-125→Cys) by PCR.Next, this mutated keratin 14 is subcloned to pIND/V5-His vector which has heat shock promoter and ecdysone/glucocorticod response element. When double transfection of pIND/V5-His K14 (Arg-125→Cys) and pVgRXR was conducted, keratin aggregates were observed in the transfected cells. When transfected cells were stained with anti-ubiquitin antibody, the keratin aggregates were revealed to be ubiquitinated These results indicated that the ubiquitinproteasome pathway may play an important role in the pathophysiology of keratin diseases.

最近，在许多遗传性皮肤病中发现了角蛋白基因的突变，这些皮肤病现在被称为角蛋白疾病，但这些角蛋白疾病的病理生理学仍有许多未解决的问题，例如，我们不知道为什么会出现棘皮症。角蛋白基因突变导致表皮过度角化的原因尚不清楚。为了解决这些悬而未决的问题，我们尝试制作角蛋白疾病角蛋白细胞模型。首先，我们通过PCR将突变引入角蛋白14 cDNA（Arg-125→Cys）。接下来，将突变的角蛋白14亚克隆到pIND/V5-His载体中。双转染时具有热休克促进剂和蜕皮激素/糖皮质激素反应元件。进行pIND/V5-His K14 (Arg-125→Cys)和pVgRXR，在转染的细胞中观察到角蛋白聚集体，当转染的细胞用抗泛素抗体染色时，显示角蛋白聚集体被泛素化。泛素蛋白酶体途径可能在角蛋白疾病的病理生理学中发挥重要作用。

项目成果

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