Construction of keratin disease keratinocyte model

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。然而，在这些角蛋白疾病的病理生理学研究中，仍有许多问题尚未解决。例如，我们还不知道为什么角蛋白基因突变后会导致表皮棘皮病。一些角蛋白疾病的过度角化的合理原因还没有得到证实。为了获得解决这些悬而未决的问题的线索，我们尝试制作角蛋白病角质形成细胞模型。然后，将突变的角蛋白14亚克隆到含有热休克启动子和蜕皮激素/糖皮质激素反应元件的载体pIND/V5-→中。当pIND/V5-His K14(Arg-125→半胱氨酸)和pVgRXR双转染后，细胞内出现角蛋白聚集体。抗泛素抗体染色显示，泛素蛋白聚集体被泛素化，提示泛素蛋白酶体途径可能在角蛋白疾病的病理生理过程中起重要作用。

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.