Cx26基因D50N和V27I双杂合突变导致KID综合征的分子机制研究

Keratitis–ichthyosis–deafness (KID) syndrome is a rare ectodermal disease. Mutations in the Cx26 gene encoding gap junction protein are the basis etiology of the disease. Single heterozygous mutation of Cx26 results in increased connexin hemichannels activity on the cell membrane. Recently we have found KID patients carrying compound heterozygous mutations: D50N and V27I. How these mutations affect the function of connexin channels and hemichannels ? And how abnormal hemichannel activity leads to pathological changes in the epidermis ? This study will make a deep understanding on the mechanisms whereby mutated Cx26 gene(D50N +V27I )affects connexin channels and hemichannels using the polar dye nuclear technology, patch clamp technique and dye dispersion test technology. Meanwhile, the effect which abnormal hemichannel activity on the cell proliferation and differentiation of skin keratinocytes will be learned using tissue engineering technology. After that, siRNA sequence will be designed to intervene mutated Cx26 gene. This study is to provide theoretical basis for elucidating the molecular.mechanism of KID syndrome pathogenesis and provide technical and theoretical support for future attempts to develop gene therapy of KID syndrome.

角膜炎- 鱼鳞病- 耳聋综合征(KID 综合征)是一种罕见的外胚层发育缺陷性疾病，缝隙连接蛋白中的Cx26基因突变是该病的发病基础。单点Cx26杂合突会导致细胞膜上的缝隙连接蛋白半通道活性增加。最近我们发现的KID综合征患者Cx26基因为D50N和V27I双杂合突变。双杂合突变是如何影响缝隙连接蛋白通道和半通道功能呢？缝隙连接蛋白半通道活性异常又是如何影响正常的表皮角化的呢？本研究将运用极性染料染核技术、膜片钳技术、染料弥散试验等技术深入探讨Cx26基因D50N + V27I双杂合突变对缝隙连接通道和半通道功能的影响，以及运用组织工程技术研究缝隙连接蛋白半通道功能异常对皮肤角质形成细胞增殖和分化的影响，并设计有效的siRNA序列进行干预。旨在为阐明KID综合征的分子发病机制提供理论依据，为未来尝试开展KID综合征的基因治疗提供技术和理论支撑。

角膜炎-鱼鳞病-耳聋综合征(KID综合征)是一种罕见的外胚层发育缺陷性疾病，缝隙连接蛋白中的Cx26基因突变是该病的发病基础。我们成功构建包含野生型Cx26基因、突变型Cx26V27I、Cx26D50N和双杂合突变的基因表达载体。获得了表达Cx26蛋白并于细胞膜上形成半通道的转染细胞模型。发现了Cx26-D50N基因突变导致其半通道蛋白通透性的增加，并受细胞外钙离子浓度的调节。发现Cx26V27I基因突变形成的半通道蛋白的通透性与野生型半通道蛋白的通透性差异无统计学意义。之后通过检测CX26不同位点突变对Hela细胞内钙含量的影响，发现D50N+V27I双位点突变和D50N细胞内钙含量无显著差异，说明D50N突变为主要致病位点。