维生素A及其核受体与睑板腺上皮角化的相关性及其机制研究

Chalazion is one of the most common eye disease in young children in China. It is well known that the blockage of meibomian gland ducts is the main cause leading to a chalazion. Previous studies showed that Chalazia were closely associated with hyperkeratosis of meibomian gland epithelia. However, the mechanism is unclear. Recent studies found that the hyperkeratosis of the epithelial cells had an association with Vitamin A (VA) deficiency. Our previous study found that the average serum vitamin A levels of children with chalazia were significantly lower than that of normal children. Further, we found that the meibomian gland epithelia of the mouse model with VA deficiency existed hyperkeratosis in our preliminary experiment. So we speculate that VA deficiency may lead to abnormal expression of VA orphan nuclear receptors in the meibomian gland and influence further the expression of the downstream genes involving in keratinization of meibomian gland epithelium. This process mentioned above may be a new mechanism leading to hyperkeratosis of meibomian gland epithelial. To vertify the hypothesis, we tend to research the meibomian gland of mouse models with VA deficiency, using immunohistochemistry, Western blot, and gene chip technology. We will observe the expression and distribution of different subtypes of VA orphan nuclear receptors in the meibomian gland, and detect the downstream genes related to keratinization. This projects aim to investigate the molecular mechanism involving in keratinization of meibomian gland epithelia and the possible signal pathway from a new perspective. Our study may offer a novel approach for prevention and treatment of chalazia in children.

霰粒肿是我国低龄儿童最常见的眼病之一。睑板腺导管开口阻塞是导致霰粒肿的主要原因，与睑板腺上皮过度角化关系密切，但具体机制尚不清楚。近期研究发现上皮细胞的过度角化与维生素A（Vitamin A, VA)缺乏相关。本课题组前期研究发现霰粒肿儿童血清维生素A水平明显低于正常儿童，并在预实验中发现VA缺乏小鼠模型的睑板腺上皮存在过度角化现象。故我们推测VA缺乏造成其核受体在睑板腺上皮中表达异常进而对下游某些维持上皮正常角化基因的表达产生影响。这一过程可能是睑板腺上皮发生过度角化的一个新机制。为证实这一假说，我们将对正常与VA缺乏小鼠模型睑板腺进行研究，采用免疫组化、Western blot、基因芯片等技术，观察不同类型的VA核受体亚型在睑板腺的表达及分布，并对下游与角化相关的基因进行筛选。本课题将从新的角度阐明睑板腺上皮角化的分子机制及可能存在的信号通路，为儿童霰粒肿的防治提供新靶点。

霰粒肿是我国低龄儿童最常见的眼病之一。睑板腺导管开口阻塞是导致霰粒肿的主要原因，与睑板腺上皮过度角化关系密切，但具体机制尚不清楚。近期研究发现上皮细胞的过度角化与边缘性维生素A（Vitamin A, VA)缺乏相关。本课题组前期研究发现霰粒肿儿童血清维生素A水平明显低于正常儿童，并在前期实验中发现VA缺乏小鼠模型的睑板腺上皮存在过度角化现象。故我们推测VA缺乏造成其核受体在睑板腺上皮中表达异常进而对下游某些维持上皮正常角化基因的表达产生影响。.为证实这一假说，我们在成功建立边缘性维生素A缺乏（marginal Vitamin A deficiency, MVAD）幼鼠动物模型基础上，对正常幼鼠及MVAD幼鼠模型睑板腺进行研究，为明确睑板腺上VA的靶受体，首先采用PCR检测其RAR（α、β、γ）、RXR（α、β、γ）各亚型受体mRNA的表达，同时采用免疫荧光及Western blot观察各亚型受体蛋白的定位及表达水平。结果发现，在正常幼鼠睑板腺中，RXRα的mRNA及蛋白表达水平是各亚型受体中最高的，且在维生素A缺乏时，其mRNA及蛋白表达水平均明显降低。.采用免疫荧光双标con-focal技术检测了VA核受体上游结合蛋白（ CRABP2 、 FABP5）蛋白表达水平，结果发现在MVAD幼鼠睑板腺中两组上游蛋白表达量均明显增加，且CRABP2/FABP5的相对表达量明显增高。Illumina高通量RNA测序技术对与角化相关的基因进行筛选，筛选目标基因：KRT1,KRT7, KRT8, KRT10, KRT16, KRT17并用qRT-PCR验证，结果发现：除KRT17明显上调外，其余均为下调基因。故与影响睑板腺上皮角化的靶基因可能为KRT17。之后我们对MVAD幼鼠补充维生素A至正常血清水平后与MVAD幼鼠进行对照研究。在给予VA补充后，免疫荧光双标con-focal技术检测的CRABP2/FABP5的相对表达量明显下降；RNA测序技术及PCR验证发现，基因KRT17表达水平明显下降。.综上课题结果，发现VA缺乏引起的CRABP2/FABP5的相对表达量的提高，从而引起KRT17上调这一途径可能VA缺乏导致睑板腺上皮角化的信号通路。本研究结果为使用维生素A治疗儿童霰粒肿提供理论依据。