囊肿相关差异表达蛋白对初级纤毛结构和蛋白分布的影响及机制研究

Autosomal dominant polycystic kidney disease （ADPKD） is the most common monogenic renal disease, and it is the forth cause of end-stage renal disease. Primary cilia is an organ of tubular epithelial cell of kidney . It is the common pathway of the pathogenesis of autosomal dominant polycystic kidney disease. The applicant of this project successfully founded Pkd1 gene knock-in mouse model　（PNAS,2007）. The former study results indicated that renal cysts are formed in the homozygote mice and several different expression proteins were found by the proteomics study. To further explore the role of the different expression protein on kidney cyst formation, the applicant plans to study the expression and distribution of the cyst-related different expression proteins on the epithelial cell and primary cilia using the cell model, mouse model and human kidney cyst lining epithelial cells by Western Blot, double immunofluorescence staining,electric microscope, etc. Screening 1-2 cyst-related different expression proteins form them, and observing the effect on the structure and protein distribution of primary cilia through upregulation and downregulation of the different expression protein. Also study the pathogenesis to the further. More information about the pathogenesis of PKD and therapy target may be got from this project.

常染色体显性多囊肾病是一种最常见的单基因遗传性肾病，占肾衰竭病因的第4位。初级纤毛是肾小管上皮细胞的一种细胞器，在多囊肾病的发病机制中起着共同通路的作用。本项目申请者成功建立了I型多囊肾病基因敲入小鼠模型（PNAS，2007），前期研究发现纯合子小鼠双侧肾脏囊肿形成，通过蛋白组学的研究获得了多个囊肿相关差异表达蛋白。为进一步深入研究差异表达蛋白在囊肿形成中的作用、寻找治疗靶标，本项目拟在细胞模型、小鼠模型和人多囊肾病肾脏组织标本上，运用免疫印迹、免疫荧光双染色、激光共聚焦、电子显微镜等方法研究前期获得的囊肿相关差异表达蛋白在细胞和初级纤毛上的表达、分布，从中筛选出1-2种囊肿相关差异表达蛋白，通过上调和下调差异蛋白的表达观察对初级纤毛的结构和蛋白分布的影响，并研究其发生机制，为明确囊肿相关差异蛋白在细胞和初级纤毛中的分布及表达差异，探索多囊肾病的发病机制、寻找治疗靶标奠定一定的理论基础。

常染色体显性多囊肾病是最常见的单基因遗传性肾病，由PKD1或PKD2基因突变引起编码的PC1和PC2表达异常所致，二种蛋白均表达于肾小管上皮细胞的初级纤毛。初级纤毛致病假说是目前最为关注的多囊肾病共同发病通路。前期课题组成功建立了多囊肾病PKD1基因敲入小鼠模型，通过蛋白组学的研究获得了多个囊肿相关差异表达蛋白，并通过观察发现野生型和纯合子小鼠肾小管上皮细胞的纤毛结构存在差异。本项目为进一步研究多囊肾病初级纤毛发病机制，选取其中一个囊肿差异蛋白cordon-bleu进行深入研究。通过western-blot和免疫荧光等方法发现，人多囊肾囊肿衬里上皮细胞及多囊肾病小鼠模型中均存在细胞骨架蛋白成核剂Cobl表达上调，以及初级纤毛结构和功能异常。并且，Cobl表达异常可影响F-actin的聚合作用，导致细胞骨架结构紊乱，从而抑制基体正常迁移至细胞膜顶端，最终导致初级纤毛结构与功能的改变。本课题的研究结果将丰富多囊肾病中初级纤毛的发病机制，为寻找干预靶标奠定一定的理论基础。