【作者】 付玉龙；

【导师】 吴冠青； 詹启敏； 林晨；

【作者基本信息】 中国协和医科大学 ， 遗传学， 2009， 博士

【摘要】 多囊肾疾病(polycystic kidney disease, PKD)是一组常见的以双肾形成多个进行性增大囊肿为主要特征的单基因遗传病。根据遗传特征不同,可以分为常染色体显性多囊肾(Autosomal dominant polycystic kidney disease, ADPKD)和常染色体隐性多囊肾(Autosomal recessive polycystic kidney disease, ARPKD)。ADPKD主要由PKD1和PKD2两个基因突变引起,基因产物分别为Polycystin-1 (PC1)和Polycystin-2 (PC2); ARPKD则由PKHD1基因突变引起,基因产物为Fibrocystin/Polyductin (FPO)。PKD1、PKD2和PKHD1的小鼠同源基因PMd1、Pkd2和Pkhd1都已被克隆,两者之间具有高度的保守性,这些基因的突变也能导致小鼠多囊肾。本研究首先建立了一个新型的Pkhd1基因敲除小鼠模型,该模型能很好的模拟人类ARPKD。结合实验室前期已经建立的Pkd2基因敲除小鼠,发现Pkhd1和Pkd2的双突变小鼠比单个基因突变表现出更为严重的症状,提示ARPKD和ADPKD这两类具有不同遗传特征的疾病可能具有分子水平的相互关系。进一步研究发现,FPC和PC2在体内能形成一个分子复合体,这一复合体的形成是通过FPC的胞内羧基末端和PC2胞内氨基末端直接相互作用实现的。FPC的缺失能引起PC2蛋白表达的下降,并能显著降低PC2的离子通道活性,然而PC2的缺失并不影响FPC的表达。这一研究表明,FPC和PC2在体内形成一个分子复合物,并于同一分子通路参与肾内管道结构的形成。除了PKD1, PKD2和PKHD1,目前已在人和其他动物体内克隆了至少20个多囊肾相关基因。Bicc1,果蝇双尾C (Bic-C)基因的同源基因,是一个小鼠多囊肾致病基因。Bpk (BALB/c polycystic kidneys)和Jcpk (Juvenile congenital polycystic kidney),这两个分别通过自发突变和人工诱变产生的多囊肾动物模型,都由该基因突变引起。Biccl普遍存在于从线虫到人类的各个物种,并且其序列高度保守。在果蝇等低等动物的研究发现,Bic-C是一个具有RNA结合活性的转录后基因表达调控因子。尽管Biccl的多囊肾动物模型为其体内功能研究提供了一些线索,然而Biccl的细胞生物学功能还完全未知。本研究通过建立IMCD细胞的稳定干涉细胞株,发现Biccl为IMCD细胞在三维培养条件下形成管状分支结构所必须,抑制Biccl的表达导致以E-cadherin为基础的细胞连接异常以及纤毛发生障碍,同时细胞的增殖、凋亡和细胞骨架也出现异常。这一研究初步揭示了Biccl的细胞生物学功能,为进一步探寻该基因在体内的功能及多囊肾的发病机制奠定了基础。更多还原

【Abstract】 Polycystic kidney disease (PKD) is a group of monogenic disorders that result in renal cyst development. Based on different patterns of inheritance, there are two kinds of PKD, Autosomal dominant PKD(ADPKD) and autosomal recessive PKD(ARPKD). Approximately 85% of ADPKD patients have mutations in the PKD1 gene, which encodes polycystin-1 (PC-1). About 15% of ADPKD cases have mutations in the PKD2 gene, which encodes polycystin-2 (PC-2). Fibrocystin or polyductin(FPC), the poduct of PKHD1 gene, is the only identified ARPKD disease gene. Pkdl, Pkd2 and Pkhdl is the mouse homolog of PKD1, PKD2 and PKHD1 respectively, whose mutation also cause mouse polycystic kidney disease.In the current study, we generate a new mouse model by disrupting Pkhdl, which perfectly recapitulates the human ARPKD phenotype. Transmutant mice for Pkhd1 and Pkd2 displayed a significantly more severe renal cystic phenotype than single mutant mice, indicating a molecular relationship between ADPKD and ARPKD. More detailed research found that FPC physically interacts with PC2 and that lack of FPC destabilizes PC2 expression in vivo but not vice versa, suggesting that PC2 functions immediately downstream of FPC. In addition, inhibition of FPC expression reduces Pkd2-channel activity, we conclude that a functional and molecular interaction exists between FPC and PC2 in vivo.Except PKD1/2 and PKHD1, more than 20 genes have currently been identified to be able to induce PKD phenotypes in human or various animal mutant models. Biccl is a mouse homologue of Drosophila Bicaudctl-C (dBic-C) and the ortholog of dBic-C in many other species (from C.elegans to humans) can be found to be very conserved. In Drosophila, the gene product of dBic-C is currently considered as an RNA-binding molecule and believed to function in regualtion of mRNA post-transcription. Although the mutant mouse models provide some knowledge on functional roles of Biccl in mammalian development, its cellular functions still remain mostly unknown. To this end, we established stable Biccl-silenced MCD cell lines and characterized its functions in cell biology. Our results indicate that the expression of normal Biccl is required to sustain renal tubulogenesis in 3-D cultures of IMCD cells. Lack of this protein resulted in obvious abnormalities in E-cadherin-based cell-cell contact, which is essential for epithelial polarization and regulation of tubulomorphagenesis. These in vitro findings indicate that Biccl functional roles in renal epithelial differentiation and renal tubule development. The observations made in vitro may illuminate Biccl cellular functions in vivo.更多还原