【作者】 胡建新；

【导师】 贺林；

【作者基本信息】 上海交通大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 A1型短指/趾症是人类群体中发现的第一例符合孟德尔遗传规律的常染色体遗传病,患者特征为手/足第二指节的指骨严重缩短或者缺失。近年来,在A1型短指/趾症家系患者中陆续鉴定到了Indian hedgehog (IHH)基因上的多个点突变。目前,人们对于A1型短指/趾症的致病机理仍不清楚。除此之外,人们对Ihh在指骨发育早期所执行的功能的了解也非常有限。为了揭示A1型短指/趾症患者指骨发育异常的原因,并理解E95K突变所导致的分子水平和发育水平变化,我们利用实验室构建的BDA1小鼠模型,从发育生物学和分子生物学角度对A1型短指/趾症致病机理进行了研究。BDA1模型小鼠在Ihh基因上携带等同人类E95K的突变。E95K纯合突变小鼠表现出个体矮小和典型的A1型短指/趾症表型——第二至第四指中指节严重缩短,第五指中指节缺失。同时,和人类患者中一样,E95K突变以显性方式作用于小鼠的指骨表型,使得携带E95K突变的杂合子小鼠也表现出轻微的短指表型。我们利用体外和体内的实验手段研究了E95K突变的分子基础。结果显示,E95K-Ihh的信号能力有一定程度的下降。更为有趣的是,通过体内实验我们发现在突变小鼠的指骨前体和侧肢长骨中,E95K-Ihh的信号作用距离显著增加。在长骨发育中,E95K-Ihh作用距离的改变扰乱了Ihh-PthrP负反馈环,从而影响了软骨细胞的增殖与分化;在指骨前体中,E95K-Ihh作用距离的增加使得更多的Ihh信号进入到中间区,并刺激了PthrP——一种指骨前体中Ihh表达的潜在调控因子——在中间区的表达。进一步的研究表明,E95K突变导致BDA1小鼠指骨前体远端的Ihh信号水平下降,并导致指放线远端间叶细胞吸纳的减缓,从而阻碍了指骨前体向远端的生长。这种生长减缓导致指骨前体尺寸减小,从而使得突变小鼠第二指节和第三指节分节异常。另外,我们观察到BDA1小鼠指骨关节中间区发育的延迟和异常。Ihh基因敲除小鼠的指骨表型表明,在Ihh功能缺失的情况下,指骨远端生长严重受损,中间区发育失败。我们的结果说明Ihh信号在指骨发育早期调控指放线的远端生长,并可能参与指骨关节中间区的早期发育调控。更多还原

【Abstract】 Brachydactyly type A1 (BDA1; MIM 112500), characterized by shortening or absence of the middle phalanges in digits, is the first recorded disorder of the autosomal dominant Mendelian trait in human. Recently, heterozygous missense mutations in the Indian hedgehog gene (IHH) were reported for BDA1. However, the underlying pathogenesis of BDA1 and the role of Ihh during early digit morphogenesis were remained unclear. As it is a developmental abnormality, we carefully studied a mouse model carrying a human equivalent E95K missense mutation to address the molecular and developmental consequences. Homozygous mice for the E95K allele displayed mild dwarfism and typical characteristics of BDA1 with severe shortening (digit II-IV) or missing (digit V) of the middle phalanges. Moreover, like in human cases, this mutation exerts a dominant effect in mice, causing mild brachydactyly type A1 phenotype in Ihh+/E95K mice. We studied the molecular basis of the E95K mutation in vitro and in vivo. Our results suggest that there is a slight reduction in the signaling capacity of the E95K Ihh protein, but the effective signaling range in the cartilage element of the developing digits and long bones is broader. In long bones, the alteration in the signaling range of Ihh signal disturbs the Ihh-PthrP negative feedback loop and impairs chondrocyte proliferation and differentiation. In digits, this increased Ihh signaling range allows additional signals extend into the interzone and enhance PthrP expression, a potential negative feedback regulator of Ihh expression. We further showed that distal Ihh signal is reduced in BDA1 mice, impairing mesenchymal recruitment and growth of the distal cartilage element, contributing to the abnormal segmentation between the middle and distal phalanges causing BDA1. In addition, inter-phalangeal interzone development is abnormal in BDA1 mice. In support of these observations, we show that the distal outgrowth and interzone development of digital primordia are almost completely abolished in the digital context correspond to Ihh signaling. Thus, our results suggest that IHH signaling is needed in regulating elongation of distal digit elements during early digit patterning.更多还原