【作者】 王敏；

【导师】 廖二元； 周后德；

【作者基本信息】 中南大学 ， 内科学， 2006， 博士

【摘要】 第一部分 晚发型脊柱骨骺发育不良伴进行性骨关节病（SEDT-PA）致病基因WISP3突变体的构建 目的：Wnt诱导分泌蛋白3（Wnt-inducible secreted protein 3，WISP3）是晚发型脊柱骨骺发育不良伴进行性骨关节病（spondyloepiphyseal dysplasia tarda with progressive arthropathy，SEDT-PA）的致病基因，构建WISP3基因致病型（1000T-C，840delT）的突变体，为研究突变基因的表达、定位和功能及SEDT-PA的发病机制奠定基础。 方法：用RT-PCR方法从正常人软骨细胞获得野生型WISP3基因的全长cDNA（WT-WISP3），用定点突变方法获得我们已报道的两种WISP3基因突变类型的全长cDNA(MUT^1000T/C和MUT^840delT)；经T载体克隆，测序证实后，将它们再分别亚克隆至真核表达载体pcDNA3.1（+）和带绿色荧光蛋白靶标的真核表达载体pEGFP-C2中，酶切和测序对插入片段进行分析和鉴定。 结果：构建的野生型WISP3基因和突变体的全长cDNA序列分别与文献及SEDT-PA患者WISP3基因突变类型一致；测序和酶切鉴定证实所有的插入片段序列正确。 结论：运用基因重组技术成功构建了SEDT-PA致病基因WISP3突变体，为进一步探讨SEDT-PA的发病机制创造条件。更多还原

【Abstract】 Part One The Construction of Mutants of SEDT-PA’s Pathogenic GeneObjectives: Wnt-inducible secreted protein 3 （WISP3） was spondyloepiphyseal dysplasia tarda with progressive arthropathy （SEDT-PA）’s pathogenic gene.To construct two types of WISP3 gene’s mutants （1000T-C,840delT） found in SEDT-PA patients.Methods: Full-length cDNAs of wild type WISP3 gene（WT-WISP3） was amplified from human chondrocytes by RT-PCR, and site-directed mutagenesis was used to obtain full-length cDNAs of the mutated WISP3 genes (MUT^1000T/C and MUT^840delT) . The products were cloned into pEGM-T-easy vector and their sequences were analysed, then subcloned into eukaryotic expression vector pcDNA3.1 （+） and pEGFP-C2, the inserted fragments were analysed and reconfirmed by restriction endonuclease analysis and sequencing.Results: By restriction endonuclease analysis and sequencing, the sequences of WT-WISP3, MUT^1000T/C and MUT^840delT were consistent with that in literature and mutated in SEDT-PA, and the open reading frames were matched with the vector sequence.Conclusion: WISP3 gene’s mutants of SEDT-PA were successfully更多还原