中文摘要：

目的：通过构建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）致病型（1000T／C，840delT）Wnt诱导分泌蛋白3（Wnt-inducible secreted protein 3，WISP3）的真核表达载体，并将其表达于真核表达系统COS-7细胞系，为研究WISP3突变致SEDT-PA的机制奠定基础。方法：从正常人软骨细胞获得野生型WISP3基因的全长cDNA（WT-WISP3）；用定点突变方法构建携带WISP3基因致病型的重组真核表达质粒MUT^1000T/C／pcDNA3．1（＋）和MUT^840delT／pcDNA3．1（＋）；以空白载体pcDNA3．1（＋）为对照，脂质体转染法将重组质粒瞬时转染COS-7细胞，48h后提取转染细胞的总RNA和总蛋白；半定量RT-PCR和免疫印迹方法检测WISP3基因的表达。结果：经测序证实，构建的野生型和突变型WISP3基因的全长cDNA序列分别与文献及前期报道的SEDT-PA患者WISP3基因突变类型一致；酶切鉴定证实，成功构建了3个重组真核表达质粒[WT-WISP3／pcDNA3．1（＋），MUT^1000T/C／pcDNA3．1（＋）及MUT^840delT／pcDNA3．1（＋）]；半定量RT-PCR和免疫印迹示，重组质粒在COS-7细胞中均高效表达。结论：成功构建了SEDT-PA致病基因WISP3的突变体并在COS-7细胞中得到表达，为进一步探讨SEDT-PA的发病机制创造了条件。

英文摘要：

Objective To construct two types of Wnt-inducible secreted protein 3 （WISP3） gene＇s mutants（ 1000T/C, 840delT） found in spondyloepiphyseal dysplasia tarda with progressive anthopathy （SEDT-PA） patients, and to observe their expression in COS-7 cells. Methods Fulllength eDNA 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 recombined plasmids WT-WISP3/pcDNA3. 1 （ ＋ ）,MUT^1000T/C/pcDNA3. 1 （ ＋ ） and MUT^840delT/pcDNA3. 1 （ ＋ ） were transfected transiently into COS- 7 ceils by liposome-mediated method, and pcDNA3. 1 （ ＋ ） vector was used as a control. The total RNA and protein of the transfected COS-7 ceils were extracted after 48 hours of transfection. The expression of WISP3 gene in the transfected COS-7 ceils was detected by semi-quantitative RT-PCR and Western blot. Results By restriction endonuclease analysis and sequencing, the sequence of MUT^1000T/C and MUT^840delT were consistent with that mutated in SEDT-PA, and the open reading frames matched with the vector sequence. Semi-quantitative RT-PCR and Western blot showed that the recombined plasmids were highly expressed in COS-7 ceils. Conclusion WISP3 gene＇ s mutants of SEDT-PA are successfully constructed by genetic recombination, and expressed in COS-7 ceils, which lays the foundation for the further study on its molecular functions in SEDT-PA.