骨关节病关节软骨细胞线粒体DNA突变检测

Detection of Mitochondrial DNA Mutations in Articular Chondrocytes of Osteoarthrosis

目的 :探讨线粒体DNA突变在退行性疾病骨关节病发病中的意义。方法 :采用PCR -SSCP技术 ,结合线粒体DNA测序方法 ,检测了 8例骨关节病和 3例正常人的关节软骨细胞线粒体DNA在一个特点区域 (np:80 0 0— 90 0 0 )内的突变改变。结果 :在 8例骨关节病例中 ,有 4例出现了线粒体DNA突变 (不包含中性突变 ) ,其中单有点突变并导致氨基酸发生改变的有 2例 ,单有缺失突变的有 1例 ,既有点突变又有缺失突变的有 1例 ;此外 ,发现中性突变 (不改变氨基酸 )的有 4例 ;1例正常标本发现有线粒体DNA点突变存在 ;所有进行了DNA测序检测的标本均发现线粒体DNA886 0位点AG的突变。结论 :骨关节病与线粒体DNA突变有密切关系 ,但其因果关系尚待进一步研究。

Objective Studies of diseases caused by mitochondrial DNA(mtDNA) mutations suggest that a variety of degenerative processes may be associated with defects in OXPHOS(oxidative phosphorylation). Osteoarthrosis is an age-related degenerative di- sease. The energy supply of mitochondria is essential in arti cular motion. The objective of this study is to probe mtDNA changes of articular chondrocytes in osteoarthrosis to further clarify its pathogenetic mechanism. Methods Test group included 8 specimens of articular cartilage taken from osteoarthrosis patients diagnosed by clinical manifestations, radiology, operative finding and hematoxylin and eosin histology. Control group included 3 specimens of normal articular cartilage taken from healthy adults subject to amputation because of traffic accident. Total DNA(including nuclear and mtDNA) was prepared using standard proteinase K/SDS lysis followed by phenol/chloroform extraction of DNA. Four pairs of primers were designed to assay mtDNA mutations and the range of detection placed at nucleotide positions:7978-9014.PCR amplification gave four different fragments of 144.306.347 and 369 bp.PCR pro- ducts were detected by 10% polyacrylamide gel electrophoresis(PAGE) and silver staining.SSCP was performed using precasted 6 4% polyacrylamide gels on a electrophoresis apparatus.The osteoarthrosis patients were screened for mtDNA variants by PCR-SSCP.The positions of the mtDNA mutations were detected by DNA sequencing. Results In 8 test specimens,4 revealed mtDNA mutations. Among them, 2 specimens revealed point mutations only, one specimen revealed just deletion and the other one revealed both point mutations and deletion. In addition, four specimens revealed neutral mutations(no changes of amino acid). All specimens detected by mtDNA sequencing revealed the mutation A/G at np:8860.Conclusion Osteoarthrosis is related to mtDNA mutations, but it is not clear about their causal relationship.