中文摘要：

本实验旨在研究异染色质蛋白1（heterochromatin protein 1,HP1）在Zmpste24基因敲除早老小鼠胚胎成纤维细胞（mouse embryonic fibroblasts,MEFs）中的表达量和磷酸化水平,探索异染色质功能异常与早老发病机制的内在联系.首先取雄雌Zmpste24杂合子小鼠胚胎,原代培养MEFs;分别用PCR和Western印迹检测MEFs基因型和A型核纤层蛋白（laminA）表达以区分野生型与Zmpste24-缺陷型早老细胞;用与衰老相关的β-半乳糖苷酶染色法（senescence associated-β-galactosidase assay,SA-β-gal）确定早老细胞出现衰老表型的传代数.用Western印迹和phos-tagWestern印迹分别检测HP1在Zmpste24＋/＋和Zmpste24-/-MEFs中表达量和磷酸化水平的差异.实验结果显示,Zmpste24-/-MEFs中存在异常的LaminA,且在传代培养第5代出现明显的细胞衰老现象.选用培养至第3代或第4代的MEFs细胞进行下述实验发现,Zmpste24-/-MEFs中HP1α表达量明显高于Zmpste24＋/＋MEFs,而HP1β未发现明显升高;Zmpste24＋/＋MEFs中HP1α以非磷酸化状态为主,但在Zmpste24-/-MEFs中磷酸化HP1α比例明显升高;2种细胞中均未检测到磷酸化HP1β.本研究结果证明,HP1α在Zmpste24-缺陷型早老小鼠传代早期MEFs中的表达量和磷酸化水平均有升高,提示HP1α参与A型核纤层蛋白相关的早老小鼠发病机制.

英文摘要：

With the attempt to elucidate the mechanism by which heterochromatin functional abnormality may be involved in the premature aging,this study focused on the expression and phosphorylation of heterochromatin protein 1（HP1） in mouse embryonic fibroblasts（MEFs） deficient of Zmpste24,an enzyme known to process the prelamin A protein.MEFs were isolated from E13.5d embryos generated by mating Zmpste24＋/-male and female mice,Zmpste24＋/＋ and Zmpste24-/-MEFs were selected based on PCR genotyping and lamin A/C expression.Cultured MEFs were stained for senescence associated β-galactosidase activity to determine the passage when premature senescence occurs.The HP1 expression and phosphorylation were examined by Western blotting with and without a phos-tag,respectively.It was showed that Zmpste24-/-MEFs expressed abnormal lamin A and had distinctive features of premature senescence in passage 5 of ex vivo primary culture condition.Compared to Zmpste24＋/＋ MEFs,Zmpste24-/-MEFs expressed higher level of HP1α as early as passage 3 and 4,while the expression of HP1β was not notably changed.Furthermore,the ratio of phosphorylated HP1α was significantly increased in Zmpste24-/-MEFs whereas the unphosphorylated HP1α was found to be the main form in Zmpste24＋/＋ MEFs.No phosphorylated HP1β was detected in either Zmpste24＋/＋ or Zmpste24-/-MEFs.These results showed that the total amount and phosphorylated form of HP1α protein were up-regulated when Zmpste24 was deficient,suggesting that HP1α might be involved in the pathogenesis of the lamin A-associated premature aging.