Abstract: Background DJ-1 gene is a causative gene which contributes to the onset of autosomal recessive early-onset parkinsonism (AREP). Many research suggest that DJ-1 protein may change expression of certain genes through regulate its transcriptional activity, which play a role in the pathogenesis of Parkinson's disease (PD). In our previous study, we found a new mutation of DJ-1 which we named as L10P. DJ-1 gene encodes the first frame 29 bp from the thymine (T)→cytosine (C), so that the leucine on the 10th locus of DJ-1 protein was replaced by proline (L10P). To elucidate the effect of the L10P mutation, we identify genes for which expressions are abnormally regulated by L10P mutant DJ-1 protein using DNA microarray analysis. Methods Human embryonic kidney cell 293 (HEK293) monoclonal cell strains which can stably express pCMV-Tag2A-Flag, pCMV-Tag2A-Flag-DJ-1 and pCMV-Tag2A-Flag-DJ-1-L10P were selected by screening, and identified on the basis of DNA, RNA and protein levels to confirm whether the acquired HEK293 monoclonal cell strains can stably express empty vector, wild-type DJ-1 protein and L10P mutant DJ-1 protein. Gene chip technique was used to perform differential gene screening for different groups of HEK293 monoclonal cell strains. Results Compared with the expression in empty vector group, the expression of 14 genes was up-regulated and 28 genes was down-regulated in wild-type group; and the expression of 14 genes was up-regulated and 9 down-regulated in expressing L10P mutant group respectively. Comparison of the expression in wild-type group, expression of 59 genes was up-regulated and 27 genes down-regulated in L10P mutant group. These differential genes all took part in the biological processes including signal transduction, transcriptional regulation, cell cycle, apoptosis, oxidative stress and so on. Conclusion L10P mutant DJ-1 protein may directly or indirectly influence the singal transduction and play a role in the mechanism of PD.

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摘要： 研究背景 DJ-1 基因是常染色体隐性遗传性早发性帕金森综合征（AREP）的致病基因之一，其作用机制尚不十分清楚。既往研究提示，DJ-1 蛋白可以通过其转录调控活性调控某些基因的表达，从而在帕金森病的发病机制中发挥作用。本研究小组发现一种新的DJ-1 基因突变位点——L10P，即DJ-1基因编码框第29 位碱基胸腺嘧啶（T）→胞嘧啶（C），从而使DJ-1 蛋白第10 位上的亮氨酸被脯氨酸替代。探讨DJ-1 基因L10P 突变在帕金森病发病机制中的作用，可以明确其通过转录调控活性调控相关基因表达的具体方式，继而参与帕金森病之发病机制。方法 筛选稳定表达pCMV-Tag2A-Flag、pCMV-Tag2A-Flag-DJ-1 和pCMV-Tag2A-Flag-DJ-1-L10P 的人胚肾细胞293（HEK293）单克隆细胞株，通过DNA、RNA 和蛋白质水平鉴定，以证实获得的HEK293 单克隆细胞株是否能够稳定表达空载体、野生型DJ-1 蛋白和L10P 突变型DJ-1 蛋白，基因芯片检测技术对各组HEK293 单克隆细胞株进行差异基因筛选。结果 基因芯片技术检测显示，与空载体组相比，野生型组共有14 个基因表达上调、28 个基因表达下调，L10P 突变型组有14 个基因表达上调、9 个基因表达下调；与野生型组相比，L10P 突变型组共有59 个基因表达上调、27 个基因表达下调。这些差异基因分别参与信号转导、基因转录调控、细胞周期调节、细胞凋亡、氧化应激等生物学过程。结论 L10P 突变型DJ-1 蛋白可能通过直接或间接方式对差异基因进行表达调控，从而影响这些信号转导通路的正常功能，进而参与帕金森病的发病机制。

关键词: 帕金森病, 基因表达谱, 突变, 转染, 免疫印迹法, DNA 探针, 细胞, 培养的