转染DJ-1和DJ-1^(L166P)基因的NIH3T3细胞中tau基因表达的研究  

Study on Expression of tau in DJ-1 and DJ-1^(L166P) Transfected NIH3T3 Cells

作　　者：张梅英[1] 蔺美娜[1] 杨葳[1] 董婉维[1] 李兆阳[1] 王惟[1] 郑志红[1] 王禄增[1] 

机构地区：[1]中国医科大学实验动物部,辽宁省转基因重点实验室,沈阳110001

出　　处：《实验动物与比较医学》2008年第6期361-366,共6页Laboratory Animal and Comparative Medicine

基　　金：辽宁省重点实验室专项资金[辽科发(2005)36号]

摘　　要：目的在转染pEGFP-DJ-1和pEGFP-DJ-1L166P质粒的NIH3T3细胞中进行tau基因表达与DJ-1基因相关性的研究,为进一步建立DJ-1和DJ-1L166P转基因小鼠模型及在动物整体水平研究DJ-1基因功能和帕金森病发病机制提供实验基础。方法采用RT-PCR的方法从人神经母细胞瘤细胞(SH-SY5Y)总RNA反转录获得DJ-1cDNA片段,采用突变试剂盒将第166位氨基酸突变,分别构建pEGFP-DJ-1和pEGFP-DJ-1L166P重组表达载体,采用脂质体转染的方法分别将pEGFP-DJ-1、pEGFP-DJ-1L166P、pEGFP-C3质粒转染NIH3T3细胞并用G418压力筛选稳定克隆,对获得的三种转染细胞在转录水平和蛋白质水平进行tau基因表达的检测。结果pEGFP-DJ-1、pEGFP-DJ-1L166P、pEGFP-C3质粒转染NIH3T3细胞G418筛选后,分别获得6、2、9个阳性细胞克隆,RT-PCR及westernblot方法进行tau基因表达检测,结果均显示pEGFP-DJ-1质粒转染组tau表达低于pEGFP-C3质粒转染组,而pEGFP-DJ-1L166P质粒转染组tau表达则高于pEGFP-C3质粒转染组,实验结果统计学分析均具有明显差异(P<0.05)。结论在转录水平及蛋白质水平,DJ-1基因使tau基因的表达下降,而DJ-1L166P使tau基因表达上升。在NIH3T3细胞中tau基因表达与DJ-1基因具有一定的相关性。Objective To explore the relationship between the expression of tau and DJ-1gene in transfected NIH3T3 cell by pEGFP-DJ-1 and pEGFP-DJ-1^L166Pplasmid, and it would provide experimental basis to establish DJ-1 and DJ-1^L166P transgenic mice and to understand the function of DJ-1 and the pathogenesis of Parkinson＇s disease. Methods DJ-1 cDNA sequence were obtained from total RNA of SH-SY5Y cells by RT-PCR. Then one clone of the cDNA was treated with a mutation kit to get a mutation at the site which encodes the 166th amino acids. Recombinant pEGFP-DJ-1 and pEGFP-DJ-1^L166P plasmids were constructed in a routine way. NIH3T3 cells were transfected into pEGFP-DJ-1, pEGFP-DJ-1^L166P, or pEGFP-C3 plasmids, and then the cells were selected with G418. The successfully transfected cells were analyzed by RT-PCR and Western blot to detect the expression of tau gene. Results We gained 6, 2, and 9 clones of pEGFP-DJ-1, pEGFP-DJ-1^L166P, and pEGFP-C3 transfected cells. The results of RT-PCR and Western blot show that： the expression of tau in pEGFP-DJ-1 transfected cells is lower than in pEGFP-C3 transfected cells, and the expression of tau in pEGFP-DJ-1^L166P transfected cells is higher than in pEGFP-C3 transfected cells. The experimental result analyzed by statistics method is significant （P〈0.05）. Conclusion DJ-1 gene down-regulates the expression of tau on both transcription and translation levels, and D J-1^L166P up-regulats the expression of tau. The expression of tau is associated with DJ-1 gene in NIH3T3 cell.

关 键 词：DJ-1 TAU 帕金森氏病 基因 

分 类 号：Q78[生物学—分子生物学]