DJ-1L166P与DJ-1M26I基因对NIH3T3细胞增殖和凋亡的比较

摘要

Objective To explore the relationship between DJ-1, DMM261, DJ-1L1669' with the cell proliferation and apoptosis of NIH 3T3 cells at cellular level, and provide a basis for the construction of a transgenic animal model of Parkinson' s disease and further study on the pathogenesis of this disease. Methods Recombinant plasmidg pcDNA3. 1/ myc-His-DJ-1, pcDNA3. l/myc-His-DJ-1L166P and pcDNA3. 1/myc-His-DJ-1M261 were transfected into NIH 3T3 cells, respectively, using lipofectamine. The cells were screened with G418 at a dose of 500 μg/mL. Stable clones were identified on the DNA, RNA and protein levels. MTT assay and annexin V-FITC kit were used to detect the viability and apoptosis of those stable cell clones. Results After the G418-screening of of NIH 3T3 cells transfected with recombinant plasmids pcDNA3. l/myc-His-DJ-l, pcDNA3. l/myc-His-DJ-1L166P or pcDNA3. 1/myc-His-DJ-1M261, one, four and three positive clones were obtained, respectively, by PCR detection. RT-PCR and Western blot detected the expression of DJ-1-His in the positive clones. NIH 3T3 cells transfected with DJ-1L166P and DJ-1M261 had a higher expression of caspase-3 mKINA than normal MH 3T3 cells, while MH3T3 cells transfected with DJ-1 had a lower expression. MTT assay showed that NIH 3T3 positive cells transfected with DJ-1L166P and DJ-1 M261 had a lower proliferation rate than that of normal NIH3T3 cells (P <0.05), while the NIH 3T3 positive cells carrying DJ-1 gene did not show significant difference compared with the normal NIH 3T3 cells. Apoptosis test indicated that the apoptosis rates of DJ-1L166P and DJ-1M261 transfected cells were higher than that of normal NIH 3T3 cells, however the apoptosis rate of the DJ-1-transfected cells was significantly lower than that of normal NIH 3T3 cells (P <0. 05). Conclusions DJ-L166P and DJ-1M261 mutations reduce the proliferation of NIH 3T3 cells. DJ-1L166P and DJ-1M261 mutations also enhance apoptosis in NIH 3T3 cells. Their effects on NIH 3T3 cell proliferation and apoptosis are similar.%目的 在细胞学水平比较DJ、DJ-1M261、DJ-1L166P基因对NIH 3T3细胞增殖速率与凋亡的关系,为建立转基因动物模型及帕金森疾病发病机制研究奠定基础.方法 分别将pcDNA3.1/myc-His-DJ-1、pcDNA3.1/myc-His-DJ-1L166P和pcDNA3.1/myc-His-DJ-1M261重组质粒脂质体方法转染NIH 3T3细胞,500 μg/ml G418压力筛选稳定克隆,对3种转染细胞在DNA水平、RNA水平和蛋白质水平进行鉴定,采用MTT染色方法和Annexin V-FITC试剂盒进行转染阳性克隆细胞的细胞活力与细胞凋亡检测.结果 pcDNA3.1/myc-His-DJ-1、pcDNA3.1/myc-His-DJ-1L166P和pcDNA3.1/myc-His-DJ-1M261重组质粒转染NIH 3T3细胞经G418筛选后,PCR方法检测分别获得1个、4个、3个阳性细胞克隆,RT-PCR及Western blot方法进行DJ-1-His基因表达检测,结果均证明外源插入基因的表达,Caspase-3 RNA水平检测DJ-1L166P和DJ-1M261组表达高于正常NIH 3T3细胞组,而DJ-1组caspase-3转录水平相对最低,MTT实验结果初步证明转染DJ-1L166P和DJ-1M261基因的NIH3T3阳性细胞组细胞增殖速率均低于DJ-1组和正常NIH 3T3细胞组(P＜0.05),转染DJ-1基因的NIH 3T3阳性细胞增殖速率与正常NIH 3T3细胞相比无明显差别；细胞凋亡检测表明转染DJ-1L166P和D J-1M261基因的NIH3T3阳性细胞凋亡率均高于正常NIH 3T3细胞,转染DJ-1基因的NIH 3T3阳性细胞凋亡率低于正常NIH 3T3细胞(P＜0.05).结论 DJ-1L166P和DJ-1M261基因突变均降低NIH3T3细胞增殖速率,DJ-1L166P和DJ-1M261基因突变更易导致NIH 3T3细胞的凋亡,DJ-1L166P和DJ-1M261基因突变对NIH3T3细胞增殖速率和细胞凋亡影响是相似的.