DJ-1^(L166P)与DJ-1^(M26I)基因对NIH3T3细胞增殖和凋亡的比较

Comparison of the Proliferation and Apoptosis of NIH 3T3 cells Treated with DJ-1^(L166P) or DJ-1^(M26I)

目的在细胞学水平比较DJ、DJ-1M26 I、DJ-1L166 P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-1L166 P和pcDNA3.1/myc-His-DJ-1M26 I重组质粒脂质体方法转染NIH 3T3细胞,500μg/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-1L166 P和pcDNA3.1/myc-His-DJ-1M26 I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-1L166 P和DJ-1M26 I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-1L166 P和DJ-1M26 I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-1L166 P和DJ-1M26 I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-1L166 P和DJ-1M26 I基因突变均降低NIH3T3细胞增殖速率,DJ-1L166 P和DJ-1M26 I基因突变更易导致NIH 3T3细胞的凋亡,DJ-1L166 P和DJ-1M26 I基因突变对NIH3T3细胞增殖速率和细胞凋亡影响是相似的。

Objective To explore the relationship between DJ-1,DJ-1M26I,DJ-1L166P 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 plasmids pcDNA3.1/myc-His-DJ-1,pcDNA3.1/myc-His-DJ-1L166P and pcDNA3.1/myc-His-DJ-1M26I 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 pcDNA3.1/myc-His-DJ-1,pcDNA3.1/myc-His-DJ-1L166P or pcDNA3.1/myc-His-DJ-1M26I,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-1M26I had a higher expression of caspase-3 mRNA than normal NIH 3T3 cells,while NIH3T3 cells transfected with DJ-1 had a lower expression.MTT assay showed that NIH 3T3 positive cells transfected with DJ-1L166P and DJ-1M26I had a lower proliferation rate than that of normal NIH3T3 cells（P0.05）,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-1M26I 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（P0.05）.Conclusions DJ-1L166P and DJ-1M26I mutations reduce the proliferation of NIH 3T3 cells.DJ-1L166P and DJ-1M26I mutations also enhance apoptosis in NIH 3T3 cells.Their effects on NIH 3T3 cell proliferation and apoptosis are similar.