论文部分内容阅读
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 compare the effects of DJ, DJ-1M26 I and DJ-1L166 P gene on the proliferation and apoptosis of NIH 3T3 cells at the cytology level, and lay the foundation for the establishment of transgenic animal models and the pathogenesis of Parkinson’s disease. Methods Recombinant plasmid pcDNA3.1 / myc-His-DJ-1, pcDNA3.1 / myc-His-DJ-1L166 P and pcDNA3.1 / myc-His-DJ-1M26 I were transfected into NIH 3T3 Cells were stained with G418 at 500μg / ml, and the clones were screened by pressure. The transfected cells were identified by DNA level, RNA level and protein level. MTT staining and Annexin V-FITC kit were used to determine the cell viability Apoptosis detection. Results After NIH 3T3 cells were transfected with recombinant plasmid pcDNA3.1 / myc-His-DJ-1, pcDNA3.1 / myc-His-DJ-1L166 P and pcDNA3.1 / myc-His-DJ- PCR method to detect one, four and three positive cell clones respectively. RT-PCR and Western blot were used to detect the expression of DJ-1-His gene. The results confirmed the expression of exogenous gene and the level of Caspase-3 RNA DJ-1L166 P and DJ-1M26 I group was higher than that of normal NIH 3T3 cell group, but DJ-1 group had the lowest caspase-3 transcription level. MTT results showed that DJ-1L166 P and DJ-1M26 I gene The proliferation rate of NIH3T3 positive cells was lower than that of DJ-1 and normal NIH 3T3 cells (P <0.05). The proliferation rate of NIH 3T3 positive cells transfected with DJ-1 gene was not significantly different from that of normal NIH 3T3 cells. The apoptosis of NIH3T3 positive cells transfected with DJ-1L166P and DJ-1M26I genes was higher than that of normal NIH 3T3 cells. The apoptosis rate of NIH 3T3-positive cells transfected with DJ-1 gene was lower than that of normal NIH 3T3 cells (P <0.05). Conclusions Mutations of DJ-1L166P and DJ-1M26I reduce the proliferation rate of NIH3T3 cells. The mutations of DJ-1L166P and DJ-1M26I are more likely to lead to the apoptosis of NIH 3T3 cells. The mutations of DJ-1L166P and DJ-1M26I The effect on NIH3T3 cell proliferation rate and apoptosis is similar.