稳定表达野生型和突变型DNA聚合酶β的NIH3T3细胞系的建立及其生长特性

Establishment of NIH3T3 cell line stably expressing wild type and mutant type DNA polymerase β gene

目的:建立稳定表达野生型及突变型DNA聚合酶β(polβ)的NIH3T3细胞系。方法:用脂质体转染法分别将野生型和突变型polβ真核表达载体pcDNA3.1-PW和pcDNA3.1-PM3转染入NIH3T3细胞,经G418筛选得到稳定转染细胞系。用RT-PCR、免疫印迹法检测转染细胞polβmRNA和POLB蛋白的表达水平,MTT法测转染细胞生长曲线,免疫组化法测增殖细胞核抗原(PCNA),并用流式细胞仪测细胞周期。结果与结论:成功建立了稳定表达野生型及突变型polβ的NIH3T3细胞系。野生型polβ对NIH3T3细胞生长无明显影响,突变型polβ则使NIH3T3细胞生长速度加快,细胞周期发生改变,细胞多被阻滞在S期。

Aim： To establish NIH3T3 cell lines stably expressing wild type and mutant type DNA polymerase β gene. Methods： The eukaryotic expression vectors of wild type and mutant type DNA polymerase 13 gene were transfected into NIH3T3 cells by lipotransfection, and the stable transfectants were screened by G418. The mRNA expression was determined using RT-PCR and the POLB protein level was assayed using Western blot. The cell growth curve was obtained using MTT,PCNA expression was determined using immunohistochemistry, and the cell cycle was determined using FCM. Results and Conclusion： Wild type and mutant type DNA polymerase β genes were established successfully. Wild type polβ has no obvious effect on the growth of NIH3T3 cells. Mutant polβ accelerates the cell growth, alteres the cell cycle, and most cells remained at S phage.