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目的观察不同类型DNA聚合酶β(DNA Polymerase β,DNA pol β)在小鼠成纤维细胞(NIH3T3细胞)的表达及细胞增殖特性的变化。方法用脂质体转染的方法,将野生型(wild type,wt)和食管癌缺失突变型(delete mutional type,dmt)的polβ重组荧光载体pEGFP-C3-polβ转染NIH3T3细胞,建立稳定表达polβ的细胞系,通过荧光倒置显微镜观察其定位,四甲基偶氮噻唑蓝(MTT)比色法绘制生长曲线,流式细胞仪测定其细胞周期。结果转染2种类型polβ的NIH3T3细胞株中,均有外源基因mRNA水平和蛋白水平的表达;荧光倒置显微镜结果显示,野生型的以细胞胞核为主,缺失突变型的polβ表达以细胞胞浆为主,从第3d开始,缺失突变型的细胞生长速度较野生型和对照组明显增快(P<0.05);转染缺失突变型NIH3T3细胞的S期细胞比例为68.29%,明显高于野生型的57.18%、对照组的58.12%和未转染NIH3T3细胞的57.35%(P<0.05)。结论外源野生型polβ基因表达后对NIH3T3细胞的增殖速度无明显影响,而缺失突变型则使其增殖速度加快。
Objective To observe the expression of DNA Polymerase β (DNA polymerase β) in mouse fibroblasts (NIH3T3 cells) and the cell proliferation characteristics. Methods The recombinant plasmid pEGFP-C3-polβ with wild type (wt) and deletion mutional type (dmt) was transfected into NIH3T3 cells by lipofectamine. The stable expression polβ cells were observed by fluorescence inverted microscope. The growth curve was drawn by MTT assay, and the cell cycle was measured by flow cytometry. Results The NIH3T3 cells transfected with two types of polβ all expressed the mRNA and protein levels of exogenous genes. The results of fluorescence inverted microscopy showed that the nuclei of nucleus were predominant in the wild-type and the mutant of polβ was expressed in the cells From the third day onward, the growth rate of mutant-type cells was significantly higher than that of wild-type and control groups (P <0.05). The percentage of S-phase cells transfected with mutant NIH3T3 cells was 68.29% 57.18% in the wild type, 58.12% in the control group and 57.35% in the untransfected NIH3T3 cells (P <0.05). Conclusion The expression of exogenous wild-type polβ gene has no significant effect on the proliferation rate of NIH3T3 cells, while the deletion mutant increases the proliferation rate of NIH3T3 cells.