食管癌突变型DNA聚合酶β对EC9706细胞影响的初步研究

A Preliminary Investigation on the Effect of Overexpression of DNA Polymerase β on EC9706 Esophageal Cancer Cell Line

目的：建立过量表达食管癌突变型（点突变型和缺失突变型）DNA聚合酶β（DNA Polymeraseβ，polβ）的EC9706细胞系，并观察其生物学特性的变化。方法：根据GenBank DNA polβ的eDNA序列设计引物，运用PCR方法，从质粒peDNA3．1-polβ中扩增出点突变和缺失突变型的DNA polβ基因，作为目的片段，定向克隆至pEGFP—N3真核绿色荧光蛋白表达载体，获得点突变和缺失突变型的重组真核表达载体pEGFP—N3-polβ。采用脂质体转染的方法，将两种突变型的pEGFP—N3-polβ转染EC9706细胞系，荧光倒置显微镜观察其细胞定位，绘制生长曲线，流式细胞仪测定细胞周期。结果：成功构建了点突变型和缺失突变型pEGFP—N3-polβ重组真核表达载体，荧光倒置显微镜结果显示缺失突变型的DNA polβ表达以细胞胞浆为主，点突变型以细胞胞核为主，而且缺失突变型的细胞生长较对照组明显减慢（P〈0．05），点突变型与对照组相比则无明显差异（P〉0．05）；缺失突变型的DNA polβ还可使EC9706细胞的S期细胞明显减少（P〈0．05），而点突变型轻度减少（P〈0．05）。结论：成功建立了稳定高表达人点突变型和缺失突变型DNA polβ的EC9706细胞系，外源点突变型和缺失突变型DNA polβ在食管癌EC9706细胞表达后可以改变其生物学特性，对研究食管癌的发病机制具有重要意义。

Objective： To establish an EC9706 esophageal cancer cell line that overexpresses the point mutation and deletion repair variants of DNA polymerase β （polβ） in esophageal cancer and to investigate the changes in the biological characteristics of the cell line. Methods： The point mutation and deletion repair variants of DNA polymerase β were amplified from recombinant expression plasmid pcDNA3.1-polβ using PCR. The primers were designed according to the cDNA sequence of DNA polymerase β （Genbank）. The point mutation and deletion repair variants of the DNA polymerase β gene were cloned into the pEGFP-N3 vector. The recombinants were then transfected into the EC9706 cell line using lipofectamine. The localization of proteins encoded by the 2 variants was detected by inverted fluorescent microscopy. The growth curves were drawn using results from MTT assay and the percentage of cells in each phase of the cell cycle was detected by flow cytometry. Results： The sequences of the DNA polymerase β variants were correct and they were successfully transfected into the EC9706 cell line. The point mutation repair DNA polymerase β protein was mostly inside the nucleus, but the deletion repair DNA polymerase β protein could be seen in the whole cell. The growth of EC9706 cells transfected with the deletion repair variant was obviously slower than that of the controls （P〈0.05）. The number of cells in S phase was 36.72 % in the cells transfected with point mutation and deletion repair DNA polymerase β, and 26.82% in the cells transfected with the point mutation repair DNA polymerase β alone （P〈0.05）. Conclusion： An EC9706 cell line overexpressing the point mutation or deletion repair variants of DNA polymerase β has been successfully established. The biological characteristics of the cell line changed after transfection with the DNA polymerase β variants. This study warrants further investigation into the pathogenesis of esophageal cancer.