论文部分内容阅读
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 Polymerase β,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cDNA序列设计引物,运用PCR方法,从质粒pcDNA3.1-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 EC9706 cell line overexpressing esophageal cancer mutant (point mutation and deletion mutant) DNA polymerase β (polβ), and to observe its biological characteristics. Methods: Primers were designed according to the cDNA sequence of GenBank DNA polβ, and the point and mutant polβ genes were amplified from plasmid pcDNA3.1-polβ by PCR. The polβ gene was cloned into pEGFP-N3 eukaryotic vector Green fluorescent protein expression vector, to obtain a point mutation and deletion mutant recombinant eukaryotic expression vector pEGFP-N3-polβ. Two kinds of mutant pEGFP-N3-polβ were transfected into EC9706 cell line by lipofectamine. The cell location was observed by fluorescence inverted microscope and the growth curve was drawn. The cell cycle was determined by flow cytometry. Results: The recombinant eukaryotic expression vector pEGFP-N3-polβ was constructed successfully. Fluorescence inverted microscopy showed that the expression of mutant DNA polβ was mainly cytoplasm, (P <0.05). However, there was no significant difference between the point mutant group and the control group (P> 0.05). The deletion mutant DNA polβ also inhibited the growth of EC9706 cells S phase cells decreased significantly (P <0.05), while point mutations slightly reduced (P <0.05). CONCLUSION: The EC9706 cell line stably overexpressed in human point mutation and deletion mutant DNA polβ was successfully established. The exogenous point mutation and deletion mutant DNA polβ could change the biological characteristics of esophageal carcinoma EC9706 cells. The pathogenesis of esophageal cancer is of great significance.