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目的:观察过表达的DNA聚合酶β对食管癌EC9706细胞系的影响.方法:运用PCR方法,从质粒pcDNA3.1-polβ中扩增出野生型和突变型的DNA聚合酶β基因,作为目的片段,定向克隆至pEGFP-C3真核绿色荧光蛋白表达载体,获得野生型和突变型的重组真核表达载体pEGFP—C3-polβ.分别将野生型和突变型的pEGFP—C3-polβ转染EC9706细胞,荧光显微镜观察其定位,绘制生长曲线,测定细胞周期.结果:DNA序列分析证实了重组载体中的DNA聚合酶β序列正确,荧光显微镜结果显示野生型的DNA聚合酶β表达以细胞核为主,突变型的DNA聚合酶β则表达在整个细胞中,明显与野生型的DNA聚合酶β的核定位不同.而且,野生型的细胞生长较对照组缓慢(P<0.05),野生型的还能抑制EC9706细胞的生长和使S期细胞减少(22.11±0.12vs44.86±0.03,P<0.05),而突变型的则不能促进EC9706细胞的生长,S期细胞增殖不明显(P>0.05).结论:过表达的野生型食管癌DNA聚合酶β可以降低EC9706细胞的增殖.
Objective: To observe the effect of overexpressed DNA polymerase β on esophageal carcinoma EC9706 cell line.Methods: Wild-type and mutant DNA polymerase β genes were amplified from plasmid pcDNA3.1-polβ by PCR, The wild-type and mutant pEGFP-C3-polβ were transfected into pEGFP-C3 eukaryotic expression vector and wild-type and mutant recombinant eukaryotic expression vector pEGFP-C3- Cells were observed by fluorescence microscopy and the growth curve was drawn to determine the cell cycle.Results: DNA sequence analysis confirmed that the DNA polymerase β sequence in the recombinant vector was correct, and the results of fluorescence microscopy showed that the expression of wild-type DNA polymerase β was mainly in the nucleus , While the mutant DNA polymerase β was expressed in the whole cell and distinctly different from that of the wild-type DNA polymerase β. Moreover, the wild-type cells grew more slowly than the control group (P <0.05) (22.11 ± 0.12vs44.86 ± 0.03, P <0.05), while the mutant type could not promote the growth of EC9706 cells and the proliferation of S phase cells was not obvious (P> 0.05) Conclusion: Overexpression of the wild DNA polymerase β may reduce esophageal EC9706 cell proliferation.