摘要
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.
AIM: To observe the effect of over-expressed DNA poly-merase β on the malignant degree of EC9706 cells of esophageal cancer. METHODS: The wild and mutant type DNA polp gene were amplified by polymerase chain reaction (PCR) and cloned into pEGFP-C3 vector to obtain wild and mutant pEGFP-C3-polβ. Then pEGFP-C3-polp was transfected into EC9706 cells using lipofectamine method. The location of DNA polβ gene-encoded protein was observed under fluorescent microscope. The growth of the cells was detected by MTT assay and the cycle of the cells was examined by flow cytometry. RESULTS: The sequences of the two recombinants were confirmed and they were transfected into the EC9706 cells successfully. The wild DNA polp protein was mostly located inside the nuclear, but the mutant DNA Polp protein was distributed in the whole cell. The proliferation of EC9706-wtPolβ cells was significantly slower than that of control cells (P<0.05). Furthermore, the S-period frequency (SPF) was significantly decreased in EC9706-wtPolβ cells (22.11 ± 0.12 vs 44.86 ± 0.03, P<0.05), but not in EC9706-mtpolβ ones (P>0.05). CONCLUSION: Over-expression of wild type DNA poly-merase β can decrease the malignant degree of esoph-ageal cancer.
出处
《世界华人消化杂志》
CAS
北大核心
2005年第12期1377-1381,共5页
World Chinese Journal of Digestology
基金
国家自然基金资助项目
No.39870287教育部科学技术研究重点项目
No.02088~~
