CAJ | 학술논문

目的：建立敲除DNA聚合酶β（ DNA polβ）基因的人食管癌Eca 9706细胞株，观察基因敲除细胞生物学行为的变化。方法：采用同源重组基因敲除方法，首先构建食管癌Eca9706细胞DNA polβ基因敲除载体，并将其导入Eca9706细胞，构建DNA polβ基因敲除细胞株。 PCR、RT-PCR、Western blot法检测DNA polβ基因敲除区段DNA存在及表达情况。流式细胞术和MTT法检测基因敲除细胞的细胞周期和生长速度。结果：筛选得到具有neo抗性的DNA polβ基因敲除Eca9706细胞，其基因组中DNA polβ基因区段已被敲除；RT-PCR和Western blot 检测不到DNA polβmRNA和蛋白表达。敲除DNA polβ基因的细胞生长速度缓慢，G2～M期细胞增多，S期细胞减少（ t＝3．882、3.869，P均＜0．05）。结论：成功构建了食管癌Eca9706 DNA polβ基因敲除细胞模型。
목적：건립고제DNA취합매β（ DNA polβ）기인적인식관암Eca 9706세포주，관찰기인고제세포생물학행위적변화。방법：채용동원중조기인고제방법，수선구건식관암Eca9706세포DNA polβ기인고제재체，병장기도입Eca9706세포，구건DNA polβ기인고제세포주。 PCR、RT-PCR、Western blot법검측DNA polβ기인고제구단DNA존재급표체정황。류식세포술화MTT법검측기인고제세포적세포주기화생장속도。결과：사선득도구유neo항성적DNA polβ기인고제Eca9706세포，기기인조중DNA polβ기인구단이피고제；RT-PCR화Western blot 검측불도DNA polβmRNA화단백표체。고제DNA polβ기인적세포생장속도완만，G2～M기세포증다，S기세포감소（ t＝3．882、3.869，P균＜0．05）。결론：성공구건료식관암Eca9706 DNA polβ기인고제세포모형。
Aim:To construct a DNA polymerase β( DNA polβ) gene knockout model in human esophageal carcinoma cell Eca9706 and investigate its biological character .Methods:Based on the homologous recombination principle ,the gene tar-geting vector was constructed to delete DNA polβgene.The vector was introduced into Eca 9706 cell.PCR,RT-PCR and West-ern blot were used to detect the expression of DNA polβgene at DNA, mRNA and protein level in DNA polβknockout EC9706 cell.Flow cytometry and MTT were used to detect cell cycle and cell growth velocity .Results:In the targeting cells, the DNA,mRNA and protein expression of DNA polβcould not be detected .Compared with normal Eca9706 cells,G2-M phase cells increased,and S phase cells decreased in gene knockout Eca 9706 cells(t=3.882,3.869,P<0.05).Conclusion:The DNA polβgene knockout EC9706 cell line has been constructed successfully .