ING4基因真核表达载体的构建及其功能

Construction of pcDNA3. 0-ING4 eukaryotic expression vector and study on its function

目的:构建真核表达载体pcDNA3.0-ING4,观察ING4基因对人肝癌SMMC7721细胞周期及凋亡的影响。方法:小鼠肝组织经RT-PCR扩增,构建真核表达载体pcDNA3.0-ING4,分别用双酶切、PCR、基因测序进行鉴定,将其转导进入人肝癌SMMC7721细胞,检测ING4基因的表达情况及其对细胞周期的影响,应用荧光显微镜和激光扫描共聚焦显微镜观察细胞凋亡情况。结果:RT-PCR产物为约750 bp的条带,基因测序正确,转导进入人肝癌细胞株SMMC7721后可延长G2期,其凋亡率(23.66%)明显高于对照组(13.75%)。结论:成功分离得到了小鼠ING4基因并成功构建真核表达载体pcDNA3.0-ING4,该质粒转染人肝癌SMMC7721细胞后可延长G2期并可促使细胞凋亡。

To construct pcDNA3.0-ING4 recombinant eukaryotic expression plasmid and explore the effect of INCA on cell cycle and cell apoptosis of SMMC7721. Methods： Using RT-PCR method, the eDNA encoding the mouse ING4 was isolated and total RNA was extracted from mouse liver tissue. The eDNA fragment was subcloned into the eukaryotic expression vector peDNA3.0. Analysis by PCR, restricting enzyme digestion and DNA sequencing were carried out to demonstrate the sequence of the plasmid, then the plasmid was transfected into SMMC7721 cell lines by lipofectamine-mediated transfection. Cell cycle was examined by flow cytometry after transfection with pcDNA3.0-ING4; apoptosis was deteced by fluorescence microscope with Hoechst 33258 staining and laser scanning confocal microscope. Results： RT-PCR product was a 750 bp specific fragment. DNA sequencing revealed that ING4 cloning was successful. Flow cytometric analysis displayed an accumulation of cells in the G2 phase of cell cycle after transfection with pcDNA3.0-ING4. With Hoechst fluorescence staining, we found that the apoptotic rate in SMMC7721 cells transfected with pcDNA3.0- ING4 （23.66%） was higher than that in HeLa cells transfected with pcDNA3.0 （13.75%, P〈0.01）. Conclusion： The gene encoding mouse ING4 and pcDNA3.0-ING4 eukaryotic expression vector are obtained. An accumulation of cells in the G2 phase of cell cycle was achieved after transfection with pcDNA3.0-ING4, and ING4 can enhance apoptosis of SMMC7721 cells.