早期B细胞因子3对HepG2细胞周期的影响

The effect of early B cell factor 3 on HepG2 cell cycling

目的:克隆人早期B细胞因子3(EBF3)基因,构建真核表达载体pEGFP/EBF3,研究人EBF3对肝癌细胞株HepG2细胞周期的影响。方法:提取人胎盘组织总RNA,采用RT-PCR技术克隆人EBF3基因,并将其克隆到真核表达载体pEGFP-N1,重组质粒双酶切鉴定,并进行序列测定。将重组质粒转染HepG2细胞,Western blot确证EBF3-EGFP融合蛋白的表达及亚细胞定位,流式细胞术分析转染细胞周期。结果:成功获得全长人EBF3基因,经双酶切和序列测定鉴定,真核表达质粒pEGFP/EBF3构建正确,将pEGFP/EBF3导入HepG2细胞24小时后,在倒置荧光显微镜下可观察到EBF3-EGFP融合蛋白主要表达于核内。Western blot证实转染pEGFP/EBF3后24小时和48小时细胞质和细胞核中均检出相对分子质量(Mr)为87 000的EBF3-EGFP融合蛋白。转染pEGFP/EBF3后48小时和72小时,S期细胞比例均明显高于pEGFP-N1转染细胞组,表明EBF3基因的导入可诱导细胞从G1期向G2期发展,从而促进细胞增殖。结论:成功构建了真核表达质粒pEGFP/EBF3,pEGFP/EBF3转染组S期细胞比例高于pEGFP-N1转染组,提示EBF3对人肝癌细胞株HepG2细胞有促进增殖作用。

Objective：To clone the encoding sequence of human EBF3 gene, construct recombinant eukaryotic expression plasmid vector pEGFP/EBF3, and study the effect of EBF3 on HepG2 cell cycling.Methods： Total RNA was isolated from placental tissue.Full-length human EBF3 cDNA was amplified by RT-PCR, cloned into eukaryotic expression plasmid vector pEGFP-N1 and sequenced. The expression and sub-cellular localization of the fusion protein EBF3-EGFP in HepG2 cells were analyzed by Western blot. Cell cycles were analyzed with flow cytometry analysis. Results： Obtained full encoding sequence of early B cell factor 3 was identical with that included in GeneBank, and the eukaryotic expression plasmid vector pEGFP/EBF3 was constructed correctly. 24 h after transfected by pEGFP/EBF3, the fusion protein EBF3- EGFP was observed mainly in the cellular nucleus under the inverted fluorescence microscope. Western blot analysis confirmed that the EBF3- EGFP fusion proteins of Mr 87 0（30 were detected in both cytoplasmic and nuclear protein of the HepG2 transfected by pEGFP/EBF＇3 for 24 h or 48 h. Flow cytometry analysis revealed that the percentage of cells in the S phase was markedly increased in HepG2 cells transfected by pEGFP/EBF3 as compared with that in pEGFP-N1 transfected cells. These findings suggested that transfection of EBF3 gene into HepG2 induced cell proliferation by increasing the number of cells from G1 phase to G2 phase. Conclusion： The recombinant eukaryotic expression plasmid vector pEGFP/EBF3 is successfully established. The percentage of ceils in the S phase is markedly increased in pEGFP/EBF3 transfected cells as opposed to pEGFP-N1 transfected cells. It is likely that EBF3 promotes HepG2 ceils proliferation through DNA replication.