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目的构建带Myc(p XJ-40)标签的CCAAT增强子结合蛋白α(CCAAT enhancer binding proteinα,C/EBPα)的真核表达载体,并检测其对骨肉瘤细胞生长的影响。方法应用聚合酶链式反应(polymerase chain reaction,PCR)技术从人乳腺文库中扩增出C/EBPα全长编码区基因;将扩增基因克隆到Myc载体中,并将重组质粒转染人胚胎肾293T细胞;Western blotting检测表达情况;另将重组Myc-C/EBPα质粒转染人骨肉瘤细胞系U2OS(实验组)、Myc空载体质粒转染U2OS细胞(对照组),生长实验检测C/EBPα对肿瘤细胞生长的影响。结果成功构建Myc-C/EBPα真核表达质粒,重组质粒转染人胚胎肾293T细胞后成功表达。生长实验显示Myc-C/EBPα转染的U2OS细胞的生长明显受限,随着培养时间延长,受抑制的程度逐渐增大,培养至第5天,Myc-C/EBPα转染的实验组细胞生长抑制率为58%,且细胞的生长速率OD=0.495明显低于空白对照组1.179,差异有统计学意义(P<0.01)。结论 C/EBPα对骨肉瘤细胞的生长具有抑制作用。
Objective To construct an eukaryotic expression vector carrying CCAAT enhancer binding protein α (C / EBPα) tagged with Myc (p XJ-40) and detect its effect on the growth of osteosarcoma cells. Methods The C / EBPα full-length coding region gene was amplified from human breast cDNA library by polymerase chain reaction (PCR) technique. The amplified gene was cloned into Myc vector and transfected into human embryo The expression of C / EBPα was detected by Western blotting. The recombinant Myc-C / EBPα plasmid was transfected into human osteosarcoma cell line U2OS (experimental group), and Myc empty vector plasmid was transfected into U2OS cells (control group) Effect on tumor cell growth. Results The eukaryotic expression plasmid Myc-C / EBPα was successfully constructed. The recombinant plasmid was successfully transfected into human embryonic kidney 293T cells. Growth experiments showed that the growth of Myc-C / EBPα-transfected U2OS cells was obviously limited. With the prolongation of culture time, the degree of inhibition was gradually increased. On the 5th day after culture, Myc-C / EBPα transfected cells The growth inhibition rate was 58%, and the cell growth rate OD = 0.495 was significantly lower than that of the blank control group 1.179, the difference was statistically significant (P <0.01). Conclusion C / EBPα can inhibit the growth of osteosarcoma cells.