人CCAAT增强子结合蛋白α基因影响骨肉瘤细胞生长活性研究

A study on the inhibitory effects of human CCAAT/enhancer binding protein-α on osteosarcoma cell growth

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 the eukaryotic expression vector of CCAAT / enhancer binding protein-α （ C/EBPα ） labeled with Myc tag （ pXJ-40 ）, and to examine the inhibitory effects of C/EBPα on osteosarcoma cell growth.Methods Human C/EBPα coding gene region was amplified from mammary gland cDNA library by polymerase chain reaction （ PCR ）, which was inserted into the pXJ-40-myc vector. Myc-C/EBPα was constructed and transfected into 293T cells. Western blotting analysis was carried out to detect their expressions. In addition, U2OS cells transfected with Myc-C/EBPα were divided into the experimental group, and U2OS cells only transfected with Myc were divided into the control group. The growth of U2OS cells in both groups was identiifed using growth curve experiment.Results The coding region of C/EBPα was successfully ampliifed by PCR and cloned into pXJ-40-myc vector. Myc-C/EBPα was successfully expressed in human 293T cells. Growth curve experiment showed that the growth of U2OS cell lines transfected with Myc-C/EBPα was apparently inhibited. With the extension of the culture time, the inhibitory effects were gradually enhanced. At the 5th day, the inhibition ratio of U2OS cell growth was 58%in the experimental group. The growth rate of U2OS cell lines transfected with Myc-C/EBPα [ optical density （ OD ）=0.495 ] was apparently lower than that of the control group （ OD=1.179 ）, and the differences between them were statistically signiifcant （P＆lt;0.01 ）.Conclusions The eukaryotic expression vector Myc-C/EBPα can effectively inhibit osteosarcoma cell growth.