【摘要】 白血病抑制因子 (LIF)是一种多功能细胞因子 ,在生物发育及维持正常生理功能中发挥着重要的作用。以成人血细胞的总RNA为模板 ,利用RT PCR方法从成人外周血细胞中扩增人白血病抑制因子 ,而后将其克隆到真核表达载体pcDNA3,在哺乳动物细胞中成功表达。将分泌到细胞培养基中的LIF因子收集 ,通过LIF信号转导通路下游蛋白质STAT3磷酸化水平的检测、EMSA实验以及荧光酶报告系统检测 ,发现其具有激活STAT3信号通路的生物学活性。同时 ,[3H] TdR参入实验的结果也表明 ,所表达LIF因子能显著抑制鼠骨髓白血病细胞系M1的增殖。更多还原

【Abstract】 Leukemia inhibitory factor (LIF), a cytokine belonging to IL-6 family, which was discovered to inhibit the proliferation of murine myeloid leukemic cell line M1, has multiple functions in various biological processes. This factor is highly glycosylated when it binds to receptor to activate the signal transduction. Therefore, expression of LIF through eukaryotic system is the best way to obtain the correct glycosylation. In this study, human LIF cDNA with the sequence of signal peptide was cloned from adult blood cells by RT-PCR, and then subcloned into pcDNA3 for expression in HEK-293T cells. After transfection of the recombinant plasmid pcDNA3/LIF into HEK-293T cells, the conditioned medium containing the secreted LIF was obtained. The activity of the secreted LIF in the conditioned medium was detected through the phosphorylation of STAT3, EMSA and luciferase reporter (pGL2-APRE-luc) assays in Hep3B and 293T cells. Furthermore, to investigate the biological functions of the overexpressed recombinant LIF, a [ 3H]-thymedine incorporation assay was performed, and the results showed that the recombinant LIF inhibited the growth of M1 cells strongly. Taken together, all the function assays suggest that the recombinant LIF has the normal functions, suggesting that the recombinant LIF could be used for further studies.更多还原