论文部分内容阅读
PCR扩增人吲哚胺2,3-双加氧酶1(indoleamine 2,3-dioxygenase 1,IDO1)基因启动子上游区域1 245 bp基因片段,将其插入到p GL4.20-basic载体中构建了p GL4-IDO1-luc荧光素酶重组质粒。基于双荧光素酶报告基因方法建立IDO1抑制剂筛选模型,筛选能够下调肿瘤细胞中IDO1表达的天然活性小分子化合物。采用MTT、Western blotting和乳酸脱氢酶(LDH)等方法探讨阳性化合物的抗肿瘤作用及其对IDO1的调控机制。化合物川楝素(toosendanin,NS-180)能显著下调IFN-γ诱导的肿瘤细胞中IDO1表达。在A549细胞中,NS-180可抑制STAT1和STAT3的磷酸化,从而下调IFN-γ诱导的IDO1蛋白表达。LDH释放实验表明,NS-180可促进NK细胞对A549细胞的杀伤作用。综上所述,筛选获得的天然小分子NS-180是一类新型高效的IDO1抑制剂,可能成为靶向IDO1的肿瘤免疫治疗候选药物。
PCR was used to amplify the 1 245 bp gene fragment in the upstream region of the indoleamine 2,3-dioxygenase 1 (IDO1) gene promoter and insert it into the pGL4.20-basic vector A pGL4-IDO1-luc luciferase recombinant plasmid was constructed. IDO1 inhibitor screening model was established based on dual luciferase reporter gene screening and screening of natural active small molecule compounds that could down-regulate IDO1 expression in tumor cells. The anti-tumor effect of positive compounds and its regulation on IDO1 were investigated by MTT, Western blotting and lactate dehydrogenase (LDH). Toosendanin (NS-180), a compound, significantly down-regulates IDO1 expression in tumor cells induced by IFN-γ. In A549 cells, NS-180 inhibits phosphorylation of STAT1 and STAT3, thereby down-regulating IFN-γ-induced IDO1 protein expression. LDH release experiments showed that, NS-180 can promote NK cell killing of A549 cells. In summary, the natural small molecule NS-180 obtained by screening is a new type of highly effective IDO1 inhibitor, which may become a candidate drug for immunotherapy of tumors targeting IDO1.