發(fā)布時(shí)間：2018-10-12 12:54

【摘要】：PCR擴(kuò)增人吲哚胺2,3-雙加氧酶1(indoleamine 2,3-dioxygenase 1,IDO1)基因啟動(dòng)子上游區(qū)域1 245 bp基因片段,將其插入到p GL4.20-basic載體中構(gòu)建了p GL4-IDO1-luc熒光素酶重組質(zhì)粒�；陔p熒光素酶報(bào)告基因方法建立IDO1抑制劑篩選模型,篩選能夠下調(diào)腫瘤細(xì)胞中IDO1表達(dá)的天然活性小分子化合物。采用MTT、Western blotting和乳酸脫氫酶(LDH)等方法探討陽性化合物的抗腫瘤作用及其對(duì)IDO1的調(diào)控機(jī)制�；衔锎ㄩ�(toosendanin,NS-180)能顯著下調(diào)IFN-γ誘導(dǎo)的腫瘤細(xì)胞中IDO1表達(dá)。在A549細(xì)胞中,NS-180可抑制STAT1和STAT3的磷酸化,從而下調(diào)IFN-γ誘導(dǎo)的IDO1蛋白表達(dá)。LDH釋放實(shí)驗(yàn)表明,NS-180可促進(jìn)NK細(xì)胞對(duì)A549細(xì)胞的殺傷作用。綜上所述,篩選獲得的天然小分子NS-180是一類新型高效的IDO1抑制劑,可能成為靶向IDO1的腫瘤免疫治療候選藥物。
[Abstract]:The 1 245 bp gene fragment of the upstream region of the promoter of the human indoleamine 23-dioxygenase 1 (IDO1) gene was amplified by PCR and inserted into the p GL4.20-basic vector to construct the recombinant plasmid of p GL4-IDO1-luc luciferase. A screening model of IDO1 inhibitor was established based on double luciferase reporter gene method to screen natural active small molecular compounds which can down-regulate the expression of IDO1 in tumor cells. MTT,Western blotting and lactate dehydrogenase (LDH) were used to investigate the antitumor effect of positive compounds and its regulatory mechanism on IDO1. Toosendanin (toosendanin,NS-180) could significantly down-regulate the expression of IDO1 in tumor cells induced by IFN- 緯. In A549 cells, NS-180 inhibited the phosphorylation of STAT1 and STAT3 and down-regulated the expression of IDO1 protein induced by IFN- 緯. LDH release assay showed that NS-180 could promote the cytotoxicity of NK cells to A549 cells. In conclusion, the obtained natural small molecule NS-180 is a novel and efficient IDO1 inhibitor, which may be a candidate for tumor immunotherapy targeting IDO1.
【作者單位】： 中國醫(yī)學(xué)科學(xué)院北京協(xié)和醫(yī)學(xué)院醫(yī)藥生物技術(shù)研究所;吉林省藥品檢驗(yàn)所;
【基金】：國家自然科學(xué)基金面上資助項(xiàng)目(81473248) 中國醫(yī)學(xué)科學(xué)院重大協(xié)同創(chuàng)新項(xiàng)目-重大前沿研究(2016-12M-1-011)
【分類號(hào)】：R73-36

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