天然活性多不饱和脂肪酸羟基和环氧两类衍生物的合成，抗白血病作用及机制研究

EPA-derived hydroxyl metabolite has been recently reported to target leukemia stem cells.However, naturally occurring oxylipins from polyunsaturated fatty acid(PUFA) were low content and complicated ingredients. A reliable and easy chemical method for synthesis of hydroxyl derivatives of LA has been reported by proposer.In the preliminary research, we found that the hydroxyl derivatives of LA were more effective than lead compound (linoleic acid and conjugated linoleic acid) against several cancer cell lines, and they were safety. Based on the study above, this project is to synthesize hydroxyl and epoxy derivatives from PUFA and investigate the effect of derivatives on leukemia (stem) cells and its mechanism in vitro and in vivo. The PUFA-derivatives will be evaluated for their cytotoxicity using MTS assay on leukemia cells and leukemia stem cells (LSC). The apoptosis and colony-forming ability of LSC will be assessed by flow cytometry and colony-forming assay. Western blot will be used to determine the regulation of ATM/p53 signal pathway by derivatives at the levels of protein. Finally, molecular modeling will be applied to study the quantitative structure and anti-leukemia activity relationship of hydroxyl and epoxy derivatives of PUFA for further generating of new derivatives and predicting their anti-leukemia activities for drug development.

最新研究报道EPA羟基代谢产物（Δ12-PGJ3）能靶向杀伤白血病干细胞，防止肿瘤复发。但是，天然来源的多不饱和脂肪酸的活性羟脂类代谢产物含量低，成分复杂，难以获取。项目申请人在国际杂志上首次报道了通过化学方法可一步合成亚油酸的羟基类衍生物，该方法方便，快捷，可用于工业化生产。并且我们的前期研究也发现亚油酸的羟基衍生物比先导化合物的体外抗肿瘤效果更强，且安全无毒。在此基础上，本课题拟以四种多不饱和脂肪酸（LA，LNA，DHA，EPA）为原料，合成一系列的羟基和环氧类衍生物，通过体外白血病细胞和白血病干细胞的增殖、凋亡和克隆化等实验，并构建小鼠白血病模型进一步考察多不饱和脂肪酸羟基和环氧类衍生物抗白血病的作用及作用机制，深入探讨它们对ATM/p53信号途径的干预作用；最后拟用分子模拟进行分析，阐明其构效关系，为进一步优化结构，寻找新的高效低毒化合物，开发具有自主知识产权的新药研究提供基础。

近年来，研究发现天然来源的多不饱和脂肪酸的活性羟脂类代谢产物具有多种重要的生理活性，如抗肿瘤，抗炎等。其中最重要的两大类衍生物为羟基衍生物和环氧衍生物。它们可通过诱导细胞凋亡产生抗肿瘤作用，但其作用机制尚不明确。本项目旨在优选出抗白血病活性较强的衍生物，并探讨其抗白血病作用机制，为开发新药提供理论依据。.【方法】选取K562，HL-60白血病细胞模型，筛选羟基衍生物和环氧衍生物体外抗白血病作用，优选出活性较强的化学物作为候选药物。采用二维凝胶电泳（2-DE）和质谱鉴定联用的蛋白质组学研究方法从蛋白质水平探讨9S-HOD抗白血病作用的机制。采用microRNA芯片技术从基因水平探讨9S-HOD抗白血病作用的靶点，最后采用生物信息学分析可能的作用通路，阐明其作用机制。【结果】首先通过体外抗白血病活性筛选出了活性较强的化合9S-HOD，流式实验结果和显微镜观察表明9S-HOD对HL-60细胞生长的抑制作用与诱导细胞凋亡有关。蛋白质组学研究鉴定出23个差异表达蛋白质。将这些差异蛋白质进行生物信息学分析表明，9S-HOD可能通过影响多种途径如mRNA的代谢过程中，蛋白质结合，DNA复制和细胞凋亡，从而发挥抗白血病作用效果。microRNA芯片结果也证实了9S-HOD抗白血病作用与抑制致癌因子microRNA-361-3p密切相关。【结论】9S-HOD可通过影响多种途径如mRNA的代谢过程中，蛋白质结合，DNA复制和细胞凋亡，从而发挥抗白血病作用效果。9S-HOD可作为一个有效的抗白血病候选药物。