【作者】 李文；

【导师】 章幼玉；

【作者基本信息】 厦门大学 ， 电子与通信工程， 2022， 硕士

【摘要】 人造麝香如二甲苯麝香(Musk Xylene,MX)与吐纳麝香(AHTN)因为其香味独特且廉价,被当做天然麝香的廉价替品,其可以通过皮肤渗入以及其他途径进入人体,从而在人体中产生富集。目前人造麝香已经成为一种新型的污染物,对于其与癌症诱发的关系已经有相关研究证明,但是对于其在致癌通路中对于其关键蛋白的靶向作用分子机制尚未明确。在前期研究中,我们发现经由二甲苯麝香暴露后,L02肝细胞发生了恶性转化,表现出恶性增殖以及强迁移侵袭能力等肿瘤细胞特性。同时对其信号通路进了基因表达分析,结果显示,二甲苯麝香暴露对于MAPK信号通路激活产生了靶向影响,其中EGFR关键蛋白表现出高表达。MAPK信号通路的激活与多种癌症的发生相关包括肝癌,因此,我们推断人造麝香经各种途径进入人体后,可能经由MAPK信号通路,靶向作用于MAPK信号通路上游基因EGFR,导致信号传导级联的激活。本研究以正常肝上皮细胞(L02)和正常皮肤表皮细胞(HACAT)为研究对象,对其进行吐纳麝香暴露,观察其恶性转化趋势,并对恶性转化细胞进行模型构建,对其恶性增殖能力以及迁移侵袭能力进行实验验证。此外,我们对二甲苯麝香/吐纳麝香暴露后MAPK信号通路高表达蛋白的靶向结合能力进行了 Biacore体外实验验证并通过分子对接预测了二甲苯麝香/吐纳麝香与EGFR的结合位点,结果显示二甲苯麝香/吐纳麝香与EGFR胞外配体结合域存在一定强度的结合。此外,我们通过分子动力学进一步研究二甲苯麝香/吐纳麝香结合后,EGFR胞外区域的构象激活情况,结果显示,二甲苯麝香/吐纳麝香结合于EGFR胞外区域不同位点对于其构象激活产生了差异性影响,其中二甲苯麝香/吐纳麝香在同一位点的结合促进了其不同程度的变构激活。通过分子动力学模拟我们发现,二甲苯麝香/吐纳麝香在EGFR胞外区域上均存在自主性选择结合能力,且在EGF内源性生长因子配体重要结合位点上,二者的结合均对EGFR胞外区域产生了类似EGF结合产生的二聚化变构趋势。本研究通过对吐纳麝香的恶性转化诱导能力进行了验证,对二甲苯麝香/吐纳麝香在MAPK致癌通路中靶向作用EGFR的分子机制进行了系统性的分析,并对其作用位点进行了揭示,这一研究结果对建立分析和评价二甲苯麝香/吐纳麝香对人体健康的影响是至关重要的,此外也对因MAPK信号通路激活继而诱导相关癌症的发生的防治与药物设计有重大意义。更多还原

【Abstract】 Artificial musk such as Musk Xylene(MX)and Tonalide(AHTN)is regarded as cheap substitutes for natural musk for their unique fragrance,easy synthesis and low price.For the lipophilicity and chemical stability,artificial musk can enter into human body and enriched through co nsumption seafood,drinking water as well as the use of personal-care products and perfumed household products,then poses a serious threat to human health.At present,artificial musk has become a new pollutant,and some relevant studies have proved the relatio nship between cancer induction and artifical exposure.However,the molecular mechanism has not been clearly studied for the past years.In our previous study,unlimited proliferation,strong migration and invasion were observed in MX-treated cells,suggesting that exposure of MX could lead to malignant tra nsformation of L02 cells.Meanwhile,gene expression analysis showed that EGFR,a key protein of MAPK signaling pathway,was strong up-regulated.MAPK signaling pathway has proved to be related to the occurrence of variety of cancers,including liver cancer.Thus we speculate that MX might activate the MAPK signaling pathway by binding with EGFR,and then resulting in the activation of signal tra nsduction cascade.In this study,normal liver epithelial cells(L02)and normal skin epidermal cells(HACAT)were exposed to AHTN,and the trend of malignant tra nsformation was observed.The ability of malignant proliferation,migration and invasion were also test.In addition,the binding ability between MX/AHTN and EGFR in the MAPK signaling pathway was quantified by Biacore technology.And the binding sites between MX/AHTN and EGFR were predicted through molecular docking.Results showed that MX/AHTN had a certain binding ability with the extracellular ligand binding domain of EGFR.In addition,We further studied the conformational change of EGFR extracellular domain after binding with MX/AHTN binding through molecular dynamics simulation.Results showed that MX/AHTN binded to different sites of EGFR extracellular region lead to differential effects on its conformational change.Meanwhile,the binding of MX/AHTN at the same site promoted its allosteric activation to varying degrees.According to molecular dynamics simulation results,we found that MX/AHTN have spontaneous binding ability,and their binding on the important binding sites of EGF endogenous growth factor ligands has a dimerization allosteric trend similar to that produced by EGF binding.In this study,the malignant tra nsformation induction ability of AHTN was verified,the molecular mechanism of MX/AHTN targeting EGFR in MAPK signaling pathway was systematically analyzed,and the binding sites were revealed.These researches were very important to establish,analyze and evaluate the impact of MX/AHTN on human health,In addition,it is also of great significance for the prevention and treatment of cancer via MAPK signaling pathway and the design of relative drugs.更多还原