【作者】 王为；

【导师】 邓慧华；

【作者基本信息】 东南大学 ， 学习科学， 2021， 硕士

【摘要】 头发中糖皮质激素和内源性大麻素含量正被越来越多地应用于慢性应激、临床和神经内分泌等领域的研究。其中,皮质醇（F）、可的松（E）、脱氢表雄酮（DHEA）、花生四烯酸乙醇氨（AEA）、2-花生四烯酸甘油（2AG）、十六酰胺乙醇（PEA）、油酰乙醇胺（OEA）和硬脂酰胺（SEA）这8种激素对人类的认知、压力、情绪调节和免疫反应等有着重要作用。然而,对这八种内源性物质进行同时检测的方法尚欠缺,并且目前尚未有研究关注HIV感染者中内源性大麻素系统的状态。由此,本研究旨在开发同时检测上述八种内源性物质的方法,并用于探究HIV感染者内源性大麻素系统状态及其与HPA轴的关系。本研究基于LC-MS/MS系统首次建立了一种用于同时检测人体头发中3种糖皮质激素和5种内源性大麻素的快速、灵敏和可靠的方法。该方法使用甲醇对头发待测物进行提取;使用梯度洗脱对待测物进行分离;使用APCI离子化源对待测物进行离子化,使待测物分离较好的同时拥有较高机器响应。该方法的低中高三个浓度的日内日间变异系数都小于15%,但在LOQ点小于20%;回收率都在100±15%以内。待测物的归一化基质因子在0.88～1.10之间;残留效应满足要求;长期、室温、冻融和高温稳定性结果也符合要求。该方法的检出限和定量限分别为0.4-10 pg/mg和1.0-25 pg/mg,足够检测出头发中的8种激素。89名受试者从广西被招募,其中艾滋病病毒（HIV）感染者58名,非HIV感染者31名作为对照组。人群分析结果显示,HIV感染者头发F含量以及F与DHEA比值(R_F/DHEA)比对照组的更高（p<0.05;p=0.07）,感染者头发AEA含量与AEA在PEA、OEA、SEA这三个内源性大麻素含量之和中的占比(R_AEA/POSEA)比对照组更低（ps<0.01）。感染组中,被试按CD4细胞计数的数值（200个/mm³）再次划分为重病症和轻病症组,CD4细胞计数较高的轻病症组头发AEA含量和R_AEA/POSEA更高（ps<0.05）,F和R_F/DHEA无显著差异。这表明HIV的感染会使感染者HPA轴功能活性长期处于较高状态,且内源性大麻素系统也随之改变;AEA的减少表明HPA负反馈调节机制受到削弱,这可能是造成感染者HPA活性长期升高的部分原因。更多还原

【Abstract】 Glucocorticoid and endocannabinoid contents in human hair are increasingly used in the fields of chronic stress,neuroendocrine and clinical research.Cortisol（F）,cortisone（E）,dehydroepiandrosterone（DHEA）,anandamide（AEA）,2-arachidonoylglycerol（2AG）,palmitoyl ethanolamide（PEA）,oleoylethanolamide（OEA）and stearoylethanolamide（SEA）play some important roles in human cognition,mood regulation,stress and immune response.However,there is still a lack of methods for simultaneously detecting the eight endogenous substances in hair.There is no research focusing on the state of the endocannabinoid system in HIV-infected patients.Therefore,the present study aimed to develop a method for simultaneously detecting the eight endogenous substances and then explore the status of the endocannabinoid system in HIV-infected individuals and its relationship with the HPA axis.Based on the LC-MS/MS system,the present study established a fast,sensitive and reliable method for the simultaneous detection of 3 glucocorticoids and 5endocannabinoids in human hair.Methanol was used for target substances extraction in hair,gradient elution for analytes material separation and the APCI ionization source for analytes ionization.The method showed that the limits of detection and quantification were 0.4-10 and 1.0-25 pg/mg,which were enough to quantitatively detect 8 hair compounds.The intra-day and inter-day coefficients of variation were less than 15%at three desired concentrations and less than 20%at LOQ and the recovery was within 100±15%.The normalized matrix factors of all analytes were between 0.88 and 1.10.The carryover and the long-term,room temperature,freeze-thaw and high temperature stability also met the requirements of measurements.Eighty-nine subjects were recruited from Guangxi including 58HIV-infected patients and 31 non-HIV healthy persons for control.The population analysis revealed that HIV-infected patients showed higher F and the ratio of F to DHEA (R_F/DHEA)in hair than the controls（p<0.05 and p=0.07）.They showed significantly lower AEA and the ratio of AEA to the sum of PEA,OEA and SEA concentration(R_AEA/POSEA)than the controls（ps<0.01）.Moreover,the HIV-infected group was divided into heavy and light groups according to the cutoff value in the CD4 cells（200/mm³）.The light group showed significantly higher AEA content and R_AEA/POSEA in hair（ps<0.05）,but there were no significant differences between them in F and R_F/DHEA.The results supported that the HIV-infected patients showed the activated HPA axis and the altered endocannabinoid system and the weakened regulation of the HPA negative feedback mechanism through the decrease of AEA.更多还原