【作者】 张帆；

【导师】 谢浩；

【作者基本信息】 武汉理工大学 ， 药学， 2019， 硕士

【摘要】 神经退行性疾病严重威胁着人类健康,其致病假说主要包括自由基损伤、神经炎症反应、蛋白异常聚集等。根据其致病假说,神经退行性疾病的治疗药物包括抗氧化剂、抗炎剂和抗蛋白聚集剂等,但尚无一种药物能逆转疾病的进程,因此,治疗药物有待更深层次的开发。近来,天然产物以其多靶点、不良反应少等优点,被应用于神经退行性疾病的治疗。同时,纳米材料具有良好的透过性、靶向性、控释性等特点,已被用于癌症和血管疾病等的临床治疗。本文将天然产物视黄酸（RA）和金纳米粒子（AuNPs）与神经退行性疾病细胞模型共培养,探究其对模型细胞的生物学效应,为疾病的预防和治疗提供新的思路,为药物的神经毒理提供依据。研究内容如下:（1）构建阿尔茨海默病（AD）细胞模型、帕金森病（PD）细胞模型、氧化损伤细胞模型、青光眼细胞模型和兴奋性毒性细胞模型,将RA和AuNPs分别与细胞共培养。CCK-8细胞活性检测结果表明,浓度低于10μM的RA无毒性,能加速β-淀粉样蛋白（Aβ）诱导的SH-SY5Y细胞凋亡,能降低1-甲基-4-苯基吡啶离子（MPP⁺）诱导SH-SY5Y细胞的损伤,但对氧化损伤和兴奋性毒性损伤模型无作用。浓度低于100 mg·L^-1的AuNPs无毒性,能极显著地协同硝普钠（SNP）损伤RGC-5细胞,能显著升高AD和PD模型细胞的活性,但对氧化损伤和兴奋性毒性损伤模型无明显作用。（2）探究RA协同Aβ40损伤SH-SY5Y细胞的机制。荧光动力学实验、原子力显微镜（AFM）形貌测试、动态光散射粒径测试和蛋白二级结构测试结果表明,RA能显著抑制Aβ40纤维化,并影响Aβ40构象转变。荧光探针结果显示RA能显著诱导阿尔茨海默病模型细胞产生活性氧（ROS）和一氧化氮（NO）。实验表明RA抑制Aβ40单体纤维化,使高毒性的Aβ寡聚体和原纤维含量升高,致使胞内ROS和NO含量升高,造成氧化损伤,最终加速细胞凋亡。（3）探究AuNPs协同SNP损伤RGC-5细胞的机制。CCK-8、线粒体膜电位和细胞凋亡检测结果表明,SNP和10 mg·L^-1 AuNPs协同诱导细胞损伤的放大倍数为80倍。场发射环境扫描电镜和透射电子显微镜检测结果表明,AuNPs能改变细胞形貌,并在细胞内代谢产生直径在500 nm左右的球状物。荧光探针和蛋白质印迹法实验结果显示,AuNPs能使青光眼模型细胞中ROS、NO、p-TRKB和p-ERK的含量升高。上述实验结果表明,AuNPs能改变细胞形貌,导致细胞对低浓度的SNP耐受能力降低,并增加胞内ROS和NO的含量,破坏氧化-抗氧化系统平衡,诱导产生氧化损伤,同时MAPK信号通路也被激活,触发级联反应,最终导致细胞凋亡。更多还原

【Abstract】 Neurodegenerative diseases are serious threats to human health,of which the pathogenic hypothesises include free radical damage,neuroinflammatory response,and abnormal protein aggregation.Based on the pathogenic hypothesis,the therapeutic drugs for neurodegenerative diseases are classified into antioxidants,anti-inflammatory drugs and anti-aggregation agents,but no drug can reverse the progress of the disease.Therefore,the development of drugs needs further research.Recently,natural products have been used for the treatment of neurodegenerative diseases because of their multiple targets and few adverse reactions.Furthermore,nanomaterials have good permeability,targeting and controlled release,and have been used in clinical treatment of cancer and vascular diseases.In this paper,retinoic acid（RA）and gold nanoparticles（AuNPs）were co-cultured respectively with cell models of neurodegenerative diseases to explore their biological effects,to provide new ideas for the treatment of diseases and to reveal the neurotoxicity of drugs.The detailed experimental studies and main results are as follows:（1）Cell models were constructed including Alzheimer’s disease（AD）cell model,Parkinson’s disease（PD）cell model,oxidative damage cell model,glaucoma cell model,and excitotoxic cell model.RA and AuNPs were co-cultured with cell models,respectively.The results of CCK-8 assay showed that RA with concentration less than10μM was non-toxic,accelerated the apoptosis of SH-SY5Y cells induced byβ-amyloid（Aβ）,and decreased the damage of SH-SY5Y cells induced by MPP⁺.However,it has no obvious effect on oxidative damage and excitotoxic damage.AuNPs with concentration less than 100 mg·L^-1 were non-toxic and could significantly synergize with sodium nitroprusside（SNP）to damage RGC-5 cells,could significantly increase the viability of AD and PD model cells,but had no significant effect on oxidative damage and excitotoxic damage.（2）The mechanism that RA cooperates with Aβ40 to damage SH-SY5Y cells was explored.The results showed that RA could significantly inhibit Aβ40 fibrillation and affect the transformation of Aβ40 through ThT fluorescence,atomic force microscopy（AFM）,dynamic light scattering and circular dichroism.RA could significantly induce reactive oxygen species（ROS）and nitric oxide（NO）in AD model cells by fluorescent probes.A series of experiments had shown that RA inhibited Aβ40 monomer fibrillation,which increased the content of high-toxic Aβoligomers and protofibrils,resulting in increased intracellular ROS and NO levels,induced oxidative damage and ultimately accelerated apoptosis.（3）The mechanism that AuNPs synergize with SNPs to damage RGC-5 cells was studied.The results showed that with the synergy of 10 mg·L^-1 AuNPs,SNP induced cell damage by 80 times.The field emission scanning electron microscope and transmission electron microscopy showed that AuNPs could change the cell morphology and were metabolized in the cells to produce spheres with a diameter of about 500 nm.Fluorescence probes and Western blot experiments showed that AuNPs increased the levels of ROS,NO,p-TRKB and p-ERK in glaucoma model cells.The above experimental results showed that AuNPs could change the cell morphology,resulting in decreased tolerance of cells to low concentrations of SNP,increased intracellular ROS and NO content,disrupted the oxidation-antioxidant system balance,induced oxidative damage,and simultaneously MAPK signals was also activated,triggering a cascade of reactions,ultimately lead to apoptosis.更多还原