【作者】 刘润泽；

【导师】 田维明；

【作者基本信息】 哈尔滨工业大学 ， 生物医学工程， 2019， 硕士

【摘要】 阿尔兹海默病（AD）是一种起病隐匿的进行性发展的神经退行性疾病。2009年全球登记的AD病例达到3560万,而估计到2050年这一数字将翻番。研究表明,β淀粉样蛋白（Amyloidβpeptides,Aβ）聚集形成的淀粉样纤维在神经组织中沉积是引发阿尔兹海默病的重要因素之一,脑内慢性炎症反应对AD的发生发展也具有重要影响。AD及相关类型痴呆仍无法治愈,目前的治疗方法只能达到中等程度的症状减轻,具有较高的副作用,而且不能从根本上治愈。研究表明,脑内微环境中透明质酸（HA）的大量降解,Neuregulin1（NRG1）表达下调与AD发展密切相关。裸鼹鼠（Heterocephalus glaber,NMRS）是寿命最长的啮齿类动物,其脑内微环境中富集的大量高分子量透明质酸（HMW-HA）和NRG1的高表达使其有耐受高水平Aβ_1-42聚集和抵抗Aβ_1-42神经毒性的能力,从而拮抗了AD相关表型的出现,甚至29岁的模型中仍未见斑块形成。本论文将裸鼹鼠HAS2基因克隆到HEK293细胞系,得到可分泌裸鼹鼠HMW-HA的HEK293-nmrHAS2稳转细胞系。对HMW-HA进行产物鉴定,证明了已得到裸鼹鼠HMW-HA。为防止HMW-HA降解,将其进行巯基化和PE化改性。为了对AD引发的脑组织炎症作出响应,本论文选择了在炎症条件下可被酯酶和高表达的基质金属蛋白酶（MMPs）分解的一种炎症响应型水凝胶三聚甘油单硬脂酸酯（Triglycerol Monostearates,TM）。将改性后的HMW-HA与NRG1一同封装到TM水凝胶网络中形成TM-HA-NRG1炎症响应水凝胶。对TM-HA-NRG1水凝胶进行材料表征,优化各组分浓度,使其最大限度的模拟了裸鼹鼠脑内微环境。将TM-HA-NRG1水凝胶分别在酯酶、基质金属蛋白酶、激活的小胶质细胞培养上清液中进行培养,TM-HA-NRG1水凝胶表现出能根据炎症环境强弱的不同缓释出不同量的HMW-HA和NRG1。将TM-HA-NRG1水凝胶与激活的小胶质细胞共培养,检测到炎症相关因子NO、IL-1β、IL-6、TNF-α和iNOS表达量显著下调。在小胶质细胞环境中,将TM-HA-NRG1水凝胶与神经元共培养,Aβ低聚物刺激条件下,TM-HA-NRG1水凝胶在炎症环境中对神经元具有显著的保护作用,拮抗炎症引起的细胞凋亡。本论文利用裸鼹鼠抗AD机制,模拟裸鼹鼠脑内微环境,构建炎症响应型水凝胶,为AD治疗提供了新的思路和方法。更多还原

【Abstract】 Alzheimer’s disease（AD）is a progressive neurodegenerative disease with insidious onset.In 2009,the number of AD cases registered worldwide reached 35.6 million,and it is estimated that this number will double by 2050.Studies have shown that the accumulation of amyloid fibers formed by the aggregation of amyloid beta proteins（A beta）in nervous tissues is one of the important factors causing Alzheimer’s disease.Chronic inflammation in the brain also plays an important role in the occurrence and development of AD.AD and related types of dementia are still incurable.Current treatment methods can only achieve moderate relief of symptoms,with high side effects,and can not be fundamentally cured.Studies have shown that hyaluronic acid（HA）is degraded in a large amount in brain microenvironment,and the downregulation of Neuregulin1（NRG1）expression is closely related to the development of AD.Nude mole（Heterocephalus glaber,NMRS）is the longestlived rodent.The high expression of HMW-HA and NRG1 in its brain microenvironment makes it able to tolerate high levels of Aβ_1-42 aggregation and resist Aβ_1-42 neurotoxicity,thus antagonizing the appearance of ADrelated phenotypes,and even plaque formation is not seen in the 29 years old model.In this study,the HAS2 gene of nude mole was cloned into HEK293 cell line,and stable HEK293-nmrHAS2 cell line secreting HMW-HA of nude mole was obtained.The product identification of HMW-HA proved that the nude mole HMW-HA had been obtained.To prevent the degradation of HMW-HA,the HMW-HA was modified by sulfhydrylation and PE.In order to respond to ADinduced brain inflammation,an inflammationresponsive hydrogel triglycerol monostearates（TM）,which can be decomposed by esterase and highexpression matrix metalloproteinase（MMPs）,was selected in this study.The modified HMW-HA and NRG1 were encapsulated into TM hydrogel network to form TM-HA-NRG1 inflammation response hydrogel.The TM-HA-NRG1 hydrogel was characterized and the concentration of each component was optimized to simulate the brain microenvironment of nude mole.TM-HA-NRG1hydrogel was cultured in the supernatant of esterase,matrix metalloproteinase and activated microglia culture respectively.TM-HA-NRG1 hydrogel showed that it could release different amounts of HMW-HA and NRG1 according to the intensity of inflammation environment.When TM-HA-NRG1 hydrogel was cocultured with activated microglia,the expressions of inflammatory related factors NO,IL-1β,IL-6,TNF-αand iNOS were significantly downregulated.In microglial environment,TM-HA-NRG1 hydrogel was cocultured with neurons.Under the stimulation of A beta oligomer,TM-HA-NRG1 hydrogel had significant protective effect on neurons in inflammatory environment and antagonized apoptosis induced by inflammation.In this study,we used the antiAD mechanism of nude mole mice to simulate the brain microenvironment of nude mole mice and construct an inflammationresponsive hydrogel,which provided a new idea and method for the treatment of AD.更多还原