【作者】 刘波；

【导师】 刘慧霞；

【作者基本信息】 中南大学 ， 临床医学， 2014， 博士

【摘要】 非酒精性脂肪肝病(nonalcoholic fatty liver disease, NAFLD)是指除酒精以及其他明确损肝因素所致的肝细胞内甘油三酯过度沉积(>5%的肝细胞组织)为主要特征的一种临床病理综合征。NAFLD不仅与2型糖尿病、肥胖有关,也是非酒精性脂肪肝炎(NASH)、肝硬化甚至肝癌发展的风险因素之一。近年来其发病率逐年增高,已经成为一个全球性的公共健康问题。目前治疗药物主要是针对改善胰岛素抵抗和抗氧化应激等,存在疗效不确切或药物副作用大等因素,尚无治疗NAFLD的特效药物问世。近年来,纳米和核酸适配体技术在载药及药物的靶向性研究中得到了越来越多的应用,是当前生物医学领域的研究热点。本文针对当前NAFLD治疗的一些难题以及治疗前景,通过多学科交叉,设计并构建了基于金纳米粒子和核酸适配体的载药载体,探索新型纳米载药载体在NAFLD治疗中的应用,为将来NAFLD进行靶向药物治疗提供新的思路。鉴于课题的设计,本论文共分为三个部分。第一部分：NAFLD细胞模型的构建、核酸适配体筛选及其特性分析目的：本部分拟建立NAFLD细胞模型,采用细胞指数级富集的配体系统进化技术(Systematic Evolution of Ligands byExponential Enrichment,Cell-SELEX)针对NAFLD细胞进行筛选,获得能特异性识别NAFLD细胞表面膜蛋白结构的核酸适配体,分析筛选到的核酸适配体NFD-1的相关特征,包括不同温度下的特异性、亲和性以及膜蛋白结合性。方法：使用100uM油酸和50uM棕榈酸诱导分化HepG2细胞1周构建NAFLD细胞模型；设计并合成随机DNA文库序列,中间25bp为随机序列,两端各有一段长16bp的引物序列,分别用异硫氰酸荧光素和生物素标记两端引物。将NAFLD细胞作为正细胞,HepG2细胞作为负细胞,将DNA文库与正负细胞反复孵育进行筛选,与正细胞结合而不与负细胞结合的文库序列采用聚合酶链法(PCR)扩增并用作下一轮筛选及鉴定。每3-4轮筛选就使用流式细胞技术对文库的富集程度进行监测、鉴定。最后采用454测序方法对富集到的文库进行测序,合成相关序列并通过流式细胞术进行鉴定；在4。C和37。C条件下将核酸适配体NFD-1与不同组织来源的细胞株孵育,流式细胞术测定其特异性,将NFD-1稀释成不同浓度后与NAFLD细胞孵育,计算其解离常数；使用Trypsin和Proteinase K两种蛋白酶检测NFD-1靶标为膜蛋白。结果：本实验成功诱导分化建立了NAFLD细胞模型。应用Cell-SELEX技术对NAFLD细胞展开筛选,经过24轮正负筛选后成功富集到针对NAFLD细胞的ssDNA文库,经过测序分析找到了4条核酸适配体与NAFLD细胞有结合,而与HepG2细胞不结合,我们选用结合能力强的1条核酸适配体进行实验,并命名为NFD-1;在4。C和37。C条件下,核酸适配体NFD-1均能与NAFLD细胞特异性结合,而与其它组织来源的细胞株几乎没有结合。解离常数测定Kd值为21.5士5.6nM,提示NFD-1能与NAFLD细胞高亲和力结合。蛋白酶检测证实NFD-1的靶标为膜蛋白。结论：通过Cell-SELEX技术针对NAFLD细胞进行筛选,获得了1条在4。C和37。C条件下能与靶细胞膜蛋白高亲和力、高特异性结合的核酸适配体NFD-1。第二部分：金纳米粒子载药载体的构建目的：合成并构建金纳米粒子载药载体,检测其特异性。方法：化学合成直径为13nm的金纳米粒子,将NFD-1和载药GC序列修饰金纳米粒子构建载药载体,检测其与NAFLD细胞结合的特异性。结果：我们合成了直径为13nm的金纳米粒子,并成功修饰了NFD-1和载药GC序列,检测结果表明修饰了NFD-1的金纳米粒子能特异性结合NAFLD细胞。结论： NFD-1与载药GC序列修饰的金纳米粒子显示出良好的结合特异性。第三部分：修饰阿霉素并检测修饰了NFD-1和Dox的金纳米载药载体的靶向载药运输目的：将阿霉素(Doxycycline,Dox)修饰到金纳米载药载体并检测Dox的效率；检测修饰了核酸适配体NFD-1和Dox的的金纳米载药载体能否特异性进入NAFLD细胞。方法：共聚焦显微镜检测修饰了核酸适配体NFD-1和Dox的金纳米载药载体特异性进入NAFLD细胞。结果：修饰了载药GC序列的金纳米载药载体能结合300倍Dox的剂量,共聚焦显微镜显示修饰了NFD-1和Dox的的金纳米载药载体能特异性地进入NAFLD细胞。结论：修饰了核酸适配体NFD-1和Dox的金纳米载药载体能特异性地进入NAFLD细胞。更多还原

【Abstract】 Non-alcoholic Fatty Liver Disease (NAFLD) is a clinical pathological syndrome occurring when excessive triglycerides is deposited in the liver (>5%of liver tissue) not due to excessive alcohol and other factors may damage liver. NAFLD is associated not only with type2diabetes, obesity,but also with non-alcoholic steatohepatitis (NASH), liver cirrhosis or even liver cancer.In recent years,the incidence of NAFLD increased and has become a global public health problem. Current drug treatment for NAFLD is mainly for insulin resistance and oxidative stress. However, the effects of treatment is uncertain and there may be some rebound in risk of treatment interruption. In recent years, nanotechnology and aptamer technology have been increasingly used in drug research and become a hot topic in the biomedical field. In this paper, to aim at some difficulties and prospects faced by NAFLD therapy, we designed and constructed drug carrier based on gold nanoparticle and aptamer, and applied its on targeted drug delivery, with the goal of exploring the potential application of NAFLD therapy and providing new ideas for NAFLD research through studies across muiti-disciplines.Given the subject of design, this paper includes three parts:(1)Construstion of Non-alcoholic Fatty Liver Disease Cell mode, Selection of its Specific Aptamer and study the characteristics of aptamer NFD-1Objective:To construct differential model of non-alcoholic fatty Liver disease cell line (NAFLD) induced from HepG2cell line and select aptamers with high affility and specificity to the membrane proteins of NAFLD cell by the Cell-based systematic evolution of ligands by exponential enrichment Cell-SELEX).Analysis of the features of aptamers NFD-1,including specificity and affinity at4℃and37℃,as well as membrane protein binding affinity.Methods:According to protocol, we induced HepG2cells into NAFLD cells with100uM oleic acid and50uM palmitic acid for one week. We designed and synthesized random DNA library sequences flanked on both sides with fixed16bases primer sequences as well as25random bases in the middle. The two primers were labeled with fluorescein isothiocyanate (FITC) and biotin, respectively. We used differentiated NAFLD cells as positive cells and HepG2cells as negative cells to perform cell-SELEX. After repeated incubation,separation steps with positive and negative cells,the enriched pools which binding to the positive cells while not binding to the negative cells were amplified by polymerase chain amplification and then used for the next round of selection. The pool enrichment was monitored by flow cytometry every three to four rounds. Last, the enriched pools were then sequenced with454sequencing technology and their binding ability was identified by performing binding test with flow cytometry. At4℃and37℃,aptamer NFD-1was synthesized and tested the binding ability to a varity of cells from different tissues by using flow cytometry; Aptamer NFD-1was diluted to different concentrations and incubated with differentiated NAFLD cells. The binding fluorescence was obtained at each point by flow cytometry used for Kd determination. Trypsin and Proteinase K were used to detect the target of NFD-1.Results:According to the protocol, we have successfully constructed the NAFLD cell mode and selected two aptamers after twenty-four rounds of positive and negative selection processes.By sequencing analysis,one aptamer named NFD-1was selected capable of binding to NAFLD cells with high affility and specificity.At4℃and37℃, aptamer NFD-1can bind to differentiated NAFLD cells with high affility and specificity while no fluorescence can be observed when incubated with cells from other tissues. Dissociation constant determination showed that NFD-1can bind to NAFLD cell with Kd values of21.5±5.6nM. Protease detection confirmed that the target of NFD-1is a membrane protein.Conclusion:Applying Cell-SELEX technology to differentiated NAFLD cells, we successfully selected one aptamer named NFD-1with high affinity and specificity to NAFLD cells. At4℃and37℃, aptamer NFD-1can bind to differentiated NAFLD cells with high affinity and specificity.(2) Construction of gold nanoparticle based drug carrierObjective:Constructed the gold nanoparticle based drug carrier according to the protocol.Method:Gold nanoparticles with diameter of13nm were synthesized and decorated with selected NFD-1and GC sequences which can hold doxorubicin (Dox). Last, we test the affinity and specificity of drug carrier based on gold nanoparticle and aptemer to differentiated NAFLD cells.Results:Gold nanoparticles with13diameter were successfully synthesized and decorated with NFD-1and GC DNA sequences.Results shown that the drug carrier based on gold nanoparticle can bind to NAFLD cells with high affinity and specificity. Conclusion:The drug carrier based on gold nanoparticle also showed binding ability to differentiated NAFLD cells with high affinity and specificity.(3) Load of Dox and the study of targeted delivery of aptamerconjugated gold nanoparticle and dox to differentiated non-alcoholic fatty liver disease cellObjective:Load Dox on gold nanoparticle drug carrier and test whether the carrier decorated with aptamer NFD-1and Dox can specifically be endocytosed to the differentiated NAFLD cell.Method:The confocal was used to check whether the gold nanoparticle drug carrier can be endocytosed to the differentiated NAFLD cells.Results:The results showed that gold nanoparticle drug carrier decorated with aptamer NFD-1can hold more than three hundred times Dox and can specifically be endocytosed to the differentiated NAFLD cells whereas not to the HepG2cells.Conclusion:The gold nanoparticle drug carrier hold high carry Dox ability and can be targeted endocytosed to the differentiated NAFLD cells compared with HepG2cells.更多还原