【作者】 张斌；

【导师】 陈春富；

【作者基本信息】 山东大学 ， 神经病学（专业学位）， 2015， 博士

【摘要】 背景与目的阿尔茨海默病(Alzheimer’s disease, AD)是发生于老年和老年前期的一种渐进性的神经退行性疾病,并且是老年人口中最常见的痴呆性疾病,它在临床上主要表现特征为慢性进行性记忆力减退、失语、失用、失认、视空间能力损害、抽象思维和计算力损害以及人格行为改变等。目前普遍认为阿尔茨海默病是由于神经元变性引起的,而后者又是由于神经纤维缠结和神经炎性斑所导致。据统计,65岁以上老年人约有5%患有AD,2006年,阿尔茨海默病在世界范围内达到2660万,到2050年,全球预计达到85个人中就有一个阿尔茨海默病患者的比例。目前,对于阿尔茨海默病的治疗只能对症,仍然没有可行的治疗来预防或延缓疾病的进程的方案。同时,对于阿尔茨海默病的发病原因目前也尚不清楚。至今为止,关于阿尔茨海默病发病原因有多种假设,其中最经典的假设为阿尔茨海默病是由一个Aβ42异常的淀粉样蛋白的沉积造成的,而淀粉样前体蛋白(amyloid precusor protein, APP)异常也会加快该疾病的进展。阿尔茨海默病神经方面的病理变化是由神经元和突触的损伤所造成的,细胞内神经原纤维呈缠结排列,细胞外出现淀粉样斑块。最近,越来越多的研究表明,神经内分泌免疫网络失调也是AD发病的机制之一。身体的免疫系统炎症与阿尔茨海默病密切相关,而先天性免疫系统对脑代谢、神经保护和修复等方面是至关重要的。一旦免疫系统和炎症信号途径被激活,机体就会产生过量的氧自由基,其次促炎性细胞因子以及前列腺素也会大量表达,进而引发炎症级联反应,从而导致神经退行性疾病和阿尔茨海默病的发生和进展。基于以上原因,关于阿尔茨海默病的发病机理目前仍然需要更深入的研究。microRNAs(微小RNA,miRNAs)是一种长度为21-25个核苷酸、成熟的内源性、非编码的单链小分子并具有高度保守的RNAs,它主要在转录后水平发挥作用,通过靶向3’端未翻译区域,干扰靶mRNA切割或抑制翻译来调控基因的表达。已经有研究证实,miRNA参与了不同的生物学进程,在多种人类疾病中发挥作用并参与几乎所有的生物学进程,包括疾病的发展、细胞分化、增殖与死亡。研究发现,miRNA的异常表达与多种疾病的发生、发展和预后都有密切联系。目前,有学者认为出现在miRNA或其结合部位的单核苷酸多态性(single nucleotide polymorphisms, SNPs)是导致miRNA遗传变异的的异常源头,从而导致癌症等疾病易感性的差别。研究者认为,基因组内特定核苷酸位置上存在两种不同的碱基,其中最少一种在群体中的频率不小于1%,这种基因组的单个位点上的碱基的差异称为SNPs,它是存在于人群中每个个体的基因序列细微差别,在整个人类基因组中分布相当广泛,其具有的生物学功能,可以通过影响成熟miRNA的二级结构、表达水平及其与靶基因的识别,使miRNA的调控网络发生异常,进而与癌症等疾病的发生发展密切相关。研究提示miRNA可能在阿尔茨海默病的发生和发展中发挥了重要作用。目前,研究报道在阿尔茨海默病患者的血液中发现多种miRNA的异常表达,其中包括miR-137, miR-181c, miR-9, miR-16和miR-29a/b等。进一步的研究表明,miR-146参与到炎症信号的上调过程中,而炎症信号的上调与朊病毒诱导的神经退行性变、类风湿及颞叶癫痫病有很大的相关性。另外,在阿尔茨海默病患者的脑组织中,发现miR-146的异常表达,这就提示异常的miR-146表达可能是导致阿尔茨海默病患者炎性老年斑病变形成的重要因素。同时研究者也发现,在阿尔茨海默病病人脑组织及五个不同的阿尔茨海默病动物模型中,miR-146a呈现异常表达的现象,其次在阿尔茨海默病的解剖神经病理组织中,也同样发现了miRNA-146a的异常表达,这些均提示miR-146a可能在阿尔茨海默病中发挥重要的作用。但是由于上述研究存在样本量少的现象,因此亟需大量的广泛的研究以明确miRNA-146a与阿尔茨海默病的相关性。在本研究中,我们对103例阿尔茨海默病患者进行pri-miR-146a基因组DNA的序列分析,虽然没有发现明显的基因组系列的变化,但是我们发现,在阿尔茨海默病患者中存在pri-miR-146a的核苷酸多态性。进一步通过qRT-PCR分析,我们发现C等位基因能够明显降低成熟的miR-146a的表达。重要的是,miR-146a表达的降低又进一步导致抑制性靶基因TLR2的高表达,而TLR2能够促进小神经胶质细胞的炎性反应,从而进一步导致阿尔茨海默病的发生。研究方法1.血液标本的收集本病例组选取作者所在医院的103例AD病例为研究对象,同时以206例健康志愿者为正常的研究对照。所有患者均为无血缘关系的独立的汉族人群。正常对照组从医院的健康检查中心随机选取,无明显的临床症状。按年龄匹配相应的对照,抽取两者外周血。研究对象必须签署知情同意书,并提供相关的完整的流行病学调查。2.细胞培养实验对HEK293T及RAW264.7细胞在含有10%肽牛血清、100IU/ml的青霉素及10 mg/mL链霉素的DMED培养基中按照常规培养方案,于37℃、5%CO2培养箱进行培养。3.RNA的提取与miR-146a的检测实验用TRIzol分别提取样本中的总RNA,反转录为cDNA,运用Quantitive RT-PCR测出样本中成熟的miR-146a与miR-146a前体的含量。4.载体构建PCR获得各突变体的片断,胶回收各突变体的片断,酶切回收片段与载体,将片段与载体进行连接反应后,转化挑克隆,进行PCR及酶切鉴定。5. miR-146a的瞬时转染实验选miR-146a表达水平低的HEK293T细胞作转染实验,HEK293T接种于12孔板中,用miR-146a mimic及miR-146a mimicNC对HEK293T细胞进行转染,然后分析转染后miR-146a对HEK293T细胞产生的相对应的影响。6.双荧光酶活性分析HEK293T接种于48孔板中,采用lipo 2000将荧光素酶报告基因与miR-146a模拟物及抑制物共同进行转染,2天之后对转染细胞进行收取,采用双荧光素报告基因系统检测基因的表达。每个实验需要重复三次。结果表示为相对荧光素酶活性。7. Western blotting通过琼脂糖凝胶电泳对蛋白进行分离,然后将琼脂糖凝胶的蛋白电转到PVDF膜上,膜经过单克隆抗体孵育后,特异性蛋白复合物再与辣根过氧化物酶偶联后,暗室曝光显影,即为所分析蛋白的表达水平。8. ELISA采用ELISA试剂盒检测TNF-a的分泌水平。9.统计学分析数据分析采用SPSS 16.0统计软件进行,进行研究数据的精确分析,计量资料采用均数±标准差表示,采取t检验比较两组间的差异,多组间比较采用单因素方差分析,P<0.05表示差别具有统计学意义。研究结果1.在阿尔茨海默病患者的外周血中rs2910164中异常C等位基因明显增加。为了分析pre-miR-146核苷酸多肽性与阿尔茨海默病的相关性,采用Taqman等位基因鉴别仪,对103例阿尔茨海默病患者与206例配对正常人血液样本中pre-miR-146a编码区核苷酸多肽性进行检测。结果发现103例阿尔茨海默病患者血液样本与配对的206例正常人血液样本比较,稀有等位基因C的表达在阿尔茨海默病患者血液样本中明显增加。2.异常C等位基因降低体内外miR-146a的表达。将不同基因型的pre-miR-146a表达载体转染HEK293T细胞,分析成熟的miR-146a的表达,结果发现,与rs2910164 G及GC杂合子基因型相比,C基因型降低成熟的]miR-146a表达量达到32%。进一步体内对阿尔茨海默病患者血清样本中pri-miR-146a不同基因型对于miR-146a表达的分析发现,与rs2910164 GG基因型携带阿尔茨海默病患者相比,CC基因型携带患者mir-146a表达水平明显降低。3. miR-146a表达的降低导致其抑制性靶基因TLR2的表达上调。为了验证TLR2是否是miR-146a的靶基因,将828bp的3’-UTR克隆至含有荧光素酶报告基因的pGL3载体中,形成双荧光系统的pGL3-TLR2质粒,将双荧光系统的pGL3-TLR2质粒与niR-146a、miR-146a模拟物及抑制物共同转染至HEK293T细胞中,结果发现,与miR-146a模拟物及抑制物共同转染至HEK293T细胞中相比,转染miR-146a组荧光强度明显降低,即miR-146a能够抑制TLR2的表达,同时也明显抑制TLR2蛋白的表达。4.RAW264.7细胞在Aβ42的刺激上,pri-miR-146a C基因型能够上调TNF-α的分泌。在细胞系水平分析异常等位基因C的功能,通过转染pri-miR-146a不同基因型载体至RAW264.7细胞,研究发现,在Aβ42的刺激上,转染pri-miR-146a-C基因型的细胞比pri-miR-146a-G基因型的细胞上清TNF-α的分泌水平增加71.1%。研究结论1.阿尔茨海默病患者存在pre-miR-146a的单核苷酸多肽性；2. miR-146a多态位点rs2910164 C等位基因可能导致阿尔茨海默病的易感性；3. miR-146a在阿尔茨海默病的发生发展中发挥重要的作用。更多还原

【Abstract】 Background and objectAlzheimer’s disease (AD) is a progressive neurodegenerative disorder that is characterized by progressive memory loss、aphasia、out of use、agnosia、 visual spatial disorder、abstract thinking and computational power and behavioral changes and the most common form of dementia among the aging population. AD is characterized by neurodegeneration that is associated with neurofibrillary tangles and neuritic plaques. There are about 5%people in the old people that is over 65 years old.In 2006, the worldwide prevalence of AD was 26.6 million, and by 2050, AD is predicted to attain 1 in 85 people globally. At present, treatment options to AD can only be used according to its symptoms, and there are no available treatments to the prevention or to delay the disease process. However, the molecular mechanisms that underlie this relationship are not fully understood.The neuropathological alterations of AD are characterized by loss of synapses and neurons, intracellular neurofibrillary tangles and extracellular amyloid plaques formation. The classical hypothesis for the cause of AD is aberrant amyloid protein deposition of Aβ42, due to abnormal amyloid precursor protein (APP) processing. Recently, an increasing number of studies indicate that dysfunctions of neuroimmune networks also contribute to the pathogenesis of AD.The immunue system of the body is related to AD, and the congenital immune system is important to cerebral metabolism,nerve protection and restoration. If the immunue system and the inflammatory signaling pathways are activated,the body can overproduct oxygen free radicals, and the promoting inflammatory cytokines and prostaglandins will express largely,the inflammatory cascade reaction may be caused. At last the result is neurodegenerative disorder and may cause AD. Based on the above,now the pathogenesis of AD is also needed to further study.MicroRNAs (miRNAs) are a family of mature,21 to 25 nucleotide-long small noncoding and highly conserved RNA gene products that regulate gene expression by base pairing with target mRNAs at the 3’-untranslated region, leading to mRNA cleavage or translational repression. It has been suggested that miRNAs are involved in various biological processes, the dysregulation of miRNA has been implicated in the development of different pathologies, including the development of disease, cell differentiation, cell proliferation and cell death.It has been reported that the abnormal expression of miRNA is related to the occurrence,the development and prognosis of many diseases.Single-nucleotide polymorphisms (SNPs) are the most common-types of genetic variation in the human genome and contribute to human phenotypic differences. Currently, some researchers believed that the single nucleotide polymorphisms (SNPs) in miRNA or it’s binding sites were the abnormal source of genetic variation, which contributed to cancer susceptibility. There were two different nucleotides on specific position in the genome; whose frequency was no less than 1% in the population. The difference of a single nucleotide of this genomic is known as SNPs, which has a specific biology, can regulate the expression of miRNA and effect its regulatory functions, thus has significant association of the tumor development. SNPs are the minor differences in gene sequence which are present in each individual human population and fairly well be distributed throughout the human genome.Dysregulated serum miRNA events, such as the down-regulation of miR-137, miR-181c, miR-9,miR-16 and miR-29a/b in the blood of AD patients,were reported. Furthermore, prior studies pointed out that miR-146 was involved in up-regulated inflammatory signaling as associated with prion-induced neurodegeneration, rheumatoid arthritis and temporal lobe epilepsy. Particularly, the upregulation of miR-146 has also been demonstrated in human AD brain, suggesting that the misregulation of miR-146 could contribute to the inflammatory senile plaques pathology observed in AD, it has also been identified to play a crucial role in AD since aberrant expression of miR-146a has been shown in five different AD transgenic mouse models and human AD brain. Notably, miRNA-146a has been found to be particularly up-regulated in anatomical regions exhibiting AD neuropathology, while unchanged in the brain regions unaffected.However,due to the small sample sizes in the above reports, there is a lack of an extensive and genomewide analysis for the diagnostic value of miRNAs in AD patients.In this study, we sequenced pri-miR-146a in the genomic DNA of 103 AD patients. Although no previously undescribed sequence changes were detected, we noticed one normal polymorphism site was related to AD. Detected by using qRT-PCR, we proved the conclusion that the rare allele C could reduce mature miR-146a expression. More importantly, the reduction of miR-146a further led to less efficient inhibition of target genes like TLR2, which enhanced microglia participated inflammation.And at last it could result in AD.Methods1. Collected specimensA total of 103 patients with AD and 206 healthy controls were recruited from the hospital of the author.. All recruited people were Han population. All of the patients are independent of unrelated han population. Normal control group randomly selected from the hospital health examination center. And no obvious clinical symptoms. Matching the corresponding control according to age. We collected their blood samples. The study was approved by Ethics Committee of the hospital of the author,and informed consent was obtained from all participants.2. Cell cultureRAW264.7, HEK293T cells were cultured in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum,100 IU/ml penicillin and 10 mg/mL streptomycin. All cells were maintained at 37℃ under an atmosphere of 5% CO2.3. RNA extraction and the detection of miR-146aTotal RNA from samples and cell lines were obtained with the TRIzol isolation reagent, and the total RNA were reverse transcribed to synthesize cDNA. qRT-PCR method was used to quantify mature miR-146a and pri-miR-146a.4. vector constructionObtaining the mutants of clips by PCR, gel extraction, enzyme digestion the mutants of clips and vector, and then connection, conversion and pick the cloning, then identified by PCR and enzyme digestion.5. Transient miRNA-146a transfectionHEK293T cell was chosen to do the transfection experiments which had the lowest expression of miR-146a. HEK293T cells in 12-well plates were transfected with miR146a precursor (miR-146a mimic) or scrambled pre-miR negative control (miR-146a mimic NC), then measured the influence of miR-146a to the HEK293T cell.6. Dual luciferase assayFor luciferase reporter assays, HEK293T cells were seeded in 48-well plates. MiR-146a mimic/inhibitor and luciferase reporter vectors were co-transfected by using lipofectamine 2000. Two days later, cells were harvested and assayed with the Dual-Luciferase Assay. Each treatment was performed in triplicate in three independent experiments. The results were expressed as relative luciferase activity.7.Western blottingThe proteins were separated by electrophoresis, and the proteins in the gels were blotted onto PVDF membranes by electrophoretic transfer. The membrane was incubated with monoclonal antibody. The specific proteinantibody complex was detected by using horseradish peroxidase conjugated goat anti rabbit or rabbit anti-mouse IgG. Detection by the chemiluminescence reaction was carried using the ECL kit. The β-actin signal was used as a loading control.8. ELISAUsing the kit of ELISA to detect the level of TNF-a in the cell supernate. 9. Statistical analysisBiostatistical analyses were performed using SPSS 16.0.Student’s t-test, one-way ANOVA, or x2-test was using to test the difference among groups. Pearson’s x2-test was used to compare the the basic situation between the case group and the control group. P< 0.05 was considered statistically significant.Results1.The rare allele C of rs2910164 is significantly increased in ADTo wander, if there were some relations between nucleotides variant of pre-miR-146 and the pathogenesis of AD, we scanned pre-miR-146a coding region in 103 AD patients and 206 healthy controls by Taqman. we noticed the rare allele C was abnormally increased in the blood of AD2.The C allele can reduce miR-146a expression in vitro and in vivoDetecting mature miR-146a amount in different genotype pri-miR-146a expression vectors transient transfected HEK293T cells by qRT-PCR. As expected, C allele reduced mature miR-146a expression to 32% relative to G allele. To understand whether this SNP affect the expression of miR-146a in vivo; we detected miR-146a expression level in the serum of Chinese-Han people between GG and CC genotype by qRT-PCR, the expression of miR-146 was used as the internal normalization control. Our data demonstrated that the miR-146a level of CC genotype in AD patients was significantly down-regulated.3.The expression of TLR2 is repressed by miR-146aTo validate whether TLR2 be the target gene of miR-146a, the full length of 828bp segment of TLR23’-UTR was cloned into the downstream of the firefly luciferase reporter gene in the pGL3 control vector (designated as pGL3-TLR2) for the dual luciferase assay. HEK293T cells were co-transfected with pGL3-TLR2 and miR-146a mimics or inhibitor. Compared with the miRNA control, the luciferase activity was significantly suppressed by the miR-146a.4.The rare allele C in pri-miR-146a up-regulates RAW264.7 cells TNF-α production when Aβ42 stimulatedWe detected the biological function of rare allele C in this cell line. RAW264.7 cells were co-transfected with different allele pri-miR-146a, As shown, compared with the cells transfected with empty vector, the supernatant TNF-α amount raised 71.1% in the cells transfected with pri-miR-146a-C when they were stimulated by Aβ42.Conclusions1. There is nucleotides variant of pre-miR-146 in ADs;2. MiR-146a nucleotides variant rs2910164 C allele is related to the risk of suffering ADs;3. MiR-146a plays an important role in the occurrence of ADs.更多还原