初探血浆外泌体中miRNA在散发性帕金森病临床诊断中的价值

发布时间：2018-08-16 12:22

【摘要】：背景:帕金森病(Parkinson's disease,PD)是世界上第二常见的神经系统变性疾病。研究发现,PD的发生与年龄密切相关,发病率在65岁以上老年人为1.7%,70岁以上老年人达到了3-5%。目前PD的临床诊断主要是根据病史、临床症状和体征及患者对多巴胺能药物的治疗反应。但因PD在疾病早期症状不典型,首发症状常为震颤、运动迟缓或肌强直,而这些临床症状出现时,事实上多巴胺能神经元变性缺失了50%-70%,这往往使患者错失了早期诊断、早期治疗的机会。且早期易与多系统萎缩、进行性核上性麻痹、皮质基底节变性和路易体痴呆等疾病相混淆,误诊率较高。文献报道,PD的临床与病理诊断符合率仅为76%左右。因此探讨寻找一种有助于提氋早期PD临床诊断的正确率、且无侵入性和创伤性的生物标志物,对于早期有效治疗PD以及延缓疾病的进展,都具有非常重要的意义。近年来,PD生物标志物的研究主要集中在脑脊液和血浆/血清,但脑脊液的获取需行腰椎穿刺,有创,病人不易接受,使用受限。血浆/血清由于取样快速、简单、成本低和微创,适合作为生物标志物的来源。目前来源于血浆/血清的PD生物标志物主要是关于DJ-1、α-突触核蛋白和miRNA,但DJ-1、α-突触核蛋白的表达水平不同研究的结果间存在差异,甚至相互矛盾;并且其浓度与PD的严重程度无相关性。近十年研究发现miRNA敏感度和特异度较高,并且部分miRNA的表达水平与PD的严重程度相关,适合作为PD的生物标志物进行研究。microRNA(miRNA)是一种小的由21-24个核苷酸组成的单链非编码RNA。研究发现在人血清中存在各种miRNAs,且这种表达谱在机体的不同状态时呈现特定的变化。已有研究表明,与正常对照健康人相比,帕金森病人的血浆/血清中miRNAs表达谱明显不同。但是不同的研究得出的结果也有所不同,且稳定性和重复性均不足。推测除了采用的方法不同、研究对象不同、验证的样本数量不同外,更重要的是以下两方面的影响:1、血液中成分复杂,获取的miRNA种类与表达水平变化较快,干扰结果的稳定性;2、由于血脑屏障的存在,血液与大脑缺少直接的联系。直接用血液的检测结果不能客观真实的反映颅内疾病的状态。目前研究显示,mirna在体外主要有三种稳定存在的形式:1、与高密度脂蛋白结合,但这种情况具有高度细胞特异性,主要是存在于肝细胞中;2、与ago2蛋白结合,但这种形式尚无令人信服的研究证明其可介导功能mirna的转运,3、包裹于外泌体等膜囊泡中,这是近年研究的热点。外泌体是一种膜性小囊泡,直径约30-100nm,可由多种细胞在生理和病理条件下分泌,并存在于多种体液中。它通过转运蛋白质、脂质、dna、mrna和mirna等物质发挥功能。最近研究表明,外泌体由于其体积小,可透过血脑屏障从中枢神经系统转移至体循环,并且外泌体由于具备双膜结构能抵抗血液中核糖核酸酶的攻击,mirna不易降解,稳定性高。这提示血浆中外泌体所携带的mirna可能更真实地反映颅内情况,具备作为帕金森病早期诊断生物标志物的潜力。目的:筛选出pd患者血浆外泌体中特异的mirna表达谱,并进行靶基因预测及kegg通路分析,qrt-pcr验证,评价其对pd诊断的价值。方法:1、收集了51例散发性pd患者血浆和50例年龄/性别匹配的健康人血浆;2、用invitrogen试剂盒从血浆中分离外泌体,通过扫描电镜观察外泌体形态、qnano测定浓度及大小分布,westernblot检测外泌体标记蛋白cd63、alix的表达以对其进行鉴定;3、使用高通道测序检测两组血浆外泌体mirna的表达差异。4、使用mirna靶基因预测软件对差异表达的mirna进行靶基因预测,并对预测的靶基因进行kegg通路分析。5、采用使用taqman做探针的qrt-pcr技术验证初筛得到的12种差异显著的mirnas。结果:1、从血浆中分离得到mode直径100nm、电镜下表现为由脂质双分子层包裹的圆形或杯口状囊泡、表达外泌体特异性标记蛋白cd63和alix的粒子,鉴定为外泌体。2、与对照组miRNA相比,PD组有66个miRNA表达明显差异(Ratio2.0,P0.05),其中27个表达上调,39个表达下调。3、预测到的与PD相关靶基因参与了ECM相互作用受体信号通路、神经因子营养通路、VEGF信号通路、TGF转化因子信号通路、白细胞跨内皮转移信号通路等信号通路。4、对初筛的表达差异显著的12种miRNAs进一步运用qRT-PCR技术验证。验证后发现有6种miRNAs(hsa-miR-195、hsa-miR-302e、hsa-mi R-15b hsa-miR-485-5p、hsa-miR-4483、hsa-miR-493-3p)在患者血浆外泌体中的表达与健康人血浆外泌体相比有显著差异;hsa-mi R-195、hsa-miR-302e显著上调,而hsa-miR-15b hsa-miR-485-5p、hsa-miR-4483、hsa-miR-493-3p显著下调。结论:1、本研究成功分离并鉴定了血浆外泌体。2、PD患者血浆外泌体中miRNA表达谱与正常人具有显著差异;我们发现PD患者血浆外泌体中mi RNA hsa-miR-195、hsa-mi R-302e显著上调,而hsa-miR-15b hsa-miR-485-5p、hsa-miR-4483、hsa-miR-493-3p显著下调;这6种miRNAs组合可能作为潜在的生物标志物进行PD诊断。
[Abstract]:BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disease in the world. It has been found that the incidence of PD is closely related to age. The incidence of PD is 1.7% in the elderly over 65 years old and 3-5% in the elderly over 70 years old. But the first symptoms of PD are often tremor, bradykinesia or myotonia because of atypical symptoms in the early stage of the disease. When these symptoms occur, in fact, the degeneration of dopaminergic neurons is lost by 50% - 70%, which often makes the patient miss the opportunity of early diagnosis and treatment. The clinical and pathological diagnostic coincidence rate of PD is only about 76%. Therefore, to explore a biomarker which is helpful to the clinical diagnosis of early PD, and is non-invasive and traumatic, for the early effective treatment. In recent years, the research on biomarkers of PD is mainly focused on cerebrospinal fluid and plasma/serum, but the acquisition of cerebrospinal fluid requires lumbar puncture, which is invasive, difficult for patients to accept and limited to use. At present, the main PD biomarkers derived from plasma/serum are DJ-1, alpha-synuclein and microRNA, but the results of different studies on the expression levels of DJ-1, alpha-synuclein are different, even contradictory, and their concentrations are not correlated with the severity of PD. MicroRNA (microRNA) is a small, single-stranded, non-coding RNA consisting of 21-24 nucleotides. Various kinds of microNAs are found in human serum, and the expression profiles are specific in different states of the body. Previous studies have shown that the expression profiles of microRNAs in plasma/serum of Parkinson's disease patients are significantly different from those of normal controls. However, the results of different studies are different, and the stability and repeatability are insufficient. The following two aspects are needed: 1. The composition of blood is complex, the type and expression of microRNAs acquired change quickly, interfering with the stability of the results; 2. Because of the existence of blood-brain barrier, blood and brain lack of direct contact. There are three main stable forms of Na in vitro: 1. binding to high-density lipoprotein, but this condition is highly cell-specific and mainly exists in hepatocytes; 2. binding to ago2 protein, but there is no convincing evidence that this form can mediate the transport of functional mirna, 3. encapsulating in the membrane vesicles such as exosomes, which is The exosome is a membrane vesicle with a diameter of about 30-100 nm. It can be secreted by a variety of cells under physiological and pathological conditions and exists in a variety of body fluids. The central nervous system metastases to the systemic circulation, and the exosome has a bimembranous structure which can resist the attack of ribonuclease in the blood, so the miRNA is not easy to degrade and has high stability. Specific miRNA expression profiles in plasma exosomes of PD patients were selected and analyzed by target gene prediction, KEGG pathway analysis and qRT-PCR to evaluate their diagnostic value. Exosome morphology was observed by electron microscopy, the concentration and size distribution of qnano were measured, and the expression of exosome marker protein CD63 and Alix were detected by Western blot for identification. 3. the expression of miRNA in plasma exosome was detected by high channel sequencing. The predicted target genes were analyzed for KEGG pathway. 5. Twelve different miRNAs were identified by qRT-PCR using TaqMan probe. Results: 1. Model was isolated from plasma with a diameter of 100 nm. Under electron microscopy, the model showed round or cup-shaped vesicles encapsulated by lipid bilayer and expressed exosome-specific marker proteins CD63 and cd63. Alx particles were identified as exosomes. 2. Compared with the control group, 66 microRNAs were significantly different in PD group (Ratio 2.0, P 0.05). 27 of them were up-regulated and 39 were down-regulated. 3. The predicted PD-related target genes were involved in ECM receptor signaling pathway, neurotrophic pathway, VEGF signaling pathway, TGF transforming factor signaling pathway. Six kinds of microRNAs (hsa-microRNAs-195, hsa-microRNAs-302 e, hsa-mi R-15b hsa-microRNAs-485-5p, hsa-microRNAs-4483, hsa-microRNAs-493-3p) were found to be expressed in the plasma of the patients and in the blood of healthy subjects. Hsa-mi R-195, hsa-mi-mi-302e were significantly up-regulated, hsa-mi-r-195, hsa-mi-15b hsa-mi-485-5p, hsa-mi-4483, hsa-mi-mi-493-3p were significantly down-regulated. CONCLUSIONS: 1. Plasma exosomes were successfully isolated and identified. 2. The expression profiles of microRNA in plasma exosomes of PD patients were significantly different from those of normal controls. MiRNA hsa-Mi-195, hsa-mi R-302 E were significantly up-regulated in the exosome, while hsa-Mi-15b hsa-Mi-485-5p, hsa-Mi-4483, hsa-Mi-493-3p were significantly down-regulated; these six microRNAs combinations may serve as potential biomarkers for PD diagnosis.
【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R742.5

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