高血压脑出血患者循环microRNAs表达谱的初步研究

发布时间：2018-05-29 16:24

  本文选题：微小核糖核酸 + 表达谱　； 参考：《南方医科大学》2014年硕士论文

【摘要】：研究背景 微小核糖核酸(microRNAs,miRNAs)是一类由约21—25个核苷酸组成的单链非编码小核糖核酸(RNA),广泛存在于植物动物和人基因组中,进化上高度保守,通过与靶mRNA3'非翻译区(3-untranslated regions,3'UTR),完全或者不完全互补配对的方式引起靶mRNA的降解或翻译抑制,从而实现在转录后水平对靶mRNA表达的调控作用,广泛参与了生物体的生长发育及许多生理及病理过程。 目前,许多研究发现,在人体的细胞外体液中,比如血浆、血清、尿液、唾液、脑脊液、腹水、胸水等中均可检测到miRNAs的存在。且已有研究表明血浆或血清中的miRNAs的性质比较稳定,它们一方面可以对抗核糖核酸酶的消化,另一方面还不受酸碱度变化、温度变化及放置时间等恶劣环境的影响。而且大部分miRNAs的序列是高度保守的。鉴于外周循环中miRNAs的稳定性、保守性,其有潜力成为比一般蛋白更为有效的生物学标记物。MiRNAs的发现成为生物学研究的一个热点。 高血压脑出血(hypertensive intracerebral hemorrhage, HICH)是以高血压为主要病因引起的原发性非外伤性脑实质内出血。在我国,随着人口老龄化的发展,HICH的发病率逐年上升。由于HICH起病急骤,且出血部位多位于脑深部,单纯外科手术难以达到满意效果,导致其具有高致残率及高致死率的特点,给个人、家庭及社会带来沉重的负担。因此,如何能够有效的预测HICH的发生成为目前的研究热点,这需要我们能够寻找到有效的分子生物学标记物,对其在HICH的发病机制、预后判断和靶向治疗中可能发挥的作用进行深入的研究和探讨。 目前,越来越多的研究表明不同疾病其外周循环(血清或血浆)中miRNAs有独特的表达改变,比如肿瘤、冠状动脉粥样硬化性心脏病、糖尿病等,并有可能作为诊断、判断预后、疗效评估的非侵入性生物学标志。关于脑梗塞与miRNAs的研究亦取得了一些成就,发现了一些miRNAs可以作为区别不同脑梗塞亚型的生物学标志,也研究了一些miRNAs在脑梗塞中的作用机制。但是在HICH中,国外尚无相关研究报道。 鉴于以上理论及实验基础,本研究将对HICH患者进行血浆miRNAs表达谱的分析,并挑选几个差异表达最明显的miRNAs进行初步验证,进一步采用生物信息学分析预测可能的靶基因推测miRNAs可能的作用机制。本研究将筛选出的差异表达的特异性miRNAs作为HICH高危人群中筛选的早期分子标记物具有非常重的意义,同时为后续的发病机制及靶向治疗的研究提供实验基础。 第一章HICH患者外周循环(血浆)microRNAs表达谱的分析 目的：研究高血压脑出血(hypertensive intracerebral hemorrhage, HICH)患者血浆与高血压患者血浆中差异性miRNAs表达谱,筛选与HICH相关的miRNAs。 实验方法： 1、研究对象的确定及研究标本的收集及处理 选取3名高血压脑出血患者与3名高血压患者作为研究对象。于清晨空腹抽取每位患者外周静脉血6ml,所有标本均使用医用EDTA抗凝管收集。所有标本均在2小时之内进行血浆分离。分离好的血浆置于-80℃保存备用。 2、血浆RNA的提取及质量检测 采用血浆/血清外泌体RNA提取试剂盒提取总RNA。总RNA的质量检测采用基于SYBR Green I的荧光定量PCR方法,以has-miR-16和has-miR-192作为质检指标。 3、miRNAs芯片检测 采用u ParaflorTM micoarray芯片筛选高血压脑出血患者与高血压患者血浆中差异表达的miRNAs。 4、统计学分析 本实验的计量资料的实验数据以均数±标准差(χ±s)来表示。计量资料的组间比较采用两独立样本的t检验,计数资料的组间比较采用χ2检验。采用SPSS13.0统计软件进行系统分析。定义P0.05为有统计学差异。 结果： 1、血浆RNA的质量检测 运用质检指标hsa-miR-16和hsa-miR-192,根据文献报道所建议的指标Ct值范围(hsa-miR-16的参考Ct值范围为16-22,hsa-miR-192的参考Ct值范围为25-30),判定此次提取的血浆RNA除了40号样品外,其余均合格,可进入下游芯片实验。40号样品提取的RNA中指标hsa-miR-16和hsa-miR-192, Ct值偏大,均超出文献规定的Ct值范围,且hsa-miR-192指标的溶解曲线出现双峰,导致该样品暂停下游实验。因40号样品为1例对照组样品,因此实际进入下游miRNAs芯片检测的总共有5个样本,即HICH组3例样本,对照组2例样本。 2、基因芯片的结果 对3例高血压脑出血组与2例高血压对照组血浆miRNAs表达谱进行比较,共筛选出9个差异性表达的miRNAs,其中表达上调的miRNAs有3个(hsa-miR-5787、hsa-miR-149-3p、hsa-miR-6786-5p),表达下调的miRNAs有6个(hsa-miR-29a-3p、hsa-miR-30c-5p、hsa-miR-5195-3p、hsa-miR-22-3p、 hsa-miR-342-3p、及hsa-miR-15b-5p)。无上调超过2倍的miRNAs,但hsa-miR-5787上调倍数最大；下调大于或等于2倍的hsa-miRNAs有3个：hsa-miR-29a-3p、hsa-miR-30c-5p、hsa-miR-5195-3p,以has-miR-29a-3p下调最为明显。 结论：初步建立起高血压脑出血患者血浆中miRNAs表达谱,相对于高血压患者,3个miRNAs在高血压脑出血患者血浆中表达上调,6个miRNAs表达下调。这些差异表达的miRNAs可能与高血压脑出血的发病相关。 第二章MicroRNAs基因芯片结果验证及生物信息学分析 目的：验证高血压脑出血患者血浆中差异性表达的miRNAs,并预测其在发病机制中可能所起的作用。 实验方法： 1、研究对象的确定及研究标本的收集及处理 选取20名高血压脑出血患者与15名高血压患者作为研究对象。于清晨空腹抽取每位患者外周静脉血6ml,使用医用EDTA抗凝管收集。所有标本均在2小时之内进行血浆分离。分离好的血浆置于-80。C保存备用。 2、血浆RNA的提取及质量检测 采用基于TRIzol法的RNA抽提技术,获取血浆总RNA。使用紫外吸收测定法及变性琼脂糖凝胶电泳法对提取的RNA进行质量检测。 3、RT-PCR 利用RT-PCR技术对筛选出的3个下调倍数最大的miRNAs: hsa-miR-29a-3p、hsa-miR-30c-5p、hsa-miR-5195-3p进行验证。 4、生物信息学分析 对筛选出的3个下调倍数最大的miRNAs:hsa-miR-29a-3p、 hsa-miR-30c-5p、hsa-miR-5195-3p进行靶基因预测。应用互联网miRNAs靶基因预测软件Pictar (http://pictar.mdc-berlin.de/)、MiRanda(http:///www.microma.org)、Targetscan (http://www.targetscan.org/)在线服务站点,输出差异表达miRNAs的预测靶基因。为了减少假阳性率,我们取至少2个软件预测到的基因作为靶基因。 5、统计学分析 本实验的计量资料的实验数据以均数±标准差(χs)来表示。计量资料的组间比较采用两独立样本的t检验,计数资料的组间比较采用χ2检验。采用SPSS13.0统计软件进行系统分析。定义P0.05为有统计学差异。 结果： 1、RNA质量检测结果 本部分实验所提取的RNA样品,用紫外分光光度计测其各自的A260和A280,得到所有样品RNA的A260/A280的值的范围均在1.8-2.1之间。变性琼腊糖凝胶电泳结果提示：所提取RNA样品的28S：18S均接近2：1,且电泳条带较清晰,无明显弥散拖尾现象及杂条带,说明所提取的RNA完整性较好,可进入下游RT-PCR实验。 2、RT-PCR结果 为了验证第一步基因芯片的结果,我们挑选了3个下调倍数最大的miRNAs:hsa-miR-29a-3p、hsa-miR-30c-5p、hsa-miR-5195-3p,在20例HICH患者和15例对照者中进行RT-PCR验证,以hsa-miR-93作为内参基因。相对于高血压患者,hsa-miR-29a-3p下调倍数仍最大,超过2倍,且比芯片下调倍数明显；hsa-miR-30c-5p和hsa-miR-5195-3p仍呈现表达下调,但下调倍数不及芯片下调倍数明显,但是下调趋势与芯片结果相同。 3、生物信息学分析结果 我们应用互联网miRNAs靶基因预测软件(Pictar、MiRanda、Targetscan)在线服务站点,对显著下调的niRNAs:hsa-miR-29a-3p、hsa-miR-30c-5p、 hsa-miR-5195-3p进行靶基因预测。为了减少假阳性率,我们取至少2个软件预测到的基因作为靶基因。hsa-miR-29a-3p共预测到103个靶基因,hsa-miR-30c-5p预测到103个靶基因。由于目前对hsa-miR-5195-3p研究较少,尚无hsa-miR-5195-3p靶基因的相关信息。其中,我们注意到,hsa-miR-29a-3p预测的靶基因中包括COL1A2。 对靶基因分析后发现,靶基因作用广泛,涉及蛋白质的合成及修饰、转录过程的调节、炎性反应、血管生成、调节信号转导通路、以及细胞的凋亡、分化、增殖等。 结论： 1、RT-PCR结果与基因芯片的结果具有一致趋势,说明基因芯片结果真实可靠。 2、实验筛选出的差异表达的miRNAs (hsa-miR-29a-3p、hsa-miR-30c-5p、 hsa-miR-5195-3p),它们可以作为HICH的分子生物标志物,也可能可以作为高血压患者中易发生HICH的高危人群的筛查指标,且有可能参与了HICH的发病机制。 3、hsa-miR-29a-3p的靶基因之一为COL1A2,与Ⅰ型胶原蛋白合成相关。hsa-miR-29a-3p可能通过对COL1A2的调控,减弱了血管壁的弹性及抗压能力,从而参与了高血压脑出血的发生发展过程。hsa-miR-30c-5p可能通过对靶基因的DLL4的调控,从而影响血管的形成及发展,进而参与HICH的发生。hsa-miR-5195-3p的作用机制需要进一步探讨。
[Abstract]:Research background
MicroRNAs (miRNAs) is a kind of single strand non coded small ribonucleic acid (RNA), which consists of approximately 21 to 25 nucleotides. It exists widely in plant and human genome. It is highly conserved in evolution. Target mRNA is caused by complete or incomplete complementarity with the target mRNA3'non translation region (3-untranslated regions, 3'UTR). Degradation or translation inhibition, thus realizing the regulatory role of post transcriptional level on the expression of target mRNA, is widely involved in the growth and development of organisms and many physiological and pathological processes.
At present, many studies have found that the presence of miRNAs in human body fluid, such as plasma, serum, urine, saliva, cerebrospinal fluid, ascites, and hydrothorax, has been detected. And studies have shown that the properties of miRNAs in plasma and serum are more stable, one side can resist the digestion of ribonuclease, on the other hand it is not acid. Most of the miRNAs sequences are highly conserved. In view of the stability and conservatism of miRNAs in the peripheral circulation, the discovery of.MiRNAs, which has the potential to be a more effective biomarker than the general protein, has become a hot spot in biological research.
Hypertensive intracerebral hemorrhage (HICH) is a primary non traumatic cerebral parenchymal hemorrhage caused by hypertension. In China, with the development of the population aging, the incidence of HICH is rising year by year. Because of the rapid onset of HICH and the location of the blood in the deep part of the brain, the simple surgical operation is difficult to reach. Satisfactory results, resulting in high disability and high mortality, bring a heavy burden to individuals, families and society. Therefore, how to effectively predict the occurrence of HICH has become a hot spot of research. It is necessary for us to find effective molecular biological markers, the pathogenesis of HICH, the prognosis and the target. To further study and explore the possible role of treatment.
At present, more and more studies have shown that miRNAs has unique expression changes in peripheral circulation (serum or plasma) of different diseases, such as tumor, coronary atherosclerotic heart disease, diabetes and so on, and may be a noninvasive biological marker for diagnosis, prognosis and evaluation of curative effect. The study of cerebral infarction and miRNAs has also been obtained. Some of the achievements have been found. Some miRNAs can be used as a biological marker to distinguish different cerebral infarction types, and some of the mechanisms of miRNAs in cerebral infarction are also studied. But there is no related research abroad in HICH.
In view of the above theoretical and experimental basis, this study will analyze the plasma miRNAs expression profile of HICH patients, and select several miRNAs with the most distinct expression for preliminary verification, further using bioinformatics analysis to predict the possible mechanism of the possible target genes to predict the possible effect of miRNAs. This study will screen the specificity of differential expression. Sexual miRNAs is of great significance as an early molecular marker selected in high risk population of HICH, and provides an experimental basis for subsequent pathogenesis and targeted therapy.
Chapter 1 Analysis of microRNAs expression profiles in peripheral blood (plasma) of patients with HICH
Objective: To study the differential miRNAs expression profiles in plasma of patients with hypertensive cerebral hemorrhage (hypertensive intracerebral hemorrhage (HICH) and hypertensive patients and to select miRNAs. related to HICH.
Experimental methods:
1, the determination of research objects and collection and processing of research specimens.
3 hypertensive intracerebral hemorrhage patients and 3 hypertensive patients were selected as subjects. The peripheral venous blood 6ml of each patient was extracted at the early morning. All specimens were collected with medical EDTA anticoagulant tube. All specimens were separated within 2 hours. The separated plasma was stored at -80 C.
2, the extraction and quality detection of plasma RNA
The quality detection of total RNA. total RNA extracted from plasma / serum exo RNA extraction kit was based on the SYBR Green I fluorescence quantitative PCR method, with has-miR-16 and has-miR-192 as the quality test index.
3, miRNAs chip detection
Screening of differentially expressed miRNAs. in patients with hypertensive intracerebral hemorrhage and hypertension by using u ParaflorTM micoarray chip
4, statistical analysis
The experimental data in this experiment were represented by mean number of standard deviation (chi square) difference (chi square s). The comparison between groups of measurement data was compared with the t test of two independent samples. The comparison between the counting data was compared with the x 2 test. The SPSS13.0 statistical software was used to make a systematic analysis. The statistical difference was defined in the definition of P0.05.
Result锛，

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