本文選題：微小核糖核酸 + 表達(dá)譜�。� 參考：《南方醫(yī)科大學(xué)》2014年碩士論文

【摘要】：研究背景 微小核糖核酸(microRNAs,miRNAs)是一類由約21—25個核苷酸組成的單鏈非編碼小核糖核酸(RNA),廣泛存在于植物動物和人基因組中,進(jìn)化上高度保守,通過與靶mRNA3'非翻譯區(qū)(3-untranslated regions,3'UTR),完全或者不完全互補(bǔ)配對的方式引起靶mRNA的降解或翻譯抑制,從而實現(xiàn)在轉(zhuǎn)錄后水平對靶mRNA表達(dá)的調(diào)控作用,廣泛參與了生物體的生長發(fā)育及許多生理及病理過程。 目前,許多研究發(fā)現(xiàn),在人體的細(xì)胞外體液中,比如血漿、血清、尿液、唾液、腦脊液、腹水、胸水等中均可檢測到miRNAs的存在。且已有研究表明血漿或血清中的miRNAs的性質(zhì)比較穩(wěn)定,它們一方面可以對抗核糖核酸酶的消化,另一方面還不受酸堿度變化、溫度變化及放置時間等惡劣環(huán)境的影響。而且大部分miRNAs的序列是高度保守的。鑒于外周循環(huán)中miRNAs的穩(wěn)定性、保守性,其有潛力成為比一般蛋白更為有效的生物學(xué)標(biāo)記物。MiRNAs的發(fā)現(xiàn)成為生物學(xué)研究的一個熱點(diǎn)。 高血壓腦出血(hypertensive intracerebral hemorrhage, HICH)是以高血壓為主要病因引起的原發(fā)性非外傷性腦實質(zhì)內(nèi)出血。在我國,隨著人口老齡化的發(fā)展,HICH的發(fā)病率逐年上升。由于HICH起病急驟,且出血部位多位于腦深部,單純外科手術(shù)難以達(dá)到滿意效果,導(dǎo)致其具有高致殘率及高致死率的特點(diǎn),給個人、家庭及社會帶來沉重的負(fù)擔(dān)。因此,如何能夠有效的預(yù)測HICH的發(fā)生成為目前的研究熱點(diǎn),這需要我們能夠?qū)ふ业接行У姆肿由飳W(xué)標(biāo)記物,對其在HICH的發(fā)病機(jī)制、預(yù)后判斷和靶向治療中可能發(fā)揮的作用進(jìn)行深入的研究和探討。 目前,越來越多的研究表明不同疾病其外周循環(huán)(血清或血漿)中miRNAs有獨(dú)特的表達(dá)改變,比如腫瘤、冠狀動脈粥樣硬化性心臟病、糖尿病等,并有可能作為診斷、判斷預(yù)后、療效評估的非侵入性生物學(xué)標(biāo)志。關(guān)于腦梗塞與miRNAs的研究亦取得了一些成就,發(fā)現(xiàn)了一些miRNAs可以作為區(qū)別不同腦梗塞亞型的生物學(xué)標(biāo)志,也研究了一些miRNAs在腦梗塞中的作用機(jī)制。但是在HICH中,國外尚無相關(guān)研究報道。 鑒于以上理論及實驗基礎(chǔ),本研究將對HICH患者進(jìn)行血漿miRNAs表達(dá)譜的分析,并挑選幾個差異表達(dá)最明顯的miRNAs進(jìn)行初步驗證,進(jìn)一步采用生物信息學(xué)分析預(yù)測可能的靶基因推測miRNAs可能的作用機(jī)制。本研究將篩選出的差異表達(dá)的特異性miRNAs作為HICH高危人群中篩選的早期分子標(biāo)記物具有非常重的意義,同時為后續(xù)的發(fā)病機(jī)制及靶向治療的研究提供實驗基礎(chǔ)。 第一章HICH患者外周循環(huán)(血漿)microRNAs表達(dá)譜的分析 目的：研究高血壓腦出血(hypertensive intracerebral hemorrhage, HICH)患者血漿與高血壓患者血漿中差異性miRNAs表達(dá)譜,篩選與HICH相關(guān)的miRNAs。 實驗方法： 1、研究對象的確定及研究標(biāo)本的收集及處理 選取3名高血壓腦出血患者與3名高血壓患者作為研究對象。于清晨空腹抽取每位患者外周靜脈血6ml,所有標(biāo)本均使用醫(yī)用EDTA抗凝管收集。所有標(biāo)本均在2小時之內(nèi)進(jìn)行血漿分離。分離好的血漿置于-80℃保存?zhèn)溆谩?2、血漿RNA的提取及質(zhì)量檢測 采用血漿/血清外泌體RNA提取試劑盒提取總RNA�？俁NA的質(zhì)量檢測采用基于SYBR Green I的熒光定量PCR方法,以has-miR-16和has-miR-192作為質(zhì)檢指標(biāo)。 3、miRNAs芯片檢測 采用u ParaflorTM micoarray芯片篩選高血壓腦出血患者與高血壓患者血漿中差異表達(dá)的miRNAs。 4、統(tǒng)計學(xué)分析 本實驗的計量資料的實驗數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(χ±s)來表示。計量資料的組間比較采用兩獨(dú)立樣本的t檢驗,計數(shù)資料的組間比較采用χ2檢驗。采用SPSS13.0統(tǒng)計軟件進(jìn)行系統(tǒng)分析。定義P0.05為有統(tǒng)計學(xué)差異。 結(jié)果： 1、血漿RNA的質(zhì)量檢測 運(yùn)用質(zhì)檢指標(biāo)hsa-miR-16和hsa-miR-192,根據(jù)文獻(xiàn)報道所建議的指標(biāo)Ct值范圍(hsa-miR-16的參考Ct值范圍為16-22,hsa-miR-192的參考Ct值范圍為25-30),判定此次提取的血漿RNA除了40號樣品外,其余均合格,可進(jìn)入下游芯片實驗。40號樣品提取的RNA中指標(biāo)hsa-miR-16和hsa-miR-192, Ct值偏大,均超出文獻(xiàn)規(guī)定的Ct值范圍,且hsa-miR-192指標(biāo)的溶解曲線出現(xiàn)雙峰,導(dǎo)致該樣品暫停下游實驗。因40號樣品為1例對照組樣品,因此實際進(jìn)入下游miRNAs芯片檢測的總共有5個樣本,即HICH組3例樣本,對照組2例樣本。 2、基因芯片的結(jié)果 對3例高血壓腦出血組與2例高血壓對照組血漿miRNAs表達(dá)譜進(jìn)行比較,共篩選出9個差異性表達(dá)的miRNAs,其中表達(dá)上調(diào)的miRNAs有3個(hsa-miR-5787、hsa-miR-149-3p、hsa-miR-6786-5p),表達(dá)下調(diào)的miRNAs有6個(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)。無上調(diào)超過2倍的miRNAs,但hsa-miR-5787上調(diào)倍數(shù)最大；下調(diào)大于或等于2倍的hsa-miRNAs有3個：hsa-miR-29a-3p、hsa-miR-30c-5p、hsa-miR-5195-3p,以has-miR-29a-3p下調(diào)最為明顯。 結(jié)論：初步建立起高血壓腦出血患者血漿中miRNAs表達(dá)譜,相對于高血壓患者,3個miRNAs在高血壓腦出血患者血漿中表達(dá)上調(diào),6個miRNAs表達(dá)下調(diào)。這些差異表達(dá)的miRNAs可能與高血壓腦出血的發(fā)病相關(guān)。 第二章MicroRNAs基因芯片結(jié)果驗證及生物信息學(xué)分析 目的：驗證高血壓腦出血患者血漿中差異性表達(dá)的miRNAs,并預(yù)測其在發(fā)病機(jī)制中可能所起的作用。 實驗方法： 1、研究對象的確定及研究標(biāo)本的收集及處理 選取20名高血壓腦出血患者與15名高血壓患者作為研究對象。于清晨空腹抽取每位患者外周靜脈血6ml,使用醫(yī)用EDTA抗凝管收集。所有標(biāo)本均在2小時之內(nèi)進(jìn)行血漿分離。分離好的血漿置于-80。C保存?zhèn)溆谩?2、血漿RNA的提取及質(zhì)量檢測 采用基于TRIzol法的RNA抽提技術(shù),獲取血漿總RNA。使用紫外吸收測定法及變性瓊脂糖凝膠電泳法對提取的RNA進(jìn)行質(zhì)量檢測。 3、RT-PCR 利用RT-PCR技術(shù)對篩選出的3個下調(diào)倍數(shù)最大的miRNAs: hsa-miR-29a-3p、hsa-miR-30c-5p、hsa-miR-5195-3p進(jìn)行驗證。 4、生物信息學(xué)分析 對篩選出的3個下調(diào)倍數(shù)最大的miRNAs:hsa-miR-29a-3p、 hsa-miR-30c-5p、hsa-miR-5195-3p進(jìn)行靶基因預(yù)測。應(yīng)用互聯(lián)網(wǎng)miRNAs靶基因預(yù)測軟件Pictar (http://pictar.mdc-berlin.de/)、MiRanda(http:///www.microma.org)、Targetscan (http://www.targetscan.org/)在線服務(wù)站點(diǎn),輸出差異表達(dá)miRNAs的預(yù)測靶基因。為了減少假陽性率,我們?nèi)≈辽?個軟件預(yù)測到的基因作為靶基因。 5、統(tǒng)計學(xué)分析 本實驗的計量資料的實驗數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(χs)來表示。計量資料的組間比較采用兩獨(dú)立樣本的t檢驗,計數(shù)資料的組間比較采用χ2檢驗。采用SPSS13.0統(tǒng)計軟件進(jìn)行系統(tǒng)分析。定義P0.05為有統(tǒng)計學(xué)差異。 結(jié)果： 1、RNA質(zhì)量檢測結(jié)果 本部分實驗所提取的RNA樣品,用紫外分光光度計測其各自的A260和A280,得到所有樣品RNA的A260/A280的值的范圍均在1.8-2.1之間。變性瓊臘糖凝膠電泳結(jié)果提示：所提取RNA樣品的28S：18S均接近2：1,且電泳條帶較清晰,無明顯彌散拖尾現(xiàn)象及雜條帶,說明所提取的RNA完整性較好,可進(jìn)入下游RT-PCR實驗。 2、RT-PCR結(jié)果 為了驗證第一步基因芯片的結(jié)果,我們挑選了3個下調(diào)倍數(shù)最大的miRNAs:hsa-miR-29a-3p、hsa-miR-30c-5p、hsa-miR-5195-3p,在20例HICH患者和15例對照者中進(jìn)行RT-PCR驗證,以hsa-miR-93作為內(nèi)參基因。相對于高血壓患者,hsa-miR-29a-3p下調(diào)倍數(shù)仍最大,超過2倍,且比芯片下調(diào)倍數(shù)明顯；hsa-miR-30c-5p和hsa-miR-5195-3p仍呈現(xiàn)表達(dá)下調(diào),但下調(diào)倍數(shù)不及芯片下調(diào)倍數(shù)明顯,但是下調(diào)趨勢與芯片結(jié)果相同。 3、生物信息學(xué)分析結(jié)果 我們應(yīng)用互聯(lián)網(wǎng)miRNAs靶基因預(yù)測軟件(Pictar、MiRanda、Targetscan)在線服務(wù)站點(diǎn),對顯著下調(diào)的niRNAs:hsa-miR-29a-3p、hsa-miR-30c-5p、 hsa-miR-5195-3p進(jìn)行靶基因預(yù)測。為了減少假陽性率,我們?nèi)≈辽?個軟件預(yù)測到的基因作為靶基因。hsa-miR-29a-3p共預(yù)測到103個靶基因,hsa-miR-30c-5p預(yù)測到103個靶基因。由于目前對hsa-miR-5195-3p研究較少,尚無hsa-miR-5195-3p靶基因的相關(guān)信息。其中,我們注意到,hsa-miR-29a-3p預(yù)測的靶基因中包括COL1A2。 對靶基因分析后發(fā)現(xiàn),靶基因作用廣泛,涉及蛋白質(zhì)的合成及修飾、轉(zhuǎn)錄過程的調(diào)節(jié)、炎性反應(yīng)、血管生成、調(diào)節(jié)信號轉(zhuǎn)導(dǎo)通路、以及細(xì)胞的凋亡、分化、增殖等。 結(jié)論： 1、RT-PCR結(jié)果與基因芯片的結(jié)果具有一致趨勢,說明基因芯片結(jié)果真實可靠。 2、實驗篩選出的差異表達(dá)的miRNAs (hsa-miR-29a-3p、hsa-miR-30c-5p、 hsa-miR-5195-3p),它們可以作為HICH的分子生物標(biāo)志物,也可能可以作為高血壓患者中易發(fā)生HICH的高危人群的篩查指標(biāo),且有可能參與了HICH的發(fā)病機(jī)制。 3、hsa-miR-29a-3p的靶基因之一為COL1A2,與Ⅰ型膠原蛋白合成相關(guān)。hsa-miR-29a-3p可能通過對COL1A2的調(diào)控,減弱了血管壁的彈性及抗壓能力,從而參與了高血壓腦出血的發(fā)生發(fā)展過程。hsa-miR-30c-5p可能通過對靶基因的DLL4的調(diào)控,從而影響血管的形成及發(fā)展,進(jìn)而參與HICH的發(fā)生。hsa-miR-5195-3p的作用機(jī)制需要進(jìn)一步探討。
[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錛，

本文編號：1951573