【摘要】：精神分裂癥(schizophrenia,SZ)是最為嚴(yán)重的精神障礙之一,其發(fā)病率約為1%,其臨床特征主要包括幻覺、妄想、精神運(yùn)動(dòng)性興奮等陽(yáng)性癥狀和(或)以情感遲鈍、情感淡漠、社交退縮、意志減退等陰性癥狀,患者個(gè)性改變,思維、情感、行為不協(xié)調(diào),社會(huì)適應(yīng)能力下降,致使患者的認(rèn)知、情感、行為和社會(huì)功嚴(yán)重受損。精神分裂癥有較高的致殘率、復(fù)發(fā)率高和殘留癥狀,不僅使患者的生活質(zhì)量水平嚴(yán)重降低,還給家庭和社會(huì)背負(fù)上了很大的經(jīng)濟(jì)花費(fèi)和照顧負(fù)擔(dān),并且在一定程度上威脅著社會(huì)的安定、和諧。然而盡管對(duì)精神分裂癥的研究在最近數(shù)十年有了很大的認(rèn)識(shí)進(jìn)展,但是在分子生物遺傳學(xué)、細(xì)胞生物病理機(jī)制尚處于探索階段。流行病學(xué)資料證實(shí)精神分裂癥是一種復(fù)雜的多基因遺傳疾病,許多基因相互作用,加上危險(xiǎn)的環(huán)境因素,共同導(dǎo)致了精神分裂癥的發(fā)生�，F(xiàn)行階段臨床醫(yī)生對(duì)于精神分裂癥的診斷仍是依靠個(gè)人臨床經(jīng)驗(yàn)和對(duì)患者諸多臨床癥狀的量表評(píng)價(jià),不能有統(tǒng)一的客觀評(píng)價(jià)方法。因此,尋找高度敏感性和特異性的生物標(biāo)志物來完善精神分裂癥的早期診斷,是臨床醫(yī)生及其科研者迫切需要解決的問題,從而可以對(duì)患者行早期的干預(yù)。lncRNA長(zhǎng)度一般超過200個(gè)核苷酸殘基,大多由RNA域轉(zhuǎn)錄,缺乏有意義的開放閱讀框架,不能編碼蛋白。既往lncRNA被認(rèn)為是轉(zhuǎn)錄過中的“噪音”,但是近年來越來越多的研究表明,lncRNA對(duì)蛋白表達(dá)具有決定性的調(diào)控作用。Lnc RNAs在轉(zhuǎn)錄后水平上可以調(diào)節(jié)基因的表達(dá),諸如蛋白質(zhì)的合成、RNA的成熟與轉(zhuǎn)運(yùn),亦可在轉(zhuǎn)錄過程的基因沉默施通過修飾染色質(zhì)結(jié)構(gòu)發(fā)揮其作用,因此在調(diào)控基因表達(dá)和信號(hào)通路網(wǎng)絡(luò)中,lncRNA往往可以成為其中的關(guān)鍵節(jié)點(diǎn);另外lncRNA還參與了疾病產(chǎn)生的基因表達(dá)調(diào)控、細(xì)胞增殖分化等廣泛的生物過程。值得注意的是,許多在大腦中高表達(dá)的lncRNA在神經(jīng)精神疾病中都有表達(dá)的異常。因此,在大腦的發(fā)育、神經(jīng)發(fā)生、神經(jīng)元成熟及突觸形成過程中以及精神疾病的發(fā)生、發(fā)展中,lncRNA也起著關(guān)鍵的調(diào)控作用。lncRNA與精神分裂癥的關(guān)系國(guó)內(nèi)外研究尚少,現(xiàn)有不多的研究表明lncRNA通過多種途徑參與了精神分裂癥發(fā)病機(jī)制和神分裂癥的發(fā)生和進(jìn)展。理想的生物標(biāo)志物,具有重復(fù)性強(qiáng)、性質(zhì)穩(wěn)定、便于采集和測(cè)量等特性。那么外周血lncRNA是否能具有這些特點(diǎn)呢?首先lncRNA的理化性質(zhì)相當(dāng)穩(wěn)定,它不能被核糖核酸酶的降解,其次,lncRNA的采集方便,只需抽取外周血即可;第三,lncRNA可以由實(shí)時(shí)熒光定量PCR方法檢測(cè),檢測(cè)費(fèi)用低廉,表達(dá)有時(shí)序性和高度的組織特異性,且便于動(dòng)態(tài)地重復(fù)檢測(cè)其表達(dá)水平;最后,更為重要的是,腦組織與外周血白細(xì)胞之間存在眾多共同的生物學(xué)通路,有許多相似的基因表達(dá);此外,PBMCs中l(wèi)ncRNA的表達(dá)譜與疾病的臨床表現(xiàn)有密切的聯(lián)系,提示PBMCs可以反映腦細(xì)胞的參與疾病的生理病理過程。因此,從外周血中分離獲取lncRNA,并以此作為精神分裂癥的生物標(biāo)志物成為了可以探索研究的課題之一�；谀壳瓣P(guān)于lncRNA的研究背景,本課題的目的在于尋找、檢測(cè)并證實(shí)在外周血中l(wèi)ncRNA能否成為精神分裂癥生物標(biāo)志物,在尋找出相關(guān)的lncRNA后,對(duì)其在精神分裂癥發(fā)生發(fā)展過程中的作用和機(jī)制進(jìn)行探討。本課題分如下四個(gè)部分進(jìn)行探索。第一部分:精神分裂癥PBMCs中表達(dá)異常的lncRNA的篩查及其驗(yàn)證 實(shí)驗(yàn)方法:采用Agilent Human lncRNA(4*180K,Design ID:062918)芯片,抽取精神分裂癥患者和正常健康對(duì)照者(各3例)的外周血樣本,分離出PBMCs并初步檢測(cè)表達(dá)的lncRNA,尋找兩組間表達(dá)異常的lncRNA。再根據(jù)結(jié)果,進(jìn)一步擴(kuò)大樣本量,選擇其中10個(gè)顯著表達(dá)異常的lncRNA在106例精神分裂癥和48例正常健康對(duì)照者中采用實(shí)時(shí)熒光定量q RT-PCR方法來驗(yàn)證已經(jīng)篩查出的這10個(gè)lncRNA結(jié)果。并用ROC曲線對(duì)這些在明顯表達(dá)異常的lncRNA進(jìn)行分析。結(jié)果:1.在lncRNA芯片檢測(cè)結(jié)果中,一共檢測(cè)出125個(gè)表達(dá)異常的lncRNA。其中62個(gè)lncRNA表達(dá)升高,63個(gè)lncRNA表達(dá)下降;2.進(jìn)一步擴(kuò)大樣本量,對(duì)其中表達(dá)顯著異常的10個(gè)lncRNA行q RT-PCR驗(yàn)證,發(fā)現(xiàn)NONHSAT089447,NONHSAT021545,NONHSAT041499等3個(gè)lncRNA的表達(dá)異常,并且具有統(tǒng)計(jì)學(xué)意義(P0.05)。對(duì)其進(jìn)行ROC曲線分析顯示,以上3個(gè)lncRNA作為聯(lián)合診斷整體,其聯(lián)合ROC曲線的AUC為0.791,敏感值為62.5%,特異值為75.0%。第二部分:抗精神病藥物干預(yù)條件下PBMC中l(wèi)ncRNA的表達(dá)變化 實(shí)驗(yàn)方法:入組的30例精神分裂癥患者采用單藥治療或聯(lián)合用藥治療的方法,以此實(shí)行抗精神病藥物的干預(yù)。采用陽(yáng)性癥狀及陰性癥狀量表(PANSS)對(duì)病人的臨床癥狀進(jìn)行評(píng)估;評(píng)估時(shí)間定在在藥物干預(yù)前以及干預(yù)8周后兩個(gè)時(shí)間點(diǎn),并在評(píng)估時(shí)分離外周血PBMC,用實(shí)時(shí)熒光定量PCR方法檢驗(yàn)lncRNA的表達(dá)數(shù)值的差異。結(jié)果:1.經(jīng)過抗精神病藥物持續(xù)干預(yù)8周后,PANSS總分和其它各項(xiàng)主因子評(píng)分均較藥物干預(yù)前有顯著下降,差異均具有統(tǒng)計(jì)學(xué)意義(P0.01);2.經(jīng)過抗精神病藥物持續(xù)干預(yù)8周后,患者外周血單核細(xì)胞中3個(gè)lncRNA中有2個(gè)lncRNA(NONHSAT089447,NONHSAT041499)的ΔCT值較治療前顯著上升,(P0.01),提示治療后這2個(gè)lncRNA的表達(dá)量較治療前顯著下調(diào);3.將治療前后lncRNA的ΔCT值之差(ΔΔCT),即ΔΔCT值與PANSS中l(wèi)ncRNA相對(duì)表達(dá)量的變化與PANSS量表所反應(yīng)其臨床癥狀的變化用Spearman相關(guān)分析,來研究?jī)烧咧g是否存在關(guān)聯(lián)。我們結(jié)果顯示,NONHSAT041499的ΔΔCT值與PANSS量表陽(yáng)性癥因子分、激活性因子分變化值呈顯著正相關(guān)((r=-0.444 and-0.423,P0.05)。第三部分:精神分裂癥PBMC中異常表達(dá)的lncRNA的生物信息學(xué)方法分析 實(shí)驗(yàn)方法:用Pearson相關(guān)分析法計(jì)算與NONHSAT089447、NONHSAT021545、NONHSAT041499共表達(dá)的m RNA,應(yīng)用DAVID軟件進(jìn)行GO功能富集分析和KEGG信號(hào)通路富集分析,然后計(jì)算Lnc RNA共表達(dá)的編碼基因集合與轉(zhuǎn)錄因子的靶基因集合的交集,得到與lncRNA顯著相關(guān)的轉(zhuǎn)錄因子,構(gòu)建lncRNA-轉(zhuǎn)錄因子-靶基因網(wǎng)絡(luò)圖。結(jié)果:1.同時(shí)與NONHSAT089447、NONHSAT021545、NONHSAT041499共表達(dá)的m RNA有89個(gè),其涉及包括多項(xiàng)與中樞神經(jīng)系統(tǒng)相關(guān)條目在內(nèi)的廣泛的GO生物學(xué)過程;2.共表達(dá)m RNA的靶基因涉及的KEGG信號(hào)通路中許多與精神分裂癥關(guān)系密切;3.NONHSAT089447、NONHSAT021545、NONHSAT041499可能在精神分裂癥的發(fā)病的病理機(jī)制中起非常重要的作用。第四部分:精神分裂癥異常表達(dá)的lncRNA與多巴胺信號(hào)通路調(diào)控機(jī)制的研究實(shí)驗(yàn)方法:在培養(yǎng)人神經(jīng)母細(xì)胞瘤細(xì)胞(SK-N-SH)的過程中加入多巴胺模擬精神分裂癥患者的神經(jīng)細(xì)胞,實(shí)時(shí)熒光定量PCR(q RT-PCR)檢測(cè)lncRNA的表達(dá)量。利用多巴胺拮抗劑奧氮平來反證NONHSAT089447是否由多巴胺所誘導(dǎo)升高,通過脂質(zhì)體轉(zhuǎn)染技術(shù)來實(shí)現(xiàn)si RNA及plasmid-447導(dǎo)入SK-N-SH,提取總RNA,q RT-PCR檢測(cè)轉(zhuǎn)染前后兩種lncRNA(NONHSAT089447,NONHSAT041499)的表達(dá)量及多巴胺受體(DRD1,DRD2,DRD3,DRD4,DRD5)表達(dá)的變化。運(yùn)用western blot技術(shù)檢測(cè)干擾及過表達(dá)NONHSAT089447多巴胺受體的下游信號(hào)的變化。結(jié)果:1.q RT-PCR結(jié)果顯示,奧氮平拮抗多巴胺使NONHSAT089447的表達(dá)受到明顯抑制(P0.001),NONHSAT041499表達(dá)抑制不明顯(P0.05)。2、小干擾RNA對(duì)NONHSAT089447表達(dá)抑制更為明顯(P0.05),過表達(dá)RNA也使NONHSAT089447具有更顯著的增長(zhǎng)(P0.01);3、干擾NONHSAT089447后發(fā)現(xiàn)DRD3和DRD5均表達(dá)降低(P0.05),過表達(dá)NONHSAT089447結(jié)果顯示DRD3,DRD5表達(dá)升高(P0.05)。4、western blot顯示干擾NONHSAT089447可使多巴胺受體下游信號(hào)減弱,過表達(dá)NONHSAT089447使多巴胺受體下游信號(hào)增強(qiáng)。結(jié)論:1.SZ患者PBMC中NONHSAT089447、NONHSAT021545、NONHSAT041499等3個(gè)lncRNA出現(xiàn)顯著性表達(dá)上調(diào),并且經(jīng)抗精神病藥物治療后,NONHSA T089447和NONHSAT041499的相對(duì)表達(dá)水平較治療前顯著下降,并且其表達(dá)水平與臨床癥狀的改善密切相關(guān),有潛力作為精神分裂癥的生物學(xué)標(biāo)志物。2.生物信息學(xué)分析發(fā)現(xiàn)了與lncRNA共表達(dá)的m RNA,發(fā)現(xiàn)與SZ有關(guān)的許多GO生物學(xué)過程和KEGG信號(hào)通路顯著富集;NONHSAT089447、NONHSAT021545、NONHSAT041499可能在SZ的病理生理機(jī)制中發(fā)揮著重要的作用。3.細(xì)胞水平研究表明:SZ患者的NONHSAT089447處于高表達(dá)狀態(tài),多巴胺受體信號(hào)通路被激活,由此進(jìn)一步促進(jìn)NONHSAT089447的表達(dá),形成一個(gè)正反饋調(diào)節(jié)通路。NONHSAT089447可能是SZ的生物標(biāo)志物,并且其表達(dá)水平可作為SZ療效評(píng)價(jià)的指標(biāo)。
[Abstract]:Schizophrenia (SZ) is one of the most serious mental disorders, with a incidence of about 1%. Its clinical features mainly include positive symptoms such as hallucinations, delusions, psychomotor excitement, and / or negative symptoms such as emotional retardation, emotional indifference, social withdrawal, and depression, patients' personality changes, thinking, emotion and behavior disharmony. The cognitive, emotional, behavioral, and social work of the patient is seriously impaired. Schizophrenia has a high rate of disability, high recurrence rate and residual symptoms, which not only seriously reduces the quality of life of the patient, but also gives the family and society a great cost and burden to the family and society, and threatens to some extent. Social stability and harmony. However, although the study of schizophrenia has made great progress in recent decades, in molecular biology, the mechanism of cell biological pathology is still at the exploratory stage. Epidemiological data confirm that schizophrenia is a complex polygenetic genetic disease, many genes interact, plus danger. The environmental factors of risk contribute to the occurrence of schizophrenia. The current stage clinicians' diagnosis of schizophrenia is still dependent on personal clinical experience and evaluation of a number of clinical symptoms, and can not have a unified objective evaluation method. Therefore, it is necessary to look for a highly sensitive and specific biomarker to improve the spirit. Early diagnosis of schizophrenia is an urgent problem to be solved by clinicians and their researchers. The early intervention of the patients is that the.LncRNA length is generally more than 200 nucleotide residues, most of which are transcribed in the RNA domain, lack of a meaningful open reading frame, and cannot encode egg white. The past lncRNA is considered to be the "noise" in the transcriptional. But in recent years, more and more studies have shown that lncRNA plays a decisive role in the regulation of protein expression,.Lnc RNAs can regulate the expression of gene at post transcriptional level, such as protein synthesis, RNA maturation and transport, and the gene silencing of transcription process can play its role by trimming the chromatin structure and therefore in the regulatory basis LncRNA can often become a key node in the expression and signaling network, and lncRNA also participates in a wide range of biological processes, such as gene expression regulation, cell proliferation, differentiation, and so on. It is worth noting that many of the high expression of lncRNA in the brain are expressed in neuropsychiatric disorders. Therefore, in the brain Development, neurogenesis, neuronal maturation and synaptic formation, as well as the occurrence of mental disorders, and the development of lncRNA also plays a key regulatory role in the relationship between.LncRNA and schizophrenia at home and abroad, and few studies have shown that lncRNA has been involved in the pathogenesis of schizophrenia and schizophrenia through a variety of ways. Occurrence and progress. Ideal biomarkers have the characteristics of reproducibility, stability, and convenience for acquisition and measurement. Then, can the peripheral blood lncRNA have these characteristics? First, the physical and chemical properties of lncRNA are fairly stable, and it can not be degraded by ribonuclease. Secondly, the collection of lncRNA is convenient, only peripheral blood can be extracted; third, LN CRNA can be detected by real time fluorescence quantitative PCR method, the detection cost is low, the expression of sometimes ordered and high tissue specificity, and it is easy to dynamically repeat the detection of its expression level. Finally, it is more important that there are many common biological pathways between brain tissue and peripheral blood white blood cells, and many similar gene expressions; in addition, PBMC The expression profiles of lncRNA in s are closely related to the clinical manifestations of the disease, suggesting that PBMCs can reflect the physiological and pathological processes of brain cells involved in disease. Therefore, it is one of the subjects to explore the separation of lncRNA from peripheral blood and take it as a biomarker of schizophrenia. Based on the present research on lncRNA Background, the purpose of this project is to find out and verify whether lncRNA can be a biomarker of schizophrenia in the peripheral blood, and to explore the role and mechanism of the lncRNA in the development of schizophrenia. This topic is divided into four parts as follows. The first part: schizophrenia PBMC The screening of abnormal lncRNA in S and its experimental method: using Agilent Human lncRNA (4*180K, Design ID:062918) chip to extract peripheral blood samples from schizophrenic patients and normal healthy controls (3 cases each), separate PBMCs and detect the expression of lncRNA, and find the lncRNA. of two groups of expressions. One step was to expand the sample size, select 10 of the significantly abnormal lncRNA in 106 cases of schizophrenia and 48 normal healthy controls by using real time fluorescence quantitative Q RT-PCR method to verify the 10 lncRNA results that had been screened. And the ROC curve was used to analyze these abnormal lncRNA. Results: 1. on the lncRNA chip. In the test results, 125 lncRNA. expressed abnormal expressions were detected, 62 lncRNA expressions were raised and 63 lncRNA expressions decreased; 2. further expanded the sample size, and 10 lncRNA rows of Q RT-PCR, which expressed significant abnormal expressions, found 3 lncRNA expressions such as NONHSAT089447, NONHSAT021545, and NONHSAT041499, and had statistical meaning. P0.05. The ROC curve analysis showed that the above 3 lncRNA as a combined diagnostic whole, the AUC of the combined ROC curve was 0.791, the sensitivity was 62.5% and the specific value was 75.0%. second: the experimental method of lncRNA expression in PBMC under the anti psychotic drug intervention: 30 schizophrenic patients in the group were treated with single drug therapy or The method of combination therapy was used to intervene in antipsychotic drugs. The positive symptoms and negative symptom scale (PANSS) were used to evaluate the clinical symptoms of the patients. The evaluation time was determined at the two time points before and after 8 weeks of intervention, and the peripheral blood PBMC was separated at the time of evaluation, and lncRNA was tested by real time fluorescence quantitative PCR method. Results: 1. after 8 weeks of continuous anti psychotic drug intervention, the scores of PANSS total and other main factors were significantly lower than those before the drug intervention, and the difference was statistically significant (P0.01); 2. after 8 weeks of persistent anti psychotic drug intervention, there were 2 lncRNA in the 3 lncRNA of the peripheral blood mononuclear cells (NO The value of delta CT in NHSAT089447, NONHSAT041499 was significantly higher than that before treatment, (P0.01), suggesting that the expression of the 2 lncRNA after treatment was significantly lower than that before treatment. 3. the difference between the delta CT value of lncRNA before and after treatment (delta delta CT), that is, the change of delta delta CT value and lncRNA relative expression in PANSS, is related to the changes in the clinical symptoms of the PANSS scale. Analysis, to investigate whether there is a correlation between the two. We have shown that the value of delta delta CT of NONHSAT041499 is positively correlated with the PANSS scale positive factor and the variation value of activation factor (r=-0.444 and-0.423, P0.05). The third part: the Bioinformatics Method of the abnormal expression of lncRNA in schizophrenia PBMC: The m RNA co expressed with NONHSAT089447, NONHSAT021545, and NONHSAT041499 was calculated by Pearson correlation analysis. The DAVID software was used to enrich the GO function and enrich the KEGG signal pathway. Then the encoding gene set co expressed by Lnc RNA was intersecting with the target gene set of the transcription factor, and the transcriptional cause associated with lncRNA was significantly related to lncRNA. The network diagram of the lncRNA- transcription factor target gene was constructed. Results: 1. there were 89 m RNA co expressed with NONHSAT089447, NONHSAT021545, and NONHSAT041499, which involved a wide range of GO biological processes including many central nervous system related items; 2. co expressed many of the KEGG signaling pathways involved in the target genes of M RNA. Cleft disease is closely related; 3.NONHSAT089447, NONHSAT021545, NONHSAT041499 may play a very important role in the pathogenesis of schizophrenia. The fourth part: the study of the abnormal expression of lncRNA and dopamine signaling pathways: the process of developing human neuroblastoma cells (SK-N-SH) Dopamine was added to simulate the nerve cells of schizophrenic patients, real-time fluorescence quantitative PCR (Q RT-PCR) was used to detect the expression of lncRNA. The dopamine antagonist olanzapine was used to verify whether NONHSAT089447 was induced by dopamine, and Si RNA and plasmid-447 were introduced into SK-N-SH by liposome transfection technology, and the total RNA, q RT-PCR was extracted. The expression of two lncRNA (NONHSAT089447, NONHSAT041499) and the changes in the expression of dopamine receptor (DRD1, DRD2, DRD3, DRD4, DRD5) were detected before and after transfection. The changes in the downstream signal of interference and overexpression of NONHSAT089447 dopamine receptor were detected by Western blot technique. The expression of 447 was obviously inhibited (P0.001), the inhibition of NONHSAT041499 expression was not obvious (P0.05).2, and the inhibition of NONHSAT089447 expression by small interference RNA was more obvious (P0.05). The overexpression of RNA also made NONHSAT089447 have a more significant increase (P0.01). 3. The expression of DRD3, DRD5 expression increased (P0.05).4, Western blot showed that interference NONHSAT089447 could weaken the downstream signal of dopamine receptor, and the over expression of NONHSAT089447 to enhance the downstream signal of dopamine receptor. Conclusion: 1.SZ patients PBMC NONHSAT089447, NONHSAT021545, and so on, 3 significant up-regulated expressions, and antipsychotic. After the treatment, the relative expression level of NONHSA T089447 and NONHSAT041499 was significantly lower than that before the treatment, and the expression level was closely related to the improvement of clinical symptoms. It has potential as a biological marker for schizophrenia,.2. bioinformatics analysis found the m RNA co expressed with lncRNA, and found many GO organisms related to SZ. The learning process and the KEGG signaling pathway are significantly enriched; NONHSAT089447, NONHSAT021545, and NONHSAT041499 may play an important role in the pathophysiological mechanism of SZ. The study of.3. cell level indicates that NONHSAT089447 in SZ patients is in high expression state, and the dopamine receptor signaling pathway is activated, thereby further promoting the expression of NONHSAT089447. A positive feedback regulatory pathway.NONHSAT089447 may be a biomarker of SZ, and its expression level can be used as an indicator of SZ efficacy.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R749.3

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1 張梁;過偉;孫欣羊;宋紅濤;趙林;仲愛芳;牛威;師征;張理義;;精神分裂癥患者血漿及單核細(xì)胞中microRNA表達(dá)水平差異的研究[J];中華行為醫(yī)學(xué)與腦科學(xué)雜志;2014年08期

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