精神分裂癥外周血單核細胞中差異性表達lncRNA的臨床和實驗研究
發(fā)布時間：2018-07-15 13:41
【摘要】：精神分裂癥(schizophrenia,SZ)是最為嚴重的精神障礙之一,其發(fā)病率約為1%,其臨床特征主要包括幻覺、妄想、精神運動性興奮等陽性癥狀和(或)以情感遲鈍、情感淡漠、社交退縮、意志減退等陰性癥狀,患者個性改變,思維、情感、行為不協調,社會適應能力下降,致使患者的認知、情感、行為和社會功嚴重受損。精神分裂癥有較高的致殘率、復發(fā)率高和殘留癥狀,不僅使患者的生活質量水平嚴重降低,還給家庭和社會背負上了很大的經濟花費和照顧負擔,并且在一定程度上威脅著社會的安定、和諧。然而盡管對精神分裂癥的研究在最近數十年有了很大的認識進展,但是在分子生物遺傳學、細胞生物病理機制尚處于探索階段。流行病學資料證實精神分裂癥是一種復雜的多基因遺傳疾病,許多基因相互作用,加上危險的環(huán)境因素,共同導致了精神分裂癥的發(fā)生。現行階段臨床醫(yī)生對于精神分裂癥的診斷仍是依靠個人臨床經驗和對患者諸多臨床癥狀的量表評價,不能有統一的客觀評價方法。因此,尋找高度敏感性和特異性的生物標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,從而可以對患者行早期的干預。lncRNA長度一般超過200個核苷酸殘基,大多由RNA域轉錄,缺乏有意義的開放閱讀框架,不能編碼蛋白。既往lncRNA被認為是轉錄過中的“噪音”,但是近年來越來越多的研究表明,lncRNA對蛋白表達具有決定性的調控作用。Lnc RNAs在轉錄后水平上可以調節(jié)基因的表達,諸如蛋白質的合成、RNA的成熟與轉運,亦可在轉錄過程的基因沉默施通過修飾染色質結構發(fā)揮其作用,因此在調控基因表達和信號通路網絡中,lncRNA往往可以成為其中的關鍵節(jié)點;另外lncRNA還參與了疾病產生的基因表達調控、細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的lncRNA在神經精神疾病中都有表達的異常。因此,在大腦的發(fā)育、神經發(fā)生、神經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也起著關鍵的調控作用。lncRNA與精神分裂癥的關系國內外研究尚少,現有不多的研究表明lncRNA通過多種途徑參與了精神分裂癥發(fā)病機制和神分裂癥的發(fā)生和進展。理想的生物標志物,具有重復性強、性質穩(wěn)定、便于采集和測量等特性。那么外周血lncRNA是否能具有這些特點呢?首先lncRNA的理化性質相當穩(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可以由實時熒光定量PCR方法檢測,檢測費用低廉,表達有時序性和高度的組織特異性,且便于動態(tài)地重復檢測其表達水平;最后,更為重要的是,腦組織與外周血白細胞之間存在眾多共同的生物學通路,有許多相似的基因表達;此外,PBMCs中l(wèi)ncRNA的表達譜與疾病的臨床表現有密切的聯系,提示PBMCs可以反映腦細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志物成為了可以探索研究的課題之一。基于目前關于lncRNA的研究背景,本課題的目的在于尋找、檢測并證實在外周血中l(wèi)ncRNA能否成為精神分裂癥生物標志物,在尋找出相關的lncRNA后,對其在精神分裂癥發(fā)生發(fā)展過程中的作用和機制進行探討。本課題分如下四個部分進行探索。第一部分:精神分裂癥PBMCs中表達異常的lncRNA的篩查及其驗證 實驗方法: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照者(各3例)的外周血樣本,分離出PBMCs并初步檢測表達的lncRNA,尋找兩組間表達異常的lncRNA。再根據結果,進一步擴大樣本量,選擇其中10個顯著表達異常的lncRNA在106例精神分裂癥和48例正常健康對照者中采用實時熒光定量q RT-PCR方法來驗證已經篩查出的這10個lncRNA結果。并用ROC曲線對這些在明顯表達異常的lncRNA進行分析。結果:1.在lncRNA芯片檢測結果中,一共檢測出125個表達異常的lncRNA。其中62個lncRNA表達升高,63個lncRNA表達下降;2.進一步擴大樣本量,對其中表達顯著異常的10個lncRNA行q RT-PCR驗證,發(fā)現NONHSAT089447,NONHSAT021545,NONHSAT041499等3個lncRNA的表達異常,并且具有統計學意義(P0.05)。對其進行ROC曲線分析顯示,以上3個lncRNA作為聯合診斷整體,其聯合ROC曲線的AUC為0.791,敏感值為62.5%,特異值為75.0%。第二部分:抗精神病藥物干預條件下PBMC中l(wèi)ncRNA的表達變化 實驗方法:入組的30例精神分裂癥患者采用單藥治療或聯合用藥治療的方法,以此實行抗精神病藥物的干預。采用陽性癥狀及陰性癥狀量表(PANSS)對病人的臨床癥狀進行評估;評估時間定在在藥物干預前以及干預8周后兩個時間點,并在評估時分離外周血PBMC,用實時熒光定量PCR方法檢驗lncRNA的表達數值的差異。結果:1.經過抗精神病藥物持續(xù)干預8周后,PANSS總分和其它各項主因子評分均較藥物干預前有顯著下降,差異均具有統計學意義(P0.01);2.經過抗精神病藥物持續(xù)干預8周后,患者外周血單核細胞中3個lncRNA中有2個lncRNA(NONHSAT089447,NONHSAT041499)的ΔCT值較治療前顯著上升,(P0.01),提示治療后這2個lncRNA的表達量較治療前顯著下調;3.將治療前后lncRNA的ΔCT值之差(ΔΔCT),即ΔΔCT值與PANSS中l(wèi)ncRNA相對表達量的變化與PANSS量表所反應其臨床癥狀的變化用Spearman相關分析,來研究兩者之間是否存在關聯。我們結果顯示,NONHSAT041499的ΔΔCT值與PANSS量表陽性癥因子分、激活性因子分變化值呈顯著正相關((r=-0.444 and-0.423,P0.05)。第三部分:精神分裂癥PBMC中異常表達的lncRNA的生物信息學方法分析 實驗方法:用Pearson相關分析法計算與NONHSAT089447、NONHSAT021545、NONHSAT041499共表達的m RNA,應用DAVID軟件進行GO功能富集分析和KEGG信號通路富集分析,然后計算Lnc RNA共表達的編碼基因集合與轉錄因子的靶基因集合的交集,得到與lncRNA顯著相關的轉錄因子,構建lncRNA-轉錄因子-靶基因網絡圖。結果:1.同時與NONHSAT089447、NONHSAT021545、NONHSAT041499共表達的m RNA有89個,其涉及包括多項與中樞神經系統相關條目在內的廣泛的GO生物學過程;2.共表達m RNA的靶基因涉及的KEGG信號通路中許多與精神分裂癥關系密切;3.NONHSAT089447、NONHSAT021545、NONHSAT041499可能在精神分裂癥的發(fā)病的病理機制中起非常重要的作用。第四部分:精神分裂癥異常表達的lncRNA與多巴胺信號通路調控機制的研究實驗方法:在培養(yǎng)人神經母細胞瘤細胞(SK-N-SH)的過程中加入多巴胺模擬精神分裂癥患者的神經細胞,實時熒光定量PCR(q RT-PCR)檢測lncRNA的表達量。利用多巴胺拮抗劑奧氮平來反證NONHSAT089447是否由多巴胺所誘導升高,通過脂質體轉染技術來實現si RNA及plasmid-447導入SK-N-SH,提取總RNA,q RT-PCR檢測轉染前后兩種lncRNA(NONHSAT089447,NONHSAT041499)的表達量及多巴胺受體(DRD1,DRD2,DRD3,DRD4,DRD5)表達的變化。運用western blot技術檢測干擾及過表達NONHSAT089447多巴胺受體的下游信號的變化。結果:1.q RT-PCR結果顯示,奧氮平拮抗多巴胺使NONHSAT089447的表達受到明顯抑制(P0.001),NONHSAT041499表達抑制不明顯(P0.05)。2、小干擾RNA對NONHSAT089447表達抑制更為明顯(P0.05),過表達RNA也使NONHSAT089447具有更顯著的增長(P0.01);3、干擾NONHSAT089447后發(fā)現DRD3和DRD5均表達降低(P0.05),過表達NONHSAT089447結果顯示DRD3,DRD5表達升高(P0.05)。4、western blot顯示干擾NONHSAT089447可使多巴胺受體下游信號減弱,過表達NONHSAT089447使多巴胺受體下游信號增強。結論:1.SZ患者PBMC中NONHSAT089447、NONHSAT021545、NONHSAT041499等3個lncRNA出現顯著性表達上調,并且經抗精神病藥物治療后,NONHSA T089447和NONHSAT041499的相對表達水平較治療前顯著下降,并且其表達水平與臨床癥狀的改善密切相關,有潛力作為精神分裂癥的生物學標志物。2.生物信息學分析發(fā)現了與lncRNA共表達的m RNA,發(fā)現與SZ有關的許多GO生物學過程和KEGG信號通路顯著富集;NONHSAT089447、NONHSAT021545、NONHSAT041499可能在SZ的病理生理機制中發(fā)揮著重要的作用。3.細胞水平研究表明:SZ患者的NONHSAT089447處于高表達狀態(tài),多巴胺受體信號通路被激活,由此進一步促進NONHSAT089447的表達,形成一個正反饋調節(jié)通路。NONHSAT089447可能是SZ的生物標志物,并且其表達水平可作為SZ療效評價的指標。
[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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R749.3
本文編號：2124262
[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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R749.3
【參考文獻】
相關期刊論文 前1條
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胞中microRNA表達水平差異的研究[J];中華行為醫(yī)學與腦科學雜志;2014年08期
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