精神分裂症外周血单核细胞中差异性表达lncRNA的临床和实验研究
发布时间：2018-07-15 13:41
【摘要】：精神分裂症(schizophrenia,SZ)是最为严重的精神障碍之一,其发病率约为1%,其临床特征主要包括幻觉、妄想、精神运动性兴奋等阳性症状和(或)以情感迟钝、情感淡漠、社交退缩、意志减退等阴性症状,患者个性改变,思维、情感、行为不协调,社会适应能力下降,致使患者的认知、情感、行为和社会功严重受损。精神分裂症有较高的致残率、复发率高和残留症状,不仅使患者的生活质量水平严重降低,还给家庭和社会背负上了很大的经济花费和照顾负担,并且在一定程度上威胁着社会的安定、和谐。然而尽管对精神分裂症的研究在最近数十年有了很大的认识进展,但是在分子生物遗传学、细胞生物病理机制尚处于探索阶段。流行病学资料证实精神分裂症是一种复杂的多基因遗传疾病,许多基因相互作用,加上危险的环境因素,共同导致了精神分裂症的发生。现行阶段临床医生对于精神分裂症的诊断仍是依靠个人临床经验和对患者诸多临床症状的量表评价,不能有统一的客观评价方法。因此,寻找高度敏感性和特异性的生物标志物来完善精神分裂症的早期诊断,是临床医生及其科研者迫切需要解决的问题,从而可以对患者行早期的干预。lncRNA长度一般超过200个核苷酸残基,大多由RNA域转录,缺乏有意义的开放阅读框架,不能编码蛋白。既往lncRNA被认为是转录过中的“噪音”,但是近年来越来越多的研究表明,lncRNA对蛋白表达具有决定性的调控作用。Lnc RNAs在转录后水平上可以调节基因的表达,诸如蛋白质的合成、RNA的成熟与转运,亦可在转录过程的基因沉默施通过修饰染色质结构发挥其作用,因此在调控基因表达和信号通路网络中,lncRNA往往可以成为其中的关键节点;另外lncRNA还参与了疾病产生的基因表达调控、细胞增殖分化等广泛的生物过程。值得注意的是,许多在大脑中高表达的lncRNA在神经精神疾病中都有表达的异常。因此,在大脑的发育、神经发生、神经元成熟及突触形成过程中以及精神疾病的发生、发展中,lncRNA也起着关键的调控作用。lncRNA与精神分裂症的关系国内外研究尚少,现有不多的研究表明lncRNA通过多种途径参与了精神分裂症发病机制和神分裂症的发生和进展。理想的生物标志物,具有重复性强、性质稳定、便于采集和测量等特性。那么外周血lncRNA是否能具有这些特点呢?首先lncRNA的理化性质相当稳定,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可以由实时荧光定量PCR方法检测,检测费用低廉,表达有时序性和高度的组织特异性,且便于动态地重复检测其表达水平;最后,更为重要的是,脑组织与外周血白细胞之间存在众多共同的生物学通路,有许多相似的基因表达;此外,PBMCs中lncRNA的表达谱与疾病的临床表现有密切的联系,提示PBMCs可以反映脑细胞的参与疾病的生理病理过程。因此,从外周血中分离获取lncRNA,并以此作为精神分裂症的生物标志物成为了可以探索研究的课题之一。基于目前关于lncRNA的研究背景,本课题的目的在于寻找、检测并证实在外周血中lncRNA能否成为精神分裂症生物标志物,在寻找出相关的lncRNA后,对其在精神分裂症发生发展过程中的作用和机制进行探讨。本课题分如下四个部分进行探索。第一部分:精神分裂症PBMCs中表达异常的lncRNA的筛查及其验证 实验方法:采用Agilent Human lncRNA(4*180K,Design ID:062918)芯片,抽取精神分裂症患者和正常健康对照者(各3例)的外周血样本,分离出PBMCs并初步检测表达的lncRNA,寻找两组间表达异常的lncRNA。再根据结果,进一步扩大样本量,选择其中10个显著表达异常的lncRNA在106例精神分裂症和48例正常健康对照者中采用实时荧光定量q RT-PCR方法来验证已经筛查出的这10个lncRNA结果。并用ROC曲线对这些在明显表达异常的lncRNA进行分析。结果:1.在lncRNA芯片检测结果中,一共检测出125个表达异常的lncRNA。其中62个lncRNA表达升高,63个lncRNA表达下降;2.进一步扩大样本量,对其中表达显著异常的10个lncRNA行q RT-PCR验证,发现NONHSAT089447,NONHSAT021545,NONHSAT041499等3个lncRNA的表达异常,并且具有统计学意义(P0.05)。对其进行ROC曲线分析显示,以上3个lncRNA作为联合诊断整体,其联合ROC曲线的AUC为0.791,敏感值为62.5%,特异值为75.0%。第二部分:抗精神病药物干预条件下PBMC中lncRNA的表达变化 实验方法:入组的30例精神分裂症患者采用单药治疗或联合用药治疗的方法,以此实行抗精神病药物的干预。采用阳性症状及阴性症状量表(PANSS)对病人的临床症状进行评估;评估时间定在在药物干预前以及干预8周后两个时间点,并在评估时分离外周血PBMC,用实时荧光定量PCR方法检验lncRNA的表达数值的差异。结果:1.经过抗精神病药物持续干预8周后,PANSS总分和其它各项主因子评分均较药物干预前有显著下降,差异均具有统计学意义(P0.01);2.经过抗精神病药物持续干预8周后,患者外周血单核细胞中3个lncRNA中有2个lncRNA(NONHSAT089447,NONHSAT041499)的ΔCT值较治疗前显著上升,(P0.01),提示治疗后这2个lncRNA的表达量较治疗前显著下调;3.将治疗前后lncRNA的ΔCT值之差(ΔΔCT),即ΔΔCT值与PANSS中lncRNA相对表达量的变化与PANSS量表所反应其临床症状的变化用Spearman相关分析,来研究两者之间是否存在关联。我们结果显示,NONHSAT041499的ΔΔCT值与PANSS量表阳性症因子分、激活性因子分变化值呈显著正相关((r=-0.444 and-0.423,P0.05)。第三部分:精神分裂症PBMC中异常表达的lncRNA的生物信息学方法分析 实验方法:用Pearson相关分析法计算与NONHSAT089447、NONHSAT021545、NONHSAT041499共表达的m RNA,应用DAVID软件进行GO功能富集分析和KEGG信号通路富集分析,然后计算Lnc RNA共表达的编码基因集合与转录因子的靶基因集合的交集,得到与lncRNA显著相关的转录因子,构建lncRNA-转录因子-靶基因网络图。结果:1.同时与NONHSAT089447、NONHSAT021545、NONHSAT041499共表达的m RNA有89个,其涉及包括多项与中枢神经系统相关条目在内的广泛的GO生物学过程;2.共表达m RNA的靶基因涉及的KEGG信号通路中许多与精神分裂症关系密切;3.NONHSAT089447、NONHSAT021545、NONHSAT041499可能在精神分裂症的发病的病理机制中起非常重要的作用。第四部分:精神分裂症异常表达的lncRNA与多巴胺信号通路调控机制的研究实验方法:在培养人神经母细胞瘤细胞(SK-N-SH)的过程中加入多巴胺模拟精神分裂症患者的神经细胞,实时荧光定量PCR(q RT-PCR)检测lncRNA的表达量。利用多巴胺拮抗剂奥氮平来反证NONHSAT089447是否由多巴胺所诱导升高,通过脂质体转染技术来实现si RNA及plasmid-447导入SK-N-SH,提取总RNA,q RT-PCR检测转染前后两种lncRNA(NONHSAT089447,NONHSAT041499)的表达量及多巴胺受体(DRD1,DRD2,DRD3,DRD4,DRD5)表达的变化。运用western blot技术检测干扰及过表达NONHSAT089447多巴胺受体的下游信号的变化。结果:1.q RT-PCR结果显示,奥氮平拮抗多巴胺使NONHSAT089447的表达受到明显抑制(P0.001),NONHSAT041499表达抑制不明显(P0.05)。2、小干扰RNA对NONHSAT089447表达抑制更为明显(P0.05),过表达RNA也使NONHSAT089447具有更显著的增长(P0.01);3、干扰NONHSAT089447后发现DRD3和DRD5均表达降低(P0.05),过表达NONHSAT089447结果显示DRD3,DRD5表达升高(P0.05)。4、western blot显示干扰NONHSAT089447可使多巴胺受体下游信号减弱,过表达NONHSAT089447使多巴胺受体下游信号增强。结论:1.SZ患者PBMC中NONHSAT089447、NONHSAT021545、NONHSAT041499等3个lncRNA出现显著性表达上调,并且经抗精神病药物治疗后,NONHSA T089447和NONHSAT041499的相对表达水平较治疗前显著下降,并且其表达水平与临床症状的改善密切相关,有潜力作为精神分裂症的生物学标志物。2.生物信息学分析发现了与lncRNA共表达的m RNA,发现与SZ有关的许多GO生物学过程和KEGG信号通路显著富集;NONHSAT089447、NONHSAT021545、NONHSAT041499可能在SZ的病理生理机制中发挥着重要的作用。3.细胞水平研究表明:SZ患者的NONHSAT089447处于高表达状态,多巴胺受体信号通路被激活,由此进一步促进NONHSAT089447的表达,形成一个正反馈调节通路。NONHSAT089447可能是SZ的生物标志物,并且其表达水平可作为SZ疗效评价的指标。
[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.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R749.3
本文编号：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.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R749.3
【参考文献】
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1 张梁;过伟;孙欣羊;宋红涛;赵林;仲爱芳;牛威;师征;张理义;;精神分裂症患者血浆及单核细胞中microRNA表达水平差异的研究[J];中华行为医学与脑科学杂志;2014年08期
，本文编号：2124262
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