【摘要】：精神分裂癥是一種病因尚未完全闡明的重性精神疾病。人群終生患病率約為1%,多于青壯年發(fā)病,嚴(yán)重威脅人類的身心健康,給家庭和社會(huì)帶來(lái)沉重負(fù)擔(dān)。 精神分裂癥是一種典型的多基因疾病,其遺傳度高達(dá)80%；但由于遺傳異質(zhì)性、基因間/位點(diǎn)間聯(lián)合作用以及基因-環(huán)境相互作用等因素的存在,加劇了該病的復(fù)雜性。精神分裂癥的多巴胺假說(shuō)是普遍接受的假說(shuō)之一,多巴胺系統(tǒng)功能紊亂被認(rèn)為是精神分裂癥發(fā)病的最終共同途徑。本組前期工作對(duì)多巴胺信號(hào)轉(zhuǎn)導(dǎo)通路相關(guān)基因篩查發(fā)現(xiàn)膽囊收縮素A型受體(Cholecystokinin type A receptor, CCKAR)基因與幻聽(tīng)相關(guān)。另一項(xiàng)前期工作初步發(fā)現(xiàn)SNAP相關(guān)蛋白(SNAP-associated protein, SNAPIN)基因與精神分裂癥相關(guān)。本文以多巴胺假說(shuō)為理論依據(jù),在前期工作的基礎(chǔ)上,應(yīng)用病例-對(duì)照的研究方法,以CCKAR、多巴胺受體1-5(Dopamine receptor D1-D5, DRD1-5)基因、多巴胺轉(zhuǎn)運(yùn)體(Dopamine transporter, DAT)基因和SNAPIN基因?yàn)楹蜻x基因,擴(kuò)大樣本驗(yàn)證基因的功能多態(tài)性位點(diǎn)與中國(guó)漢族人群偏執(zhí)型精神分裂癥的相關(guān)性,并對(duì)部分相關(guān)基因在nRNA表達(dá)、miRNA調(diào)控及相互作用蛋白等層面進(jìn)行功能學(xué)探索。 多項(xiàng)研究顯示CCKAR基因的rs1800857與精神分裂癥的幻聽(tīng)等陽(yáng)性癥狀相關(guān)。我們?cè)?22名偏執(zhí)型精神分裂癥患者和527名對(duì)照中檢測(cè)了CCKAR基因5’端調(diào)控區(qū)的rs1800857等5個(gè)單核苷酸多態(tài)性位點(diǎn)(Single nucleotide polymorphism, SNP)。盡管未能發(fā)現(xiàn)rs1800857與疾病相關(guān),但觀察到rs1800857對(duì)其上游的其他4個(gè)SNPs均存在順勢(shì)調(diào)節(jié)作用,并且存在3種5-SNP單倍型G-A-G-C-T、T-G-G-G-T和T-G-G-C-C均為疾病發(fā)生的風(fēng)險(xiǎn)因素(p=2.2×10-5,p=1.1×10-6'p=1.9×10-6),說(shuō)明CCKAR基因的等位基因異質(zhì)性參與精神分裂癥的發(fā)生。同時(shí),數(shù)量性狀分析發(fā)現(xiàn)5-SNP單倍型與PANSS量表中幻覺(jué)行為、猜疑／被害、敵對(duì)性和陽(yáng)性癥狀總分顯著相關(guān)。此外,我們采用real-time PCR檢測(cè)了抗精神分裂癥藥物氟哌啶醇(Haloperidol)和氯氮平(Clozapine)對(duì)CHP212細(xì)胞系中CCKAR基因表達(dá)的影響,發(fā)現(xiàn)36h內(nèi)氟哌啶醇使CCKAR基因的表達(dá)量出現(xiàn)先升高再恢復(fù)到初始表達(dá)水平的現(xiàn)象,而氯氮平組并未觀察到明顯變化。 對(duì)DRD1-5和DAT基因初步篩查發(fā)現(xiàn)DRD2和DAT與偏執(zhí)型精神分裂癥相關(guān)。隨后在1351名患者和1640名對(duì)照中驗(yàn)證了DRD2基因的rs1076560-T可提高精神分裂癥的發(fā)病風(fēng)險(xiǎn)(p=0.022)。同時(shí),在368名患者和420名對(duì)照中發(fā)現(xiàn)DAT基因的rs2455391與疾病相關(guān)(allelic p=0.015,genotypicp=0.018)其單倍型rs2975223(G)-rs2455391(C)在患者組中的頻率顯著高于對(duì)照組(p=0.0012)。 Snapin蛋白可結(jié)合SNAP-25以促進(jìn)SNARE復(fù)合物的形成進(jìn)而促進(jìn)突觸囊泡的釋放,其相互作用蛋白dysbindin的編碼基因DTNBP1是精神分裂癥最熱門(mén)的易感基因之一。在516名患者和532名對(duì)照樣本中我們首次發(fā)現(xiàn)SNAPIN基因3’UTR的rs7345與偏執(zhí)型精神分裂癥相關(guān)(allelic p=0.014,genotypic p=0.003)；但體外報(bào)告基因?qū)嶒?yàn)并未發(fā)現(xiàn)rs7345位點(diǎn)直接影響基因表達(dá)。隨后,通過(guò)生物信息學(xué)預(yù)測(cè)發(fā)現(xiàn)hsa-miR-30家族可能與SNAPI-3'UTR結(jié)合,經(jīng)雙熒光素酶報(bào)告基因方法檢測(cè)發(fā)現(xiàn)miR-30e可以結(jié)合SNAPIN-3'UTR,并抑制報(bào)告基因的表達(dá)。在113名患者和127名對(duì)照血漿樣本中發(fā)現(xiàn)患者組miR-30e含量較對(duì)照組明顯降低(p=0.001)。之后,我們又在蛋白質(zhì)相互作用層面應(yīng)用串聯(lián)親和純化(tandem affinity purification,TAP)結(jié)合質(zhì)譜鑒定的方法,初步找到57種Snapin的候選相互作用蛋白。 綜上所述,我們不僅在遺傳學(xué)水平上驗(yàn)證了CCKAR.DRD2.DAT和SNAPIN基因在中國(guó)漢族人群中與偏執(zhí)型精神分裂癥相關(guān),而且在藥物對(duì)基因表達(dá)水平、miRNA調(diào)控水平及蛋白相互作用水平初步探索了易感基因的功能。
[Abstract]:Schizophrenia is a major mental disorder that is not yet fully set forth in the cause of schizophrenia. The lifetime prevalence of the population is about 1%, more than the onset of the young and the middle-aged, the physical and mental health of the human being seriously threatened, and the heavy burden on the family and the society. Schizophrenia is a typical multi-gene disease, and its genetic degree is as high as 80%; however, due to the genetic heterogeneity, the combination of intergenic/ intersite and the gene-environment interaction, the complexity of the disease is aggravated. The dopamine hypothesis of schizophrenia is one of the most commonly accepted hypotheses, and the dysfunction of the dopamine system is thought to be the final common way of the onset of schizophrenia. In this study, we found that the gene of the type A receptor (CCKAR) of the cholecystokinin A receptor (CCKAR) and the auditory phase were found in the early stage of this group. On the other hand, SNAP-associated protein (SNAP-associated protein, SNAPIN) gene and schizophrenia On the basis of the hypothesis of dopamine, this paper, on the basis of the previous work, applied the case-control study to the candidate base of the gene of CCKAR, dopamine receptor 1-5 (Dopammine receptor D1-D5, DRD1-5), the dopamine transporter (DAT) and the SNAPIN gene. The relationship between the functional polymorphism site of the sample verification gene and the paranoid schizophrenia in the Chinese Han population was expanded, and some related genes were studied in the aspects of nRNA expression, miRNA regulation and interaction protein. The results showed that the rs1800857 of the CCKAR gene and the auditory hallucination of the schizophrenia were positive. In 522 paranoid and 527 controls, we tested 5 single-nucleosonic acid polymorphism sites, such as rs1800857 of the 5 '-end regulatory region of the CCKAR gene, S NP). Although rs1800857 was not found to be related to the disease, it was observed that rs1800857 had a homeopathic regulatory effect on the other 4 SNPs upstream of it, and there were three 5-SNP haplotype G-A-G-C-T, T-G-G-G-T and T-G-G-C-C all the risk factors for disease (p = 2.2)10-5, p = 1.1,10-6 'p = 1.9%10 -6), indicating that the allele heterogeneity of the CCKAR gene is involved in the schizophrenia Occurrence of hallucination, suspicion/ murder, hostility and positive symptoms in 5-SNP haplotype and PANSS scale were found in quantitative trait analysis. In addition, the effect of clozapine and clozapine on the expression of CCKAR gene in CHP212 cell line was detected by the real-time PCR, and the expression of CCKAR gene in 36 h was first raised and then returned to the initial expression level. And the chlorazepine group was not observed. Significant changes. Preliminary screening of DRD1-5 and DAT genes found DRD2 and DAT and paranoia The risk of the onset of schizophrenia (p = 0) was then increased in 1351 patients and in the 1640 control with the rs1076560-T of the DRD2 gene. .022). At the same time, the frequency of rs2455391 (G)-rs2455391 (C) of the DAT gene was found to be significantly higher in the patient group than in the control group (p = 0) in 368 patients and 420 controls. 0012). The Snapin protein can be combined with SNAP-25 to promote the formation of the SNARE complex and further promote the release of the bursal, and the coding gene DNBP1 of the interaction protein, dlybindin, is the most popular in the case of schizophrenia. One of the susceptible genes was found in 516 and 532 control samples for the first time that the rs7345 of the SNAPIN gene 3 'UTR was associated with a paranoid type of schizophrenia (allelic p = 0.014, genetic p = 0.003); however, the in vitro reporter assay did not find that the rs7345 site was straight The expression of the influence gene was affected. Subsequently, the hsa-miR-30 family was found to be bound to the SNAPI-3 'UTR by bioinformatics prediction, and it was found that the miR-30e could bind to the SNAPIN-3' UTR by the double luciferase reporter gene method and inhibit The expression of the reporter gene. In 113 patients and 127 control plasma samples, the content of miR-30e in the patient group was significantly lower in the control group (p = 0.001). After that, we applied tandem affinity purification (TAP) in the protein-interaction level to identify the 57 types of Snapin by means of tandem affinity purification (TAP) and mass spectrometry. To sum up, we have not only verified that the CCKAR. DRDD2. DAT and SNAPIN gene are related to the paranoid schizophrenia in the Chinese Han population, but also the level of gene expression, the level of miRNA regulation and the level of protein interaction in the Chinese Han population.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R749.3

【共引文獻(xiàn)】

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