本文選題：外側(cè)韁核　切入點(diǎn)：DNA甲基化　出處：《吉林大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：抑郁癥作為一種常見的情感障礙精神疾病,其發(fā)病原因及機(jī)制十分復(fù)雜。目前普遍認(rèn)為中樞神經(jīng)系統(tǒng)中單胺類神經(jīng)遞質(zhì)(如5-羥色胺、多巴胺)的缺少或不足是抑郁癥發(fā)病的重要機(jī)制。表觀遺傳研究證實(shí),情感障礙與表觀遺傳學(xué)修飾有很密切的關(guān)系[10,11]。LHb作為連接邊緣前腦和中腦的關(guān)鍵樞紐,它在包括抑郁癥在內(nèi)的情感障礙中的作用逐漸受到研究者的關(guān)注。研究顯示,LHb神經(jīng)元極度活躍可以引起抑郁,抑制其活動(dòng)度又可以起到抗抑郁的效果。目前還不清楚,LHb神經(jīng)元的這種興奮性變化是否與其DNA甲基化的改變有關(guān),以及到底哪些基因發(fā)生了甲基化改變。我們?cè)缙诘难芯孔C實(shí)[9],LHb內(nèi)有豐富的DNMTs的表達(dá),當(dāng)抑制LHb內(nèi)DNMTs活性時(shí),LHb內(nèi)DNA甲基化水平發(fā)生改變,動(dòng)物的情感關(guān)聯(lián)行為也發(fā)生明顯變化。但LHb內(nèi)發(fā)生DNA甲基化改變時(shí),核團(tuán)內(nèi)哪些基因的表達(dá)水平受到了影響以及這些基因啟動(dòng)子區(qū)域甲基化改變等具體機(jī)制還不清楚。本研究檢測(cè)了LHb內(nèi)微量注射DNMTs抑制劑5-aza D引起大鼠行為學(xué)改變,同時(shí)也檢測(cè)了LHb內(nèi)興奮關(guān)聯(lián)基因、抑制關(guān)聯(lián)基因及應(yīng)激關(guān)聯(lián)基因的表達(dá)水平,再通過甲基化DNA免疫共沉淀(methylated DNA immunoprecipitation,Me DIP)及關(guān)聯(lián)real-time PCR方法檢測(cè)基因Exon I前啟動(dòng)子區(qū)域甲基化改變情況,從而探討LHb內(nèi)甲基化的變化誘導(dǎo)行為改變的可能機(jī)制。我們以前的研究證實(shí),LHb內(nèi)微量注射DNMTs抑制劑5-aza D可明顯降低LHb基因組DNA甲基化水平,可減少大鼠在曠場(chǎng)中探索行為,增加大鼠在強(qiáng)迫游泳中的不動(dòng)時(shí)間[9]。在本研究中,我們應(yīng)用同樣的注射方法并檢測(cè)了大鼠的快感缺失行為。在糖水偏愛實(shí)驗(yàn)中,LHb內(nèi)微量注射5-aza D的大鼠對(duì)糖水的偏愛程度顯著低于對(duì)照的溶劑組,表明降低LHb內(nèi)DNA甲基化可誘導(dǎo)大鼠快感缺失。上述所有結(jié)果表明,降低LHb基因組DNA甲基化水平可誘發(fā)大鼠的抑郁樣行為。然后我們采用real-time PCR技術(shù)檢測(cè)了LHb內(nèi)微量注射DNMTs抑制劑5-aza D或?qū)φ杖軇┐笫蟮腖Hb內(nèi)興奮關(guān)聯(lián)基因(βCa MK II、αCa MK II、Glu R1)、抑制關(guān)聯(lián)基因(GABAAα1、GAD67、GAD65)及應(yīng)激關(guān)聯(lián)基因(GR)的m RNA表達(dá)情況。結(jié)果顯示:與溶劑組相比,5-aza D組大鼠LHb內(nèi)βCa MK II、Glu R1m RNA表達(dá)顯著增加,GR m RNA表達(dá)顯著減少,αCa MK II、GABAAα1、GAD67、GAD65 m RNA表達(dá)無(wú)顯著變化。我們的實(shí)驗(yàn)結(jié)果提示,降低LHb基因組DNA甲基化水平,可通過興奮關(guān)聯(lián)的βCa MK II、Glu R1表達(dá)增加,提高LHb神經(jīng)元的興奮性,誘導(dǎo)大鼠抑郁樣行為。那么,LHb給予DNMTs抑制劑5-aza D是否直接改變了上述基因Exon I前啟動(dòng)子區(qū)域的甲基化,進(jìn)而影響其m RNA表達(dá),這還不清楚。我們利用Me DIP-q PCR方法檢測(cè)了βCa MK II基因(Cam K2b)和GR基因(NR3C1)Exon I前啟動(dòng)子區(qū)域的甲基化情況。結(jié)果顯示,注藥后兩個(gè)基因的Exon I前啟動(dòng)子區(qū)域的甲基化程度無(wú)顯著變化,表明5-aza D不是通過改變Cam K2b和NR3C1基因Exon I前啟動(dòng)子區(qū)域的甲基化來(lái)影響βCa MK II和GR m RNA的表達(dá),其它啟動(dòng)子區(qū)域的甲基化情況還需要我們進(jìn)一步去探究。綜上所述,我們的研究表明:LHb內(nèi)微量注射DNMTs抑制劑5-aza D后,大鼠對(duì)糖水的偏愛程度顯著降低,引起快感缺失為特征的抑郁樣行為;同時(shí)測(cè)得5-aza D組大鼠LHb內(nèi)興奮關(guān)聯(lián)的βCa MK II、Glu R1 m RNA表達(dá)顯著增加,而糖皮質(zhì)激素受體GR m RNA表達(dá)顯著降低;Me DIP-q PCR法測(cè)得Cam K2b和NR3C1基因Exon I前啟動(dòng)子區(qū)域的甲基化程度無(wú)顯著變化。本研究從表觀遺傳學(xué)的角度揭示了LHb內(nèi)DNA甲基化改變與抑郁樣行為的聯(lián)系及機(jī)制,為更多了解情感障礙發(fā)病機(jī)制及可能的治療提供更多的研究基礎(chǔ)。
[Abstract]:Depression is a common emotional disorder of mental disease, its etiology and mechanism is very complicated. It is generally believed that the monoamine neurotransmitters in the central nervous system (such as 5- serotonin, dopamine) lack or deficiency is an important pathogenesis of depression. Epigenetic research confirms, affective disorder and epigenetic modifications of [10,11].LHb relationship very close as a key hub connecting limbic forebrain and midbrain, its role in affective disorders including depression, the researchers have been paying attention. Studies show that LHb neurons hyperactive can cause depression, inhibit its activity and can play the anti depression effect. It is unclear that LHb neurons changes in the excitability and DNA methylation changes, and what are the genes were methylated. Our early studies confirmed that [9], LHb in Abundant expression of DNMTs when inhibiting the activity of DNMTs LHb, the DNA methylation level of LHb changed, also changed significantly associated with emotional animal behavior. But LHb changed DNA methylation, expression of nuclei which genes affected by these genes and promoter methylation changes the specific mechanism is not clear. This study examined LHb microinjection of DNMTs inhibitor 5-aza D induced rat behavior change, but also examined the excitement associated gene LHb, the expression level of genes associated with inhibition and stress associated genes, and then through the methylated DNA immunoprecipitation (methylated DNA immunoprecipitation, Me DIP) detection Association of real-time gene Exon and PCR methods I promoter methylation changes, the possible mechanism to investigate the changes of LHb in methylation induced behavioral changes. Our previous study confirmed LHb, microinjection of DNMTs inhibitor 5-aza D can significantly reduce the LHb methylation level of genomic DNA, can reduce the exploratory behavior of rats in the open field, increase the rats in the immobility time of forced swimming in [9]. in this study, we used the same injection method and detected the anhedonia behavior of rats. Sucrose preference test, the degree of preference for sugar water LHb microinjection of 5-aza D rats was significantly lower than that of control group showed a decrease in solvent, LHb DNA methylation induced anhedonia. All the above results showed that the decrease of LHb genomic DNA methylation level can induce depression like behavior in rats. Excited Association then we use the real-time PCR gene was detected in LHb microinjection of DNMTs inhibitor 5-aza or D control rats in LHb solvent (beta Ca MK II MK II Glu, alpha Ca, R1), inhibition associated gene (GABAA alpha 1, GAD67, GAD65) and stress. Gene expression of M (GR) RNA. The results showed that: compared with the solvent group, 5-aza D group rats LHb Ca MK beta II, significantly increased expression of Glu R1m RNA, m RNA significantly decreased the expression of GR MK II GABAA, alpha Ca, alpha 1, GAD67 GAD65, m RNA expression did not change significantly. Our experimental results suggest that reduced LHb genomic DNA methylation level, through the association of beta Ca MK excited II, Glu increase the expression of R1, increase the excitability of LHb neurons, induced depression like behavior in rats. Then, LHb 5-aza D with the DNMTs inhibitor is directly changed the gene Exon I before the start of methyl sub region, and then influence the M Expression of RNA, it is still not clear. We use Me DIP-q PCR to detect the Ca MK beta II gene (Cam K2b) and GR gene (NR3C1) Exon I before starting the methylation status of sub region. The results showed that two genes after injection of Exon I promoter methylation level 5-aza D showed no significant change, not by changing the Cam K2b and NR3C1 Exon gene I methylation in promoter region to influence the expression of beta II and Ca MK GR m RNA, the methylation status of the promoter region of the other we also need further study. In conclusion, our research shows that: LHb microinjection DNMTs inhibitor 5-aza D, the degree of preference for sugar water rats decreased significantly, depression like behavior caused by lack of pleasure as the feature; while the measured 5-aza of rats in the D group LHb beta Ca MK II excited Association, Glu R1 m RNA expression was significantly increased, while the expression of glucocorticoid receptor GR m RNA decreased significantly Me; DIP-q PCR K2b and NR3C1 Cam were measured before I Exon gene promoter methylation level. No significant changes in the relationship and mechanism of LHb in DNA methylation and depression like behavior revealed from the view of epigenetics, to learn more about the The pathogenesis and possible treatment of affective disorders provide more research basis.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R749.4

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