外侧缰核DNA甲基化改变诱导大鼠抑郁样行为的机制探讨

发布时间：2018-03-15 06:09

本文选题：外侧缰核　切入点：DNA甲基化　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：抑郁症作为一种常见的情感障碍精神疾病,其发病原因及机制十分复杂。目前普遍认为中枢神经系统中单胺类神经递质(如5-羟色胺、多巴胺)的缺少或不足是抑郁症发病的重要机制。表观遗传研究证实,情感障碍与表观遗传学修饰有很密切的关系[10,11]。LHb作为连接边缘前脑和中脑的关键枢纽,它在包括抑郁症在内的情感障碍中的作用逐渐受到研究者的关注。研究显示,LHb神经元极度活跃可以引起抑郁,抑制其活动度又可以起到抗抑郁的效果。目前还不清楚,LHb神经元的这种兴奋性变化是否与其DNA甲基化的改变有关,以及到底哪些基因发生了甲基化改变。我们早期的研究证实[9],LHb内有丰富的DNMTs的表达,当抑制LHb内DNMTs活性时,LHb内DNA甲基化水平发生改变,动物的情感关联行为也发生明显变化。但LHb内发生DNA甲基化改变时,核团内哪些基因的表达水平受到了影响以及这些基因启动子区域甲基化改变等具体机制还不清楚。本研究检测了LHb内微量注射DNMTs抑制剂5-aza D引起大鼠行为学改变,同时也检测了LHb内兴奋关联基因、抑制关联基因及应激关联基因的表达水平,再通过甲基化DNA免疫共沉淀(methylated DNA immunoprecipitation,Me DIP)及关联real-time PCR方法检测基因Exon I前启动子区域甲基化改变情况,从而探讨LHb内甲基化的变化诱导行为改变的可能机制。我们以前的研究证实,LHb内微量注射DNMTs抑制剂5-aza D可明显降低LHb基因组DNA甲基化水平,可减少大鼠在旷场中探索行为,增加大鼠在强迫游泳中的不动时间[9]。在本研究中,我们应用同样的注射方法并检测了大鼠的快感缺失行为。在糖水偏爱实验中,LHb内微量注射5-aza D的大鼠对糖水的偏爱程度显著低于对照的溶剂组,表明降低LHb内DNA甲基化可诱导大鼠快感缺失。上述所有结果表明,降低LHb基因组DNA甲基化水平可诱发大鼠的抑郁样行为。然后我们采用real-time PCR技术检测了LHb内微量注射DNMTs抑制剂5-aza D或对照溶剂大鼠的LHb内兴奋关联基因(βCa MK II、αCa MK II、Glu R1)、抑制关联基因(GABAAα1、GAD67、GAD65)及应激关联基因(GR)的m RNA表达情况。结果显示:与溶剂组相比,5-aza D组大鼠LHb内βCa MK II、Glu R1m RNA表达显著增加,GR m RNA表达显著减少,αCa MK II、GABAAα1、GAD67、GAD65 m RNA表达无显著变化。我们的实验结果提示,降低LHb基因组DNA甲基化水平,可通过兴奋关联的βCa MK II、Glu R1表达增加,提高LHb神经元的兴奋性,诱导大鼠抑郁样行为。那么,LHb给予DNMTs抑制剂5-aza D是否直接改变了上述基因Exon I前启动子区域的甲基化,进而影响其m RNA表达,这还不清楚。我们利用Me DIP-q PCR方法检测了βCa MK II基因(Cam K2b)和GR基因(NR3C1)Exon I前启动子区域的甲基化情况。结果显示,注药后两个基因的Exon I前启动子区域的甲基化程度无显著变化,表明5-aza D不是通过改变Cam K2b和NR3C1基因Exon I前启动子区域的甲基化来影响βCa MK II和GR m RNA的表达,其它启动子区域的甲基化情况还需要我们进一步去探究。综上所述,我们的研究表明:LHb内微量注射DNMTs抑制剂5-aza D后,大鼠对糖水的偏爱程度显著降低,引起快感缺失为特征的抑郁样行为;同时测得5-aza D组大鼠LHb内兴奋关联的βCa MK II、Glu R1 m RNA表达显著增加,而糖皮质激素受体GR m RNA表达显著降低;Me DIP-q PCR法测得Cam K2b和NR3C1基因Exon I前启动子区域的甲基化程度无显著变化。本研究从表观遗传学的角度揭示了LHb内DNA甲基化改变与抑郁样行为的联系及机制,为更多了解情感障碍发病机制及可能的治疗提供更多的研究基础。
[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.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.4

【参考文献】