miR-219a-5p在甲基苯丙胺成瘾中的作用

发布时间：2018-05-10 06:51

本文选题：甲基苯丙胺 + 微小RNA　；参考：《宁波大学》2017年硕士论文

【摘要】：目的:目前甲基苯丙胺(METH)滥用依然是全球性的公共健康问题。长期使用甲基苯丙胺会导致多种精神疾病,但甲基苯丙胺成瘾神经生物学机制仍未明确。本文从甲基苯丙胺成瘾中的microRNA入手,筛选出靶向中枢血管紧张素系统的microRNAs,通过调节microRNA来探究中枢血管紧张素系统(RAS)在甲基苯丙胺成瘾中的潜在作用。方法:1、将甲基苯丙胺静脉自身主动给药组(甲基苯丙胺主动组)、甲基苯丙胺静脉被动给药组(甲基苯丙胺被动组)和生理盐水静脉主动给药组(生理盐水组)的大鼠伏隔核进行microRNA microarray,找出异常表达的microRNA;基于GO分析与KEGG分析,对中枢血管经张素系统有关的microRNA进行筛选;2、通过微注射慢病毒在大鼠脑区-伏隔核中过表达miR-219a-5p,分别通过固定频率(FR)和累进频率(PR)来检验miR-219a-5p过表达后对大鼠强迫给药和给药动机的调控作用;3、通过微注射慢病毒在大鼠脑区-伏隔核中过表达miR-219a-5p,探索miR-219a-5p在甲基苯丙胺复吸中的调控作用;4、通过酶联免疫吸附测定法来测定醛固酮合成和分泌通路中血管紧张素II受体1(AT1)、磷脂酰肌醇磷脂酶(PLCβ)和cAMP反应元件结合蛋白(CREB)等蛋白在甲基苯丙胺自身给药动物中是否有显著变化;5、根据实验四的验证,对实验二和实验三中的大鼠伏隔核脑区,在miR-219a-5p过表达后,检测主要蛋白的表达变化。结果:1、基于甲基苯丙胺主动组和甲基苯丙胺被动组以及甲基苯丙胺主动组和生理盐水组分别进行比较,筛选出显著变化的靶向中枢血管紧张素系统的microRNAs,最后,根据相对表达倍数筛选出研究目标miR-219a-5p;2、miR-219a-5p过表达后,对于固定频率给药,在0.025mg/kg的剂量中,LV1-miR-219a-5p组与LV1CN组之间没有差异,而在0.05mg/kg、0.075mg/kg和0.1mg/kg中,LV1-miR-219a-5p组相对于LV1CN组给药次数显著下降。对于累进频率给药,在0.025mg/kg的剂量中,LV1-miR-219a-5p组与LV1CN组之间没有差异,而在0.05mg/kg、0.075mg/kg和0.1mg/kg中,LV1-miR-219a-5p组相对于LV1CN组给药次数显著下降;3、miR-219a-5p过表达后,无论是线索诱导还是药物诱导,LV1-miR-219a-5p组相对于LV1CN组有效鼻触数显著下降;4、通过酶联免疫吸附测定法(ELISA),AT1、PLCβ和CREB等蛋白表达水平在甲基苯丙胺成瘾中显著上升;5、通过免疫印迹法测定实验二和实验三中的大鼠伏隔核脑区AT1、PLCβ和CREB表达情况,可以发现LV1-miR-219a-5p组相对于LV1CN组蛋白的表达量显著下降。结论:我们的研究结果表明,大鼠伏隔核中的miR-219a-5p在中枢肾素-血管紧张素-醛固酮系统(RAAS)的重要作用,揭示了调控甲基苯丙胺成瘾的分子机制。
[Abstract]:Objective: the abuse of methamphetamine methamphetamine (METHH) is still a global public health problem. Long-term use of methamphetamine can lead to many mental disorders, but the neurobiological mechanism of methamphetamine addiction remains unclear. Starting with microRNA in methamphetamine addiction, microRNAs-targeted central angiotensin system was screened to explore the potential role of central angiotensin system in methamphetamine addiction by regulating microRNA. Methods: 1, methamphetamine group (methamphetamine active group), methamphetamine group (methamphetamine passive group) and saline group (saline group) were divided into three groups: methamphetamine self-administered group (methamphetamine active group), methamphetamine group (methamphetamine passive group) and saline group (saline group). MicroRNA microRNAs were detected in the nucleus accumbens of rats, based on go analysis and KEGG analysis. The microRNA related to the tensin system in central blood vessels was screened. The overexpression of miR-219a-5p in rat brain region and nucleus accumbens was detected by microinjection of lentivirus, and forced injection of miR-219a-5p was performed by fixed frequency and progressive frequency. By microinjection of lentivirus into rat brain region and nucleus accumbens to investigate the regulatory role of miR-219a-5p in methamphetamine relapse, aldosterone was determined by enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay). Whether angiotensin II receptor 1 (AT1), phosphatidylinositol phospholipase 尾 (PLC- 尾) and cAMP response element binding protein (CREBB) have significantly changed in methamphetamine self-administered animals. After overexpression of miR-219a-5p in the nucleus accumbens of rats in experiment two and three, the expression of major proteins was detected. Results: 1, based on the comparison between the active methamphetamine group and the methamphetamine passive group, and the methamphetamine active group and the normal saline group, the microRNAss targeting the central angiotensin system were screened out. After over expression of miR-219a-5pn2miR-219a-5p, there was no difference between LV1-miR-219a-5p group and LV1CN group in the dose of 0.025mg/kg, but in 0.05mg / kg of LV1-miR-219a-5p group and 0.1mg/kg group, LV1-miR-219a-5p group was significantly lower than that of LV1CN group. For progressive frequency administration, there was no difference between LV1-miR-219a-5p group and LV1CN group in the dose of 0.025mg/kg. However, LV1-miR-219a-5p group was significantly lower than LV1CN group in the frequency of administration of LV1-miR-219a-5p in 0.05 mg / kg and 0.1mg/kg 0.075 mg / kg, compared with that in LV1CN group, the expression of LV1-miR-219a-5p was significantly lower than that of LV1CN group, and the expression of LV1-miR-219a-5p was significantly lower than that of LV1CN group. Both cue-induced and drug-induced LV1-miR-219a-5p groups significantly decreased the effective nasal contacts compared with those of LV1CN group. The expression levels of ELISAA AT1 CREB 尾 and CREB in methamphetamine addiction were significantly increased by Elisa. The expression of PLC 尾 and CREB in the nucleus accumbens of rats in experiment two and three were measured by trace method. It was found that the expression of histone in LV1-miR-219a-5p group was significantly lower than that in LV1CN group. Conclusion: our results suggest that miR-219a-5p in rat nucleus accumbens plays an important role in central renin-angiotensin-aldosterone system, which reveals the molecular mechanism of regulating methamphetamine addiction.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.64

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