IMPX977对MeCP2蛋白表达的影响以及基于IP-MS技术对不同物种大脑中MeCP2的功能研究

发布时间：2018-02-03 19:49

  本文关键词： MeCP2 Rett综合征 IMPX977 免疫共沉淀联用质谱　出处：《北京协和医学院》2017年硕士论文　论文类型：学位论文

【摘要】：Rett综合征(Rett syndrome,RTT)是一种X连锁的神经发育障碍性遗传病,在女性严重智力低下的致病因素中占主导地位。编码甲基化CpG结合蛋白2(Methyl-CpG-binding protein 2,MeCP2)的基因发生突变是导致RTT病理学改变的主要原因,MeCP2作为转录抑制因子调控基因表达。在RTT发病机制中,由于缺乏MeCP2蛋白与甲基化DNA的结合,阻碍了其对下游靶基因的调控,最终导致脑功能障碍。目前,对于MeCP2在脑发育过程中的重要作用以及如何导致RTT的发生发展,其机制尚未明确。而且目前还没有针对RTT的获批准的治疗药物上市,减轻RTT的症状将成为一个重要的治疗手段。因此本实验从RTT致病的根本原因考虑,以增加MeCP2表达水平为研究方向,从分子和基因两方面来探究IMPX977对大鼠MeCP2表达的影响,同时应用免疫共沉淀联用质谱技术研究MeCP2相互作用蛋白分子,试图探索MeCP2蛋白在体内的生物学功能。实验共分为四个部分:第一章正常雄性大鼠各组织中MeCP2的表达目的为了研究RTT的发病机制中MeCP2基因产物的功能和定位,我们进行了MeCP2蛋白大鼠体内区域分布的研究,对多种组织的裂解液进行免疫印迹分析和实时荧光定量PCR(RT-PCR)测定,以期从分子和基因水平层面研究MeCP2在野生型雄性大鼠体内的组织分布。方法将6周龄野生型雄性大鼠置于动物房饲养三天,取出用水合氯醛麻醉后断头处死,在冰浴上迅速分离海马、皮层、小脑组织,并分离心、肝、脾、肺、肾和肌肉组织,通过Western blotting技术,研究MeCP2蛋白在大鼠体内的组织分布;同时,提取多种组织的总RNA,检测完整性,逆转录成cDNA后,进行RT-PCR检测,在分子层面研究MeCP2mRNA的表达水平。结果Western blotting实验结果显示MeCP2蛋白主要在脑组织中高表达,包括小脑、皮层和海马组织,其次为脾、肺和心脏组织,在肝脏、肾脏和肌肉中含量较少。RT-PCR实验结果显示海马、皮层和小脑组织中MeCP2 mRNA高表达。结论MeCP2蛋白在大鼠的神经发育中起着重要的作用,MeCP2蛋白和mRNA水平没有明显的关联,可能原因是存在转录后调控的组织特异性。第二章IMPX977对雄性大鼠各组织中MeCP2表达量的影响目的研究不同剂量的IMPX977对雄性大鼠各组织中MeCP2表达量的影响。方法将雄性SD大鼠分为4组,空白对照组、橄榄油组(阴性对照组)、IMPX977低剂量组(10mg/kg)和IMPX977高剂量组(30mg/kg)。连续灌胃给药两周,末次给药结束后水合氯醛麻醉断头取脑,分离皮层、小脑、心、脾和肺组织。通过Western blotting技术,研究不同剂量IMPX977对多种组织中MeCP2蛋白表达的影响。同时,提取小脑和皮层组织中的总RNA,检测完整性,逆转录成cDNA后,进行RT-PCR检测,在分子层面研究IMPX977对MeCP2 mRNA表达水平的影响。结果Western blotting实验结果显示,与空白对照组相比,橄榄油组大鼠小脑和心脏组织中MeCP2蛋白表达水平均明显升高(0.05,P0.05),而IMPX977高剂量组大鼠心脏和脾脏组织中MeCP2蛋白的表达量都显著性降低(P0.01,P0.05)。同时,IMPX977低剂量组皮层组织中MeCP2蛋白表达显著上调。RT-PCR实验结果显示,相较于空白对照组或者阴性对照组,IMPX977低剂量组大鼠小脑组织中MeCP2蛋白的表达量明显升高。而与空白对照组相比,IMPX977低剂量组和高剂量组大鼠皮层组织中MeCP2基因的表达量均显著性降低。结论低剂量IMPX977(10 mg/kg)对雄性大鼠皮层组织中MeCP2蛋白的表达有显著上调的作用,但是与基因表达水平趋势并不一致。第三章IMPX977对雌性大鼠各组织中MeCP2表达量的影响目的研究不同剂量的IMPX977对雌性大鼠各组织中MeCP2表达量的影响。方法将雌性SD大鼠分为4组,空白对照组、橄榄油组(阴性对照组)、IMPX977低剂量组(10 mg/kg)和IMPX977高剂量组(30 mg/kg)。连续灌胃给药两周,末次给药结束后断头处死,冰浴上迅速分离皮层和小脑组织。通过Western blotting技术,研究不同剂量IMPX977对两种组织中MeCP2蛋白表达量的影响。同时,提取小脑和皮层组织中的总RNA,逆转录成cDNA后,进行RT-PCR分析,在分子层面研究不同剂量IMPX977对MeCP2 mRNA表达水平的影响。结果Western blotting实验结果显示,与空白对照组相比,橄榄油组、IMPX977低剂量组和高剂量组大鼠小脑组织中MeCP2蛋白表达水平均有升高,但都没有显著性差异(P0.05,P0.05)。RT-PCR实验结果显示,相较于空白对照组,其他三组大鼠小脑组织中MeCP2基因表达水平没有明显变化。而阴性对照组和IMPX977高剂量组大鼠皮层组织中MeCP2基因表达量均显著降低。结论两种剂量的IMPX977对雌性大鼠小脑、皮层组织中MeCP2蛋白的表达没有明显变化。第四章免疫共沉淀联合质谱筛选MeCP2相互作用蛋白质及蛋白质相互作用数据评价目的研究皮层组织中MeCP2相互作用蛋白质,试图探索MeCP2在皮层组织中如何发挥生理功能。方法将雄性SD大鼠、C57BL/6小鼠和雌性食蟹猴饲养一周后,用水合氯醛(0.3-0.4 mL/100g)腹腔注射麻醉大鼠和小鼠,用盐酸氯胺酮(3 mg/kg)肌肉注射麻醉食蟹猴,在冰浴上迅速分离皮层组织。通过IP-MS(免疫共沉淀联用质谱)技术、Western blotting技术、SDS-PAGE电泳、考马斯亮蓝染色、切取SDS-PAGE胶上蛋白质LTQ质谱鉴定其组成成分,探究在不同生物皮层组织中MeCP2相互作用的蛋白质,采用生物信息学方法对所获得的候选蛋白质进行评估分析。结果考马斯亮蓝染色结果显示,MeCP2蛋白呈现清晰的条带,且在大鼠、小鼠、食蟹猴样品中的颜色深浅不一,MeCP2蛋白总量高低不同。Western blotting结果显示,在小鼠input样品中,MeCP2蛋白得到了富集,且在大鼠、小鼠和食蟹猴IP样品中MeCP2的含量不一,相对分子质量也不相同。应用生物信息学对小鼠IP-MS结果进行分析,发现MeCP2相互作用蛋白候选分子肽段数最高的是KIF5B和KCL1,靶蛋白主要与信号转导通路、细胞应激反应、转录调控等过程有关,其中与EGFR信号转导通路、FGF信号转导通路、Wnt信号转导通路、Cadherin信号转导通路关系密切,并参与了帕金森病、亨廷顿舞蹈症等疾病的发病过程。结论这些相互作用蛋白质的获得为全面揭示MeCP2的生物学功能、深入研究MeCP2转录调控机制以及其参与RTT发病过程均提供了线索和研究思路,为我们下一步的深入研究提供了科学依据。
[Abstract]:Rett syndrome (Rett syndrome, RTT X) is a neurodevelopmental disorder linked to genetic diseases, the dominant pathogenic factors in women of severe mental retardation. Encoding methyl CpG binding protein 2 (Methyl-CpG-binding protein 2, MeCP2) gene mutation is a major cause of pathological changes RTT, MeCP2 as a transcription inhibitory factor to regulate gene expression. In the pathogenesis of RTT, due to the lack of MeCP2 protein and DNA methylation, hindered the regulation of downstream target genes, leading to brain dysfunction. At present, MeCP2 plays an important role in the process of brain development and how to lead to the occurrence and development of RTT, its mechanism is not clear. But there is no approved drugs for the listing of RTT, relieve the symptoms of RTT will become an important means of treatment. Therefore this experiment from the root causes of RTT disease, in order to increase The expression level of MeCP2 as the research direction, to explore the effect of IMPX977 on the expression of MeCP2 in rats from the two aspects of molecular and gene, and co immunoprecipitation combined with mass spectrometry study of MeCP2 interacting protein molecules, to explore MeCP2 protein biological function in vivo. The experiment is divided into four parts: the first chapter study the expression of MeCP2 normal male rats in different tissues in order to function and orientation of MeCP2 to study the pathogenesis of RTT gene product, we studied the distribution of MeCP2 protein in the body region of the rat, the lysate of multiple tissues by Western blot analysis and quantitative real-time PCR (RT-PCR) were determined, in order from the molecular and genetic level. Study of MeCP2 in wild type male rats. Methods the tissue distribution in real animal 6 week old wild type male rats fed for three days, remove with chloral hydrate anesthetized and decapitated, Rapid separation in an ice bath on the hippocampus, cortex, cerebellum, heart and liver, spleen, separation, lung, kidney and muscle tissue by Western Blotting Technology, the distribution of MeCP2 protein in rat tissues; at the same time, total RNA was extracted from various tissue, detection of integrity, reverse transcription into cDNA, for RT-PCR assay, expression of MeCP2mRNA in molecular level. Results Western blotting results showed that MeCP2 protein mainly expressed in the brain, including cerebellum, cortex and hippocampus, followed by the spleen, lung and heart tissues, in the liver, less content of hippocampal.RT-PCR experimental results showed that the kidney and muscle, MeCP2 high expression of mRNA the cortex and cerebellum. Conclusion MeCP2 protein in the rat nerve plays an important role in the development, no significant correlation between MeCP2 protein and mRNA levels may be due to tissue-specific existence of post transcriptional regulation. In the second chapter IMPX977 of different tissues in male rats MeCP2 expression to study the effects of different doses of IMPX977 on the expression of MeCP2 in tissues of male rats was determined. Methods male SD rats were divided into 4 groups, blank control group, olive oil group (negative control group), low dose IMPX977 group (10mg/kg) and high dose of IMPX977 group (30mg/kg). Continuous gavage for two weeks, the lastadministration ended chloralhydrate decapitated, isolated from cortex, cerebellum, heart, spleen and lung tissue by Western Blotting Technology, study on the effects of different doses of IMPX977 on the expression of MeCP2 protein in various tissues. At the same time. Total RNA was extracted from the cerebellum and cortex tissue, detection of integrity, reverse transcription into cDNA, detected by RT-PCR, at the level of the influences of IMPX977 on the molecular level on the expression of MeCP2 mRNA Western blotting. The experimental results show that compared with the control group, rats of olive oil The expression of MeCP2 protein levels were significantly higher in the cerebellum and heart tissue (0.05, P0.05), whereas the expression of MeCP2 protein in IMPX977 high dose group rat heart and spleen tissues were significantly lower (P0.01, P0.05). At the same time, IMPX977 low dose group in the cortical tissue MeCP2 protein expression was significantly up-regulated.RT-PCR experimental results show, compared with the blank control group and negative control group, the expression of MeCP2 protein in cerebellum of rats in the low dose of IMPX977 increased significantly. Compared with the control group, the expression of MeCP2 gene IMPX977 in low dose group and high dose group rat cortical tissue were significantly decreased. Conclusion: low dose IMPX977 (10 mg/kg) increased on the expression of MeCP2 protein in cortex tissue of male rats in the role, but the trend is not consistent with the gene expression level. In the third chapter, the influence of IMPX977 on target amount of MeCP2 expressed in various tissues of female rats The study of different doses of IMPX977 on the expression of MeCP2 in tissues of female rats was determined. Methods: the female SD rats were divided into 4 groups, blank control group, olive oil group (negative control group), IMPX977 low dose group (10 mg/kg) and IMPX977 high dose group (30 mg/kg) orally. For two weeks, the lastadministration ended guillotined, rapid separation of cortex and cerebellum of the ice bath. Through Western Blotting Technology, the effect of different doses of IMPX977 on the expression of MeCP2 protein in two tissues. At the same time, the total RNA extraction of cerebellum and cortex tissues, reverse transcription into cDNA, for RT-PCR analysis, at the molecular level of different doses of IMPX977 on the expression of MeCP2 on mRNA Western blotting. The experimental results show that compared with the control group, olive oil group, the expression of IMPX977 MeCP2 protein levels were low dose group and high dose group of rat cerebellum Have increased, but no significant difference (P0.05, P0.05).RT-PCR experimental results show that compared with the control group, the other three groups of MeCP2 rat cerebellar tissue gene expression levels did not change significantly. While MeCP2 negative group and IMPX977 high dose group rat cortical tissue gene expression decreased significantly. Conclusion two doses of IMPX977 on female rat cerebellum, no significant changes in the expression of MeCP2 protein in the cortical tissue. Combined mass screening of MeCP2 interacting protein MeCP2 interacting proteins and protein interaction data evaluation objective to study the cortical organization in the fourth chapter, CO immunoprecipitation, MeCP2 attempts to explore how to play a physiological function in the cortex tissue. Methods male SD rats, C57BL/6 mice and female cynomolgus monkeys fed for a week, with chloral hydrate (0.3-0.4 mL/100g) intraperitoneal injection of anesthesia in rats and mice with ketamine hydrochloride. Ketone (3 mg/kg) intramuscular injection anesthesia in cynomolgus monkeys, rapid separation of cortical tissue in ice bath. Through IP-MS (mass spectrometry immunoprecipitation) technology, Western Blotting Technology, SDS-PAGE electrophoresis, Coomassie brilliant blue staining Kaumas, cut from SDS-PAGE gel protein LTQ identified the components, explore in different biological cortex tissues the interaction of MeCP2 proteins, methods to assess the candidate protein obtained by bioinformatics. Results the Coomassie blue staining showed that MeCP2 protein showed clear bands, and the mice in the rat, and color depth of cynomolgus monkeys in the sample, the total protein level of different MeCP2.Western blotting showed that the mice in input samples, MeCP2 protein was enriched, and the content of MeCP2 in rats, mice and cynomolgus monkey IP samples in a relative molecular mass is not the same. The application of bioinformatics Mouse IP-MS results of the analysis, found that the MeCP2 interaction protein candidate peptides with the highest number of KIF5B and KCL1, target proteins and signal transduction, cell stress response, transcriptional regulation and so on, and the EGFR signal transduction pathway, FGF signaling pathway, Wnt signaling pathway, Cadherin signaling pathway is closely related and, in Parkinson's disease, Huntington's disease and other disease pathogenesis. Conclusion these protein interactions have to reveal the biological function of MeCP2, in-depth study of MeCP2 transcriptional regulation mechanism and its participation in the pathogenesis of RTT provide clues and research ideas, provide a scientific basis for further research on our next step.

【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R596

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