本文關(guān)鍵詞： MeCP2 Rett綜合征 IMPX977 免疫共沉淀聯(lián)用質(zhì)譜　出處：《北京協(xié)和醫(yī)學(xué)院》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：Rett綜合征(Rett syndrome,RTT)是一種X連鎖的神經(jīng)發(fā)育障礙性遺傳病,在女性嚴(yán)重智力低下的致病因素中占主導(dǎo)地位。編碼甲基化CpG結(jié)合蛋白2(Methyl-CpG-binding protein 2,MeCP2)的基因發(fā)生突變是導(dǎo)致RTT病理學(xué)改變的主要原因,MeCP2作為轉(zhuǎn)錄抑制因子調(diào)控基因表達(dá)。在RTT發(fā)病機(jī)制中,由于缺乏MeCP2蛋白與甲基化DNA的結(jié)合,阻礙了其對下游靶基因的調(diào)控,最終導(dǎo)致腦功能障礙。目前,對于MeCP2在腦發(fā)育過程中的重要作用以及如何導(dǎo)致RTT的發(fā)生發(fā)展,其機(jī)制尚未明確。而且目前還沒有針對RTT的獲批準(zhǔn)的治療藥物上市,減輕RTT的癥狀將成為一個(gè)重要的治療手段。因此本實(shí)驗(yàn)從RTT致病的根本原因考慮,以增加MeCP2表達(dá)水平為研究方向,從分子和基因兩方面來探究IMPX977對大鼠MeCP2表達(dá)的影響,同時(shí)應(yīng)用免疫共沉淀聯(lián)用質(zhì)譜技術(shù)研究MeCP2相互作用蛋白分子,試圖探索MeCP2蛋白在體內(nèi)的生物學(xué)功能。實(shí)驗(yàn)共分為四個(gè)部分:第一章正常雄性大鼠各組織中MeCP2的表達(dá)目的為了研究RTT的發(fā)病機(jī)制中MeCP2基因產(chǎn)物的功能和定位,我們進(jìn)行了MeCP2蛋白大鼠體內(nèi)區(qū)域分布的研究,對多種組織的裂解液進(jìn)行免疫印跡分析和實(shí)時(shí)熒光定量PCR(RT-PCR)測定,以期從分子和基因水平層面研究MeCP2在野生型雄性大鼠體內(nèi)的組織分布。方法將6周齡野生型雄性大鼠置于動(dòng)物房飼養(yǎng)三天,取出用水合氯醛麻醉后斷頭處死,在冰浴上迅速分離海馬、皮層、小腦組織,并分離心、肝、脾、肺、腎和肌肉組織,通過Western blotting技術(shù),研究MeCP2蛋白在大鼠體內(nèi)的組織分布;同時(shí),提取多種組織的總RNA,檢測完整性,逆轉(zhuǎn)錄成cDNA后,進(jìn)行RT-PCR檢測,在分子層面研究MeCP2mRNA的表達(dá)水平。結(jié)果Western blotting實(shí)驗(yàn)結(jié)果顯示MeCP2蛋白主要在腦組織中高表達(dá),包括小腦、皮層和海馬組織,其次為脾、肺和心臟組織,在肝臟、腎臟和肌肉中含量較少。RT-PCR實(shí)驗(yàn)結(jié)果顯示海馬、皮層和小腦組織中MeCP2 mRNA高表達(dá)。結(jié)論MeCP2蛋白在大鼠的神經(jīng)發(fā)育中起著重要的作用,MeCP2蛋白和mRNA水平?jīng)]有明顯的關(guān)聯(lián),可能原因是存在轉(zhuǎn)錄后調(diào)控的組織特異性。第二章IMPX977對雄性大鼠各組織中MeCP2表達(dá)量的影響目的研究不同劑量的IMPX977對雄性大鼠各組織中MeCP2表達(dá)量的影響。方法將雄性SD大鼠分為4組,空白對照組、橄欖油組(陰性對照組)、IMPX977低劑量組(10mg/kg)和IMPX977高劑量組(30mg/kg)。連續(xù)灌胃給藥兩周,末次給藥結(jié)束后水合氯醛麻醉斷頭取腦,分離皮層、小腦、心、脾和肺組織。通過Western blotting技術(shù),研究不同劑量IMPX977對多種組織中MeCP2蛋白表達(dá)的影響。同時(shí),提取小腦和皮層組織中的總RNA,檢測完整性,逆轉(zhuǎn)錄成cDNA后,進(jìn)行RT-PCR檢測,在分子層面研究IMPX977對MeCP2 mRNA表達(dá)水平的影響。結(jié)果Western blotting實(shí)驗(yàn)結(jié)果顯示,與空白對照組相比,橄欖油組大鼠小腦和心臟組織中MeCP2蛋白表達(dá)水平均明顯升高(0.05,P0.05),而IMPX977高劑量組大鼠心臟和脾臟組織中MeCP2蛋白的表達(dá)量都顯著性降低(P0.01,P0.05)。同時(shí),IMPX977低劑量組皮層組織中MeCP2蛋白表達(dá)顯著上調(diào)。RT-PCR實(shí)驗(yàn)結(jié)果顯示,相較于空白對照組或者陰性對照組,IMPX977低劑量組大鼠小腦組織中MeCP2蛋白的表達(dá)量明顯升高。而與空白對照組相比,IMPX977低劑量組和高劑量組大鼠皮層組織中MeCP2基因的表達(dá)量均顯著性降低。結(jié)論低劑量IMPX977(10 mg/kg)對雄性大鼠皮層組織中MeCP2蛋白的表達(dá)有顯著上調(diào)的作用,但是與基因表達(dá)水平趨勢并不一致。第三章IMPX977對雌性大鼠各組織中MeCP2表達(dá)量的影響目的研究不同劑量的IMPX977對雌性大鼠各組織中MeCP2表達(dá)量的影響。方法將雌性SD大鼠分為4組,空白對照組、橄欖油組(陰性對照組)、IMPX977低劑量組(10 mg/kg)和IMPX977高劑量組(30 mg/kg)。連續(xù)灌胃給藥兩周,末次給藥結(jié)束后斷頭處死,冰浴上迅速分離皮層和小腦組織。通過Western blotting技術(shù),研究不同劑量IMPX977對兩種組織中MeCP2蛋白表達(dá)量的影響。同時(shí),提取小腦和皮層組織中的總RNA,逆轉(zhuǎn)錄成cDNA后,進(jìn)行RT-PCR分析,在分子層面研究不同劑量IMPX977對MeCP2 mRNA表達(dá)水平的影響。結(jié)果Western blotting實(shí)驗(yàn)結(jié)果顯示,與空白對照組相比,橄欖油組、IMPX977低劑量組和高劑量組大鼠小腦組織中MeCP2蛋白表達(dá)水平均有升高,但都沒有顯著性差異(P0.05,P0.05)。RT-PCR實(shí)驗(yàn)結(jié)果顯示,相較于空白對照組,其他三組大鼠小腦組織中MeCP2基因表達(dá)水平?jīng)]有明顯變化。而陰性對照組和IMPX977高劑量組大鼠皮層組織中MeCP2基因表達(dá)量均顯著降低。結(jié)論兩種劑量的IMPX977對雌性大鼠小腦、皮層組織中MeCP2蛋白的表達(dá)沒有明顯變化。第四章免疫共沉淀聯(lián)合質(zhì)譜篩選MeCP2相互作用蛋白質(zhì)及蛋白質(zhì)相互作用數(shù)據(jù)評價(jià)目的研究皮層組織中MeCP2相互作用蛋白質(zhì),試圖探索MeCP2在皮層組織中如何發(fā)揮生理功能。方法將雄性SD大鼠、C57BL/6小鼠和雌性食蟹猴飼養(yǎng)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,用鹽酸氯胺酮(3 mg/kg)肌肉注射麻醉食蟹猴,在冰浴上迅速分離皮層組織。通過IP-MS(免疫共沉淀聯(lián)用質(zhì)譜)技術(shù)、Western blotting技術(shù)、SDS-PAGE電泳、考馬斯亮藍(lán)染色、切取SDS-PAGE膠上蛋白質(zhì)LTQ質(zhì)譜鑒定其組成成分,探究在不同生物皮層組織中MeCP2相互作用的蛋白質(zhì),采用生物信息學(xué)方法對所獲得的候選蛋白質(zhì)進(jìn)行評估分析。結(jié)果考馬斯亮藍(lán)染色結(jié)果顯示,MeCP2蛋白呈現(xiàn)清晰的條帶,且在大鼠、小鼠、食蟹猴樣品中的顏色深淺不一,MeCP2蛋白總量高低不同。Western blotting結(jié)果顯示,在小鼠input樣品中,MeCP2蛋白得到了富集,且在大鼠、小鼠和食蟹猴IP樣品中MeCP2的含量不一,相對分子質(zhì)量也不相同。應(yīng)用生物信息學(xué)對小鼠IP-MS結(jié)果進(jìn)行分析,發(fā)現(xiàn)MeCP2相互作用蛋白候選分子肽段數(shù)最高的是KIF5B和KCL1,靶蛋白主要與信號轉(zhuǎn)導(dǎo)通路、細(xì)胞應(yīng)激反應(yīng)、轉(zhuǎn)錄調(diào)控等過程有關(guān),其中與EGFR信號轉(zhuǎn)導(dǎo)通路、FGF信號轉(zhuǎn)導(dǎo)通路、Wnt信號轉(zhuǎn)導(dǎo)通路、Cadherin信號轉(zhuǎn)導(dǎo)通路關(guān)系密切,并參與了帕金森病、亨廷頓舞蹈癥等疾病的發(fā)病過程。結(jié)論這些相互作用蛋白質(zhì)的獲得為全面揭示MeCP2的生物學(xué)功能、深入研究MeCP2轉(zhuǎn)錄調(diào)控機(jī)制以及其參與RTT發(fā)病過程均提供了線索和研究思路,為我們下一步的深入研究提供了科學(xué)依據(jù)。
[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.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R596

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