朊病毒病相關(guān)LncRNA表達(dá)譜分析研究
發(fā)布時間：2018-08-14 10:12
【摘要】：［背景］朊病毒病是一種高致病性，高致死性的神經(jīng)退行性疾病，是由于細(xì)胞內(nèi)正常的朊蛋白發(fā)生構(gòu)象改變，形成的一種抗逆性極強(qiáng)，能夠大量聚集在神經(jīng)細(xì)胞內(nèi)的有毒蛋白PrPSc，導(dǎo)致細(xì)胞代謝紊亂，神經(jīng)細(xì)胞大量死亡。現(xiàn)已證明，人們誤食感染瘋牛病的牛肉后，會引起人類朊病毒病（克雅氏�。纱丝梢�，朊病毒病已經(jīng)打破了種間屏障，成為嚴(yán)重威脅人類健康的一種人獸共患病。經(jīng)過朊病毒學(xué)者們的不懈努力和深入研究，發(fā)現(xiàn)細(xì)胞內(nèi)可能存在與朊病毒感染、致病相關(guān)的宿主因子。LncRNA是近些年發(fā)現(xiàn)的一種重要的長鏈非編碼RNA分析，長度大于200nt，參與了生命過程中一系列重要進(jìn)程，可能是朊病毒致病的重要協(xié)助因子，它的研究給朊病毒病的發(fā)病機(jī)理帶來了希望，因此，我們對與朊病毒病相關(guān)的LncRNA分子進(jìn)行了初步的篩選與鑒定。 ［方法］本實(shí)驗(yàn)通過建立朊病毒小鼠鼠腦LncRNA和mRNA表達(dá)譜，根據(jù)分析結(jié)果，初步篩選出與朊病毒致病相關(guān)的LncRNA分子；然后使用生物信息學(xué)可高精度分辨出功能性LncRNA分子的靶向關(guān)系，參與生物學(xué)進(jìn)程等；利用熒光定量PCR和Western blotting定量分析功能性LncRNA分子對朊蛋白基因以及朊病毒復(fù)制的作用。 ［結(jié)果］建立朊病毒病模型小鼠腦部LncRNA和mRNA表達(dá)譜，并通過熒光定量PCR進(jìn)行了驗(yàn)證，最終檢測出6條LncRNA表達(dá)量相比對照組下調(diào)，13條LncRNA表達(dá)量相比對照組上調(diào)；建立了小鼠海馬組織中mRNA芯片，共檢測出170條差異表達(dá)的mRNA分子，其中25條mRNA表達(dá)下調(diào)，145條miRNA表達(dá)上調(diào)。 通過生物信息學(xué)分析預(yù)測，得到差異性表達(dá)LncRNA作用的靶基因以及靶基因的功能和參與細(xì)胞信號轉(zhuǎn)導(dǎo)通路。 熒光定量PCR分析得知過表達(dá)LncRNA-2對Prnp基因并沒有顯著影響，WesternBlotting檢測ScN2a細(xì)胞內(nèi)PrPSc含量說明，過表達(dá)LnRNA-2后能夠抑制PrPSc蛋白的復(fù)制。 ［結(jié)論］本實(shí)驗(yàn)建立了完整的朊病毒病小鼠海馬組織LncRNA以及mRNA表達(dá)譜，通過生物信息學(xué)分析初步篩選出了LnRNA-2進(jìn)行深入研究，利用熒光定量PCR和Westernblotting初步驗(yàn)證了該分子高表達(dá)之后能夠抑制朊病毒復(fù)制。為后續(xù)研究長鏈非編碼RNA以及朊病毒相關(guān)分子伴侶提供了一個理論的支持，奠定了基礎(chǔ)。
[Abstract]:[background] Prion disease is a neurodegenerative disease with high pathogenicity and high mortality. Prion disease is a highly resistant neurodegenerative disease due to the conformation change of normal prion proteins in cells. PrPSc, a toxic protein that accumulates in large numbers of nerve cells, leads to cell metabolic disorders and the death of large numbers of nerve cells. It has been proved that human prion disease (Creutzfeldt-Jakob disease) can be caused by miseating beef infected with mad cow disease, so prion disease has broken the interspecific barrier and become a serious threat to human health. Through the unremitting efforts and in-depth research of prion scholars, it is found that there may be a prion infection in cells. LncRNA, a host factor related to prion infection, is an important long strand noncoding RNA analysis discovered in recent years. It takes part in a series of important processes in the course of life and may be an important assisting factor of prion pathogenesis. Its research brings hope to the pathogenesis of prion disease. We screened and identified the LncRNA molecules associated with prion disease. [methods] the expression profiles of LncRNA and mRNA in the brain of prion mice were established. The LncRNA molecules associated with prion pathogenicity were preliminarily screened, and then the target relationship of functional LncRNA molecules could be identified by bioinformatics, which could participate in the biological process and so on. The effects of functional LncRNA molecules on prion gene and prion replication were quantitatively analyzed by fluorescence quantitative PCR and Western blotting. [results] the expression profiles of LncRNA and mRNA in the brain of prion disease model mice were established. The fluorescence quantitative PCR was used to verify that the expression of 6 LncRNA was up-regulated than that of the control group, and the mRNA microarray was established in the hippocampus of mice, and 170 differentially expressed mRNA molecules were detected. Among them, 25 mRNA down-regulated and 145 miRNA were up-regulated. By bioinformatics analysis and prediction, the function of target gene and target gene involved in cellular signal transduction pathway were obtained. Fluorescence quantitative PCR analysis showed that overexpression of LncRNA-2 had no significant effect on Prnp gene. Western blotting showed that PrPSc content in ScN2a cells was detected by Western blotting. Overexpression of LnRNA-2 can inhibit the replication of PrPSc protein. [conclusion] the complete LncRNA and mRNA expression profiles in the hippocampus of prion disease mice were established and LnRNA-2 was screened out by bioinformatics analysis for further study. Fluorescence quantitative PCR and Westernblotting showed that high expression of this molecule could inhibit prion replication. It provides a theoretical support for the further study of long chain noncoding RNA and prion-related molecular chaperone.
【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S855.3
本文編號：2182537
[Abstract]:[background] Prion disease is a neurodegenerative disease with high pathogenicity and high mortality. Prion disease is a highly resistant neurodegenerative disease due to the conformation change of normal prion proteins in cells. PrPSc, a toxic protein that accumulates in large numbers of nerve cells, leads to cell metabolic disorders and the death of large numbers of nerve cells. It has been proved that human prion disease (Creutzfeldt-Jakob disease) can be caused by miseating beef infected with mad cow disease, so prion disease has broken the interspecific barrier and become a serious threat to human health. Through the unremitting efforts and in-depth research of prion scholars, it is found that there may be a prion infection in cells. LncRNA, a host factor related to prion infection, is an important long strand noncoding RNA analysis discovered in recent years. It takes part in a series of important processes in the course of life and may be an important assisting factor of prion pathogenesis. Its research brings hope to the pathogenesis of prion disease. We screened and identified the LncRNA molecules associated with prion disease. [methods] the expression profiles of LncRNA and mRNA in the brain of prion mice were established. The LncRNA molecules associated with prion pathogenicity were preliminarily screened, and then the target relationship of functional LncRNA molecules could be identified by bioinformatics, which could participate in the biological process and so on. The effects of functional LncRNA molecules on prion gene and prion replication were quantitatively analyzed by fluorescence quantitative PCR and Western blotting. [results] the expression profiles of LncRNA and mRNA in the brain of prion disease model mice were established. The fluorescence quantitative PCR was used to verify that the expression of 6 LncRNA was up-regulated than that of the control group, and the mRNA microarray was established in the hippocampus of mice, and 170 differentially expressed mRNA molecules were detected. Among them, 25 mRNA down-regulated and 145 miRNA were up-regulated. By bioinformatics analysis and prediction, the function of target gene and target gene involved in cellular signal transduction pathway were obtained. Fluorescence quantitative PCR analysis showed that overexpression of LncRNA-2 had no significant effect on Prnp gene. Western blotting showed that PrPSc content in ScN2a cells was detected by Western blotting. Overexpression of LnRNA-2 can inhibit the replication of PrPSc protein. [conclusion] the complete LncRNA and mRNA expression profiles in the hippocampus of prion disease mice were established and LnRNA-2 was screened out by bioinformatics analysis for further study. Fluorescence quantitative PCR and Westernblotting showed that high expression of this molecule could inhibit prion replication. It provides a theoretical support for the further study of long chain noncoding RNA and prion-related molecular chaperone.
【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S855.3
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 ;Immune Responses in Wild-type Mice Against Prion Proteins Induced Using a DNA Prime-Protein Boost Strategy[J];Biomedical and Environmental Sciences;2011年05期
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