朊病毒病相关LncRNA表达谱分析研究
发布时间：2018-08-14 10:12
【摘要】：［背景］朊病毒病是一种高致病性，高致死性的神经退行性疾病，是由于细胞内正常的朊蛋白发生构象改变，形成的一种抗逆性极强，能够大量聚集在神经细胞内的有毒蛋白PrPSc，导致细胞代谢紊乱，神经细胞大量死亡。现已证明，人们误食感染疯牛病的牛肉后，会引起人类朊病毒病（克雅氏病），由此可见，朊病毒病已经打破了种间屏障，成为严重威胁人类健康的一种人兽共患病。经过朊病毒学者们的不懈努力和深入研究，发现细胞内可能存在与朊病毒感染、致病相关的宿主因子。LncRNA是近些年发现的一种重要的长链非编码RNA分析，长度大于200nt，参与了生命过程中一系列重要进程，可能是朊病毒致病的重要协助因子，它的研究给朊病毒病的发病机理带来了希望，因此，我们对与朊病毒病相关的LncRNA分子进行了初步的筛选与鉴定。 ［方法］本实验通过建立朊病毒小鼠鼠脑LncRNA和mRNA表达谱，根据分析结果，初步筛选出与朊病毒致病相关的LncRNA分子；然后使用生物信息学可高精度分辨出功能性LncRNA分子的靶向关系，参与生物学进程等；利用荧光定量PCR和Western blotting定量分析功能性LncRNA分子对朊蛋白基因以及朊病毒复制的作用。 ［结果］建立朊病毒病模型小鼠脑部LncRNA和mRNA表达谱，并通过荧光定量PCR进行了验证，最终检测出6条LncRNA表达量相比对照组下调，13条LncRNA表达量相比对照组上调；建立了小鼠海马组织中mRNA芯片，共检测出170条差异表达的mRNA分子，其中25条mRNA表达下调，145条miRNA表达上调。 通过生物信息学分析预测，得到差异性表达LncRNA作用的靶基因以及靶基因的功能和参与细胞信号转导通路。 荧光定量PCR分析得知过表达LncRNA-2对Prnp基因并没有显著影响，WesternBlotting检测ScN2a细胞内PrPSc含量说明，过表达LnRNA-2后能够抑制PrPSc蛋白的复制。 ［结论］本实验建立了完整的朊病毒病小鼠海马组织LncRNA以及mRNA表达谱，通过生物信息学分析初步筛选出了LnRNA-2进行深入研究，利用荧光定量PCR和Westernblotting初步验证了该分子高表达之后能够抑制朊病毒复制。为后续研究长链非编码RNA以及朊病毒相关分子伴侣提供了一个理论的支持，奠定了基础。
[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.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3
本文编号：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.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3
【参考文献】
相关期刊论文 前1条
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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