特发性高草酸尿症大鼠模型空肠组织的基因表达谱和蛋白组学研究
发布时间：2018-08-01 13:43
【摘要】：第一部分 特发性高草酸尿症大鼠模型空肠组织差异表达基因的筛选研究 目的特发性高草酸尿症的发生可能与内源性草酸生成增多和外源性草酸吸收增加有关,由于缺乏理想的动物模型,目前国际上对特发性高草酸尿症分子生物学发病机制的研究尚属空白。我们成功建立了特发性高草酸尿症的大鼠模型,在此基础上先期分析特发性高草酸尿症大鼠空肠组织基因表达谱的变化,筛选可能导致其发病的相关基因,探讨特发性高草酸尿症在外源性草酸来源方面的发病机制。 方法应用基因表达谱芯片,检测3只特发性高草酸尿症模型大鼠和正常大鼠空肠组织基因表达差异,对差异表达基因进行生物信息学分析。 结果在特发性高草酸尿症大鼠与正常大鼠空肠组织之间存在差异表达基因720条,其中上调基因517条,下调基因203条,包括细胞信号转导、DNA结合与转录、ATP结合、离子结合与转运、细胞受体、免疫相关、细胞周期蛋白、细胞骨架、代谢蛋白等多种基因。KEGG信号通路分析显示239个通路功能改变差异有统计学意义(P＜0.05)。 结论基因芯片能有效的筛选出特发性高草酸尿症模型大鼠空肠中的差异基因,显著差异表达基因与其发病机理可能存在相关性。 第二部分 特发性高草酸尿症大鼠模型空肠组织蛋白质组二维电泳可行性研究 目的在对样品进行正式的双向凝胶电泳实验前,需要对该样品的双向电泳进行可行性评价,为双向凝胶电泳实验的顺利进行奠定实验基础。方法取1只特发性高草酸尿大鼠和正常对照大鼠的空肠组织300mg,匀浆提取组织总蛋白,以双向电泳技术分离蛋白质,银染后扫描获得的图谱,并以ImageMasterTM2D Platinum software(Version 5.0)软件进行分析。 结果获得了清晰、稳定的凝胶蛋白图谱,正常大鼠凝胶图谱可检测到2541个蛋白点,特发性高草酸尿症大鼠凝胶图谱可检测到2654个蛋白点。结论2个蛋白质样品的二维电泳图谱显示了蛋白质点的稳定迁移,进一步保证了需要进行正式双向凝胶电泳实验的样品能具有较好的可行性和重复稳定性。 第三部分 特发性高草酸尿症大鼠模型空肠组织差异蛋白质的筛选研究 目的寻找特发性高草酸尿症大鼠空肠组织差异表达的蛋白质,探讨特发性高草酸尿症在外源性草酸来源方面的发病机制。 方法雌性特发性高草酸尿症大鼠和正常大鼠各3只,切取500mg的空肠组织后提取其总蛋白。以二维凝胶电泳(2-DE)技术分离空肠中的全部蛋白质并进行差异表达蛋白质筛选,应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)鉴定二维电泳筛选出来的差异表达蛋白质,最后应用Gene Ontology软件对差异蛋白质进行功能分类和细胞定位。 结果获得了分辨率和重复性均较好的凝胶蛋白图谱,通过2-DE筛选出差异表达的蛋白质点40个,其中在特发性高草酸尿症空肠组织中表达上调的蛋白质点有31个,表达下调的蛋白质点有9个,最终有34个差异蛋白质点被鉴定确认,高表达25个,包括Ppplr8、Psma1、Krt19、Hspb1等,低表达9个,包括Actb、Krt8、Psma5等,这些蛋白的功能涉及细胞骨架、信号转导、蛋白降解等。 结论特发性高草酸尿症大鼠和正常大鼠空肠组织中存在差异表达蛋白,这些差异蛋白可能是参与了特发性高草酸尿症的发生的关键蛋白,筛选出来的这些差异蛋白为今后特发性高草酸尿症的分子生物学发病机制研究奠定了实验基础。
[Abstract]:Part one
Screening of differentially expressed genes in jejunal tissues of rats with idiopathic high oxaluria
Objective idiopathic high oxaluuria may be associated with increased endogenous oxalic acid production and increased Exogenous Oxalic acid absorption. Due to the lack of ideal animal models, the research on the molecular biological pathogenesis of idiopathic high oxaluria is still blank. We have successfully established a rat model of idiopathic high oxaluria. On this basis, the changes of gene expression profiles in the jejunum tissue of idiopathic rats with high oxaluuria were analyzed in advance, and the related genes that might lead to its pathogenesis were screened to explore the pathogenesis of idiopathic aloxaluria in the source of Exogenous Oxalic acid.
Methods gene expression profiles were used to detect the differences in gene expression of jejunum in 3 idiopathic high oxalate model rats and normal rats, and the bioinformatics analysis of the differentially expressed genes was carried out.
Results there were 720 differentially expressed genes between the rats with idiopathic uridiuria and normal rat jejunum, of which 517 were up-regulated and 203 were down regulated, including cell signal transduction, DNA binding and transcription, ATP binding, ion binding and transport, cell receptor, immunization, cytoskeleton, cytoskeleton, metabolic protein and so on. The analysis of.KEGG signaling pathway showed that the difference in function between the 239 pathways was statistically significant (P < 0.05).
Conclusion gene chip can effectively screen out the different genes in the jejunum of the rat model of idiopathic high oxaluria, and the significant differentially expressed genes may be related to the pathogenesis.
The second part
Feasibility study of two dimensional electrophoresis of proteome in jejunal tissues of rats with idiopathic high oxaluria
Objective to evaluate the feasibility of bi-directional electrophoresis of the sample before the formal two-dimensional gel electrophoresis test of the sample, to lay the foundation for the smooth progress of the two-dimensional gel electrophoresis experiment. Methods the jejunum 300mg of 1 idiopathic high oxalate rats and normal control rats was used to extract the total tissue protein in the homogenate, and the bi-directional electricity was obtained. The proteins were separated by swimming technique and the maps obtained by silver staining were analyzed by ImageMaster TM 2D Platinum software (Version 5.0).
Results a clear and stable gel protein map was obtained. The gel Atlas of normal rats could detect 2541 protein points. The gel Atlas of idiopathic rats with high oxaluria could detect 2654 proteins. Conclusion the two-dimensional electrophoresis Atlas of 2 protein samples showed the stable migration of protein points, which further ensured the need for formal two-way. The gel electrophoresis experiments showed that the samples were feasible and reproducibility.
The third part
Screening of differentially expressed proteins in jejunal tissues of rats with idiopathic high oxaluria
Objective to find out the proteins expressed differently in the jejunum tissue of idiopathic rats with high oxaluuria, and to explore the pathogenesis of idiopathic aloxaluria in the source of Exogenous Oxalic acid.
Methods 3 female idiopathic high oxaluria rats and 3 normal rats were selected to extract the total protein after cutting the jejunum tissue. Two dimensional gel electrophoresis (2-DE) technique was used to separate all the proteins in the jejunum and to select the differential expression protein. The two dimensional electricity was identified by the matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The differentially expressed proteins were screened out by swimming. Finally, Gene Ontology software was used to classify and localize the differentially expressed proteins.
Results a good gel protein atlas of both resolution and repeatability was obtained. 40 proteins expressed differently were screened by 2-DE. Among them, there were 31 protein points up regulated in the jejunum of idiopathic high oxaluria and 9 down regulated protein points, and 34 protein points were identified and confirmed to be 25. Ppplr8, Psma1, Krt19, Hspb1, and 9 low-expression proteins, including Actb, Krt8, Psma5, are involved in cytoskeleton, signal transduction and protein degradation.
Conclusion differentially expressed proteins are found in the jejunum tissues of idiopathic and normal rats. These differential proteins may be the key proteins involved in the occurrence of idiopathic haluuria. These differential proteins are the basis for the study of molecular biological pathogenesis of idiopathic high oxaluria in the future.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R-332
[Abstract]:Part one
Screening of differentially expressed genes in jejunal tissues of rats with idiopathic high oxaluria
Objective idiopathic high oxaluuria may be associated with increased endogenous oxalic acid production and increased Exogenous Oxalic acid absorption. Due to the lack of ideal animal models, the research on the molecular biological pathogenesis of idiopathic high oxaluria is still blank. We have successfully established a rat model of idiopathic high oxaluria. On this basis, the changes of gene expression profiles in the jejunum tissue of idiopathic rats with high oxaluuria were analyzed in advance, and the related genes that might lead to its pathogenesis were screened to explore the pathogenesis of idiopathic aloxaluria in the source of Exogenous Oxalic acid.
Methods gene expression profiles were used to detect the differences in gene expression of jejunum in 3 idiopathic high oxalate model rats and normal rats, and the bioinformatics analysis of the differentially expressed genes was carried out.
Results there were 720 differentially expressed genes between the rats with idiopathic uridiuria and normal rat jejunum, of which 517 were up-regulated and 203 were down regulated, including cell signal transduction, DNA binding and transcription, ATP binding, ion binding and transport, cell receptor, immunization, cytoskeleton, cytoskeleton, metabolic protein and so on. The analysis of.KEGG signaling pathway showed that the difference in function between the 239 pathways was statistically significant (P < 0.05).
Conclusion gene chip can effectively screen out the different genes in the jejunum of the rat model of idiopathic high oxaluria, and the significant differentially expressed genes may be related to the pathogenesis.
The second part
Feasibility study of two dimensional electrophoresis of proteome in jejunal tissues of rats with idiopathic high oxaluria
Objective to evaluate the feasibility of bi-directional electrophoresis of the sample before the formal two-dimensional gel electrophoresis test of the sample, to lay the foundation for the smooth progress of the two-dimensional gel electrophoresis experiment. Methods the jejunum 300mg of 1 idiopathic high oxalate rats and normal control rats was used to extract the total tissue protein in the homogenate, and the bi-directional electricity was obtained. The proteins were separated by swimming technique and the maps obtained by silver staining were analyzed by ImageMaster TM 2D Platinum software (Version 5.0).
Results a clear and stable gel protein map was obtained. The gel Atlas of normal rats could detect 2541 protein points. The gel Atlas of idiopathic rats with high oxaluria could detect 2654 proteins. Conclusion the two-dimensional electrophoresis Atlas of 2 protein samples showed the stable migration of protein points, which further ensured the need for formal two-way. The gel electrophoresis experiments showed that the samples were feasible and reproducibility.
The third part
Screening of differentially expressed proteins in jejunal tissues of rats with idiopathic high oxaluria
Objective to find out the proteins expressed differently in the jejunum tissue of idiopathic rats with high oxaluuria, and to explore the pathogenesis of idiopathic aloxaluria in the source of Exogenous Oxalic acid.
Methods 3 female idiopathic high oxaluria rats and 3 normal rats were selected to extract the total protein after cutting the jejunum tissue. Two dimensional gel electrophoresis (2-DE) technique was used to separate all the proteins in the jejunum and to select the differential expression protein. The two dimensional electricity was identified by the matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The differentially expressed proteins were screened out by swimming. Finally, Gene Ontology software was used to classify and localize the differentially expressed proteins.
Results a good gel protein atlas of both resolution and repeatability was obtained. 40 proteins expressed differently were screened by 2-DE. Among them, there were 31 protein points up regulated in the jejunum of idiopathic high oxaluria and 9 down regulated protein points, and 34 protein points were identified and confirmed to be 25. Ppplr8, Psma1, Krt19, Hspb1, and 9 low-expression proteins, including Actb, Krt8, Psma5, are involved in cytoskeleton, signal transduction and protein degradation.
Conclusion differentially expressed proteins are found in the jejunum tissues of idiopathic and normal rats. These differential proteins may be the key proteins involved in the occurrence of idiopathic haluuria. These differential proteins are the basis for the study of molecular biological pathogenesis of idiopathic high oxaluria in the future.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R-332
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