基于iTRAQ技术的结核性挛缩膀胱及狭窄输尿管组织蛋白质组学研究及差异蛋白验证
发布时间：2018-11-25 07:26
【摘要】：目的:本研究通过蛋白质组学技术筛选正常膀胱组织与结核性挛缩膀胱组织、正常输尿管组织与结核性狭窄输尿管组织标本之间共有的差异蛋白,查阅文献并从差异蛋白中筛选可能与膀胱及输尿管纤维化病变发生、发展的相关蛋白,通过蛋白质免疫印迹技术进行差异蛋白验证,为膀胱挛缩及输尿管狭窄的分级、诊断、治疗提供新的方法及靶点。方法:第一部分:收集2015年1月至2016年12月解放军309医院泌尿外科确诊泌尿系结核患者的挛缩膀胱及狭窄输尿管组织各2块;获取我院同一时期DCD供者的膀胱及输尿管组织各2块作为正常对照。4种组织混合后用iTRAQ标记技术(相对和绝对定量同位素标记)联合LC-MSMS(液相色谱串联质谱)对各组的总蛋白进分析,将在正常膀胱与挛缩膀胱、正常输尿管与狭窄输尿管组织中表达差异在2倍及2倍以上的蛋白质定义为差异蛋白,并筛选2组之间共同表达的差异蛋白。通过Uniprot、DAVID、String、Wego数据库对共有差异蛋白进行功能聚类、细胞定位、相互作用分析。第二部分:分别选取上述4种组织各6块,进行Masson染色,验证挛缩膀胱、狭窄输尿管组织胶原纤维的表达量;从共有的差异蛋白中挑选微纤丝相关糖蛋白4(MFAP4)和纤维调节素(FMOD)进行蛋白质免疫印迹(Western blot)验证。结果:第一部分:通过蛋白质组学相关实验,在正常膀胱组织与挛缩膀胱组织组共鉴定出188种差异蛋白质,正常输尿管与狭窄输尿管组共鉴定出130种差异蛋白质,2组所共有的差异蛋白质48种,其中20种差异蛋白质下调,28种蛋白质表达上调;48种差异蛋白质在DAVID数据库中共有28种蛋白质被标记,这28种蛋白质主要涉及17种生物学过程,5种分子功能途径和13种细胞组成;48种蛋白质在String数据库中,39种蛋白质被检测到,共形成3条相互作用网络。第二部分:Masson染色提示在挛缩膀胱组织与狭窄输尿管组织中,肌层可见大量胶原纤维表达,挛缩膀胱与正常膀胱、狭窄输尿管与正常输尿管相比,胶原纤维表达明显增高,差异具有统计学意义;western blot结果提示MFAP4、FMOD在挛缩膀胱及狭窄输尿管中表达明显增高,差异具有统计学意义。结论:1、iTRAQ技术联合LC-MSMS可对样本蛋白进行定性、定量鉴定,筛选出差异蛋白,并对蛋白进行生物信息学分析;2、纤维调节素蛋白(FMOD)在病变组织中表达上调,是影响胶原纤维合成的重要因素,与膀胱及输尿管纤维化关系密切。3、人微纤丝相关糖蛋白4(MFAP4)在病变组织中高表达,可进一步行实验探讨其在结核性纤维化病变的诊断、分级及预后中的运用。
[Abstract]:Objective: to screen the common differential proteins between normal bladder tissue and tuberculous contracture bladder tissue, normal ureteral tissue and tuberculous stricture ureter tissue by proteomics. Literature was reviewed and proteins related to the occurrence and development of bladder and ureteral fibrosis were screened from the differential proteins. The differential protein was verified by Western blotting, which was the classification and diagnosis of bladder contracture and ureteral stricture. Treatment provides new methods and targets. Methods: the first part: from January 2015 to December 2016, 2 pieces of contracture bladder and 2 pieces of ureteral stricture were collected from urological department of PLA 309 Hospital. The bladder and ureter tissues of DCD donors in the same period were obtained as normal control. The four tissues were mixed with iTRAQ labeling technique (relative and absolute quantitative isotope labeling) combined with LC-MSMS (liquid chromatographic tandem substance). Analysis of the total protein in each group, The differential protein was defined as differential protein in normal bladder and contracture bladder, normal ureter and narrow ureteral tissue, and the differentially expressed proteins were screened between the two groups. Functional clustering, cellular localization and interaction analysis of common differentially expressed proteins were carried out by Uniprot,DAVID,String,Wego database. The second part: 6 pieces of each of the four tissues were selected for Masson staining to verify the expression of collagen fibers in contracture bladder and ureter. Microfibril associated glycoprotein 4 (MFAP4) and fibronectin (FMOD) were selected from common differential proteins for Western blot (Western blot) verification. Results: in the first part, 188 different proteins were identified in normal bladder tissue and contracture bladder tissue group, 130 differential proteins were identified in normal ureter and narrow ureter group. There were 48 differentially expressed proteins in both groups, of which 20 were down-regulated and 28 were up-regulated. A total of 28 proteins were labeled in the DAVID database. The 28 proteins were mainly involved in 17 biological processes, 5 molecular functional pathways and 13 cell compositions. In String database, 39 proteins were detected and 3 interaction networks were formed. In the second part, Masson staining showed that a large number of collagen fibers were expressed in the muscle layer of contracture bladder and narrow ureter, and the expression of collagen fibers in contracture bladder and normal bladder, narrow ureter and normal ureter were higher than those in normal ureter. The difference is statistically significant. Western blot results suggested that the expression of MFAP4,FMOD in contracture bladder and ureter was significantly higher than that in ureter. Conclusion: 1ITRAQ combined with LC-MSMS can identify the sample protein qualitatively and quantitatively, screen out the differential protein, and analyze the protein by bioinformatics. 2, the expression of fibronectin (FMOD) is up-regulated in the pathological tissues, which is an important factor affecting the synthesis of collagen fibers, and is closely related to the fibrosis of bladder and ureter. Human microfibril associated glycoprotein 4 (MFAP4) is highly expressed in pathological tissues, which can be used in the diagnosis, grading and prognosis of tuberculous fibrosis.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R699
本文编号：2355291
[Abstract]:Objective: to screen the common differential proteins between normal bladder tissue and tuberculous contracture bladder tissue, normal ureteral tissue and tuberculous stricture ureter tissue by proteomics. Literature was reviewed and proteins related to the occurrence and development of bladder and ureteral fibrosis were screened from the differential proteins. The differential protein was verified by Western blotting, which was the classification and diagnosis of bladder contracture and ureteral stricture. Treatment provides new methods and targets. Methods: the first part: from January 2015 to December 2016, 2 pieces of contracture bladder and 2 pieces of ureteral stricture were collected from urological department of PLA 309 Hospital. The bladder and ureter tissues of DCD donors in the same period were obtained as normal control. The four tissues were mixed with iTRAQ labeling technique (relative and absolute quantitative isotope labeling) combined with LC-MSMS (liquid chromatographic tandem substance). Analysis of the total protein in each group, The differential protein was defined as differential protein in normal bladder and contracture bladder, normal ureter and narrow ureteral tissue, and the differentially expressed proteins were screened between the two groups. Functional clustering, cellular localization and interaction analysis of common differentially expressed proteins were carried out by Uniprot,DAVID,String,Wego database. The second part: 6 pieces of each of the four tissues were selected for Masson staining to verify the expression of collagen fibers in contracture bladder and ureter. Microfibril associated glycoprotein 4 (MFAP4) and fibronectin (FMOD) were selected from common differential proteins for Western blot (Western blot) verification. Results: in the first part, 188 different proteins were identified in normal bladder tissue and contracture bladder tissue group, 130 differential proteins were identified in normal ureter and narrow ureter group. There were 48 differentially expressed proteins in both groups, of which 20 were down-regulated and 28 were up-regulated. A total of 28 proteins were labeled in the DAVID database. The 28 proteins were mainly involved in 17 biological processes, 5 molecular functional pathways and 13 cell compositions. In String database, 39 proteins were detected and 3 interaction networks were formed. In the second part, Masson staining showed that a large number of collagen fibers were expressed in the muscle layer of contracture bladder and narrow ureter, and the expression of collagen fibers in contracture bladder and normal bladder, narrow ureter and normal ureter were higher than those in normal ureter. The difference is statistically significant. Western blot results suggested that the expression of MFAP4,FMOD in contracture bladder and ureter was significantly higher than that in ureter. Conclusion: 1ITRAQ combined with LC-MSMS can identify the sample protein qualitatively and quantitatively, screen out the differential protein, and analyze the protein by bioinformatics. 2, the expression of fibronectin (FMOD) is up-regulated in the pathological tissues, which is an important factor affecting the synthesis of collagen fibers, and is closely related to the fibrosis of bladder and ureter. Human microfibril associated glycoprotein 4 (MFAP4) is highly expressed in pathological tissues, which can be used in the diagnosis, grading and prognosis of tuberculous fibrosis.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R699
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