基于iTRAQ对肾功能不全大鼠血清差异蛋白的鉴定及生物信息学分析

发布时间：2019-05-25 04:22

【摘要】：背景肾功能不全(renalinsufficiency)是各种复杂因素导致肾小球损害,肾功能降低,造成人体水电解质酸碱平衡失常,代谢紊乱的临床综合症候群。肾功能不全是一种发病率高,危害较大,严重危害患者健康与生命的病症之一。目前肾功能不全的发生进展机制都还不够完善。我们希望通过血清蛋白质组学研究发现肾功能不全大鼠与正常大鼠的差异蛋白,为肾功能不全的进展研究提供实验基础。目的运用iTRAQ技术进行蛋白质组学分析,研究大鼠肾功能不全发生进展的相关差异蛋白;运用omicsbean在线软件,通过GO分析、KEGG分析和STRING等生物信息学分析对筛选出的差异蛋白进行分析,并对与肾功能不全相关的蛋白质进行鉴定及验证。方法把44只SD雄性大鼠随机分为正常对照组(A组),模型低剂量组(B组),模型中剂量组(C组)和模型高剂量组(D组),各11只,正常对照组常规饲养,模型轻剂量组,模型中剂量组和模型高剂量组大鼠分别给予20g/d/只含0.125%、0.25%、0.5%腺嘌呤食物,连续21d。造模结束后,测验各组大鼠BUN(尿素氮)、Scr(血肌酐),体质量,24h饮水量,HE染色检测肾组织形态学改变。利用iTRAQ技术分析血清蛋白质组,鉴定出差异表达蛋白,进一步用实时定量PCR、蛋白质印迹及酶联免疫吸附实验等实验对触珠蛋白(Haptoglobin,HP),正五聚蛋白-3(Pentaxin,PTX3),血清转铁蛋白(Serotransferrin,TRF)的表达水平加以验证。结果通过对各模型组大鼠与正常对照大鼠血清的质谱分析,我们共发现416种蛋白质,并从中成功鉴定出差异表达蛋白29种。利用omicsbean生物信息学在线软件分析,发现这29种差异蛋白主要分布在细胞外区域,血液微粒和膜界囊泡中,以分子功能调节和酶调节活性等功能为主,参与炎症反应以及对外部刺激应答等生物学过程。通过查阅文献和结果分析,从29种差异表达蛋白中筛选了3种蛋白,即HP、PTX3和TRF。蛋白质印迹、实时定量PCR和酶联免疫吸附实验结果与质谱结果一致。结论成功建立了肾功能不全大鼠模型,并基于iTRAQ技术筛选出了各期肾功能不全大鼠与正常大鼠的差异蛋白,经生物信息学分析,肾功能不全的发生与多个蛋白质、生物学进程、蛋白通路及蛋白质调控网络有关,提示HP、PTX3和TRF可能与肾功能不全的发生进展密切相关,为肾功能不全的药物靶点治疗提供了一定实验基础。
[Abstract]:Background (renalinsufficiency) of renal insufficiency is a clinical syndrome with various complex factors leading to glomerular damage and decrease of renal function, resulting in abnormal acid-base balance and metabolic disorder of hydrolytic acid and base in human body. Renal insufficiency is one of the diseases with high incidence, great harm and serious harm to the health and life of patients. At present, the mechanism of the occurrence and progress of renal insufficiency is not perfect. We hope that the differential proteins between rats with renal insufficiency and normal rats will be found by serum proteome study, which will provide an experimental basis for the progress of renal insufficiency. Objective to study the differential proteins related to the progress of renal insufficiency in rats by proteome analysis with iTRAQ technique. Omicsbean online software, GO analysis, KEGG analysis and STRING bioinformatics analysis were used to analyze the screened differential proteins, and the proteins related to renal insufficiency were identified and verified. Methods 44 male SD rats were randomly divided into normal control group (group A), model low dose group (group B), middle dose group (group C) and model high dose group (group D) with 11 rats in each group. Rats in the model light dose group, the middle dose group and the model high dose group were given 20g/d/ containing only 0.125%, 0.25% and 0.5% adenine food for 21 days. After the establishment of the model, BUN (urea nitrogen), Scr (serum creatinine), body mass, 24 h drinking water and HE staining were used to detect the morphological changes of renal tissue. The serum proteome was analyzed by iTRAQ, and the differentially expressed proteins were identified. Haptoglobin,HP and Pentaxin,PTX3 were further detected by real-time quantitative PCR, Western imprinting and enzyme-linked immunosorbent assay (Elisa). The expression level of serum transferrin (Serotransferrin,TRF) was verified. Results by mass spectrometry analysis of serum of rats in each model group and normal control group, 416 kinds of proteins were found, and 29 kinds of differentially expressed proteins were successfully identified. Using omicsbean bioinformatics online software analysis, it was found that the 29 differential proteins were mainly distributed in extracellular region, blood particles and membrane vesicles, mainly in molecular function regulation and enzyme regulation activity. Participate in biological processes such as inflammatory response and response to external stimuli. Through literature review and result analysis, three proteins, HP,PTX3 and TRF., were screened from 29 differentially expressed proteins. The results of Western imprinting, real-time quantitative PCR and enzyme-linked immunosorbent assay were consistent with those of mass spectrometry. Conclusion the rat model of renal insufficiency was successfully established, and the differential proteins between rats with renal insufficiency and normal rats were screened by iTRAQ technique. The occurrence of renal insufficiency and the occurrence of multiple proteins and biological processes were analyzed by bioinformatics analysis. Protein pathway and protein regulatory network are related, suggesting that HP,PTX3 and TRF may be closely related to the occurrence and progress of renal insufficiency, which provides an experimental basis for drug target therapy of renal insufficiency.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692

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