基于iTRAQ技术的子痫前期子代血管功能紊乱的定量蛋白质组学研究

发布时间：2018-05-11 18:35

本文选题：配子-胚胎源性疾病 + iTRAQ　；参考：《浙江大学》2016年博士论文

【摘要】：第一部分基于于iTRAQ-2D-LC-MS/MS的子痫前期子代脐动脉血管的蛋白质组学分析流行病学调查显示子痫前期对母亲和胎儿的健康都有重大影响,宫内的不良环境可能导致成人心血管疾病的发生,但对其分子机制知之甚少。因此,我们利用定量蛋白质组学技术试图深入的研究子痫前期子代心血管功能紊乱的分子机制。iTRAQ结果显示我们总共鉴定到1521个蛋白质,其中1496个蛋白质具有定量信息。进一步分析得到在脐动脉血管中有53个具有显著差异表达的蛋白质,其中22个蛋白质上调表达,31个蛋白质下调表达。通过利用IPA软件进行深入的生物信息学分析,我们发现了一组与心血管密切相关的蛋白质(SLIT3,CD99,FN1,DAPK3,CRKL,FBN1,C1 QBP,CSNK2B,AKR1B1,TMOD1,LRPA P1,FBN2,PRKCA),这些蛋白质与心血管疾病和功能密切相关,功能方面主要是与血管生成(angiogenesis)最为相关,相关疾病方面则是跟高血压(hypertension)联系最为密切。我们对AKR1B1,FN1,FBN1这三个蛋白质进行了western b lot的验证,结果与蛋白质组学结果一致。在对这些心血管功能相关蛋白的上游调控分析中,我们发现miR-9-5p可以抑制AKRIB 1,FBN1,FBN2,PRKCA这四个蛋白的表达。心血管功能相关蛋白的相互作用网络显示FN1蛋白处于轴心位置。研究结果对揭示子痫前期子代的血管功能紊乱提供了新的研究视角。第二部分：基于子痫前期动物模型的成年子代血管的蛋白质组学分析心血管疾病的胚胎起源是全世界备受重视的研究领域。流行病学调查显示子痫前期所造成的宫内不良环境会导致胎儿的血管功能出现不同程度的紊乱,从而加大了子代发育过程中心血管疾病发生的风险,但其分子机制仍有待研究。在前期研究中,我们分析了子痫前期子代的脐动脉差异蛋白表达谱,为了更深入的研究子痫前期子代成年后的血管功能变化,我们建立了子痫前期的动物模型,喂养子代至成年(1年)后,我们收集其胸主动脉进行定量蛋白质组学的研究。iTRAQ结果显示我们总共鉴定到1825个蛋白质,进一步分析可知有106个具有显著差异表达的蛋白质,其中75个蛋白质上调表达,31个蛋白质下调表达。通过IPA软件的疾病与功能分析,我们发现差异表达蛋白中有20个心血管疾病和功能都有明显相关性。其中血管生成(angiogenesis)是心血管相关功能中最为密切的,相关疾病方面则是跟动脉阻塞(occlusion of artery)和动脉粥样硬化(atherosclerosis)最为相关。在对这些心血管功能相关蛋白的上游调控分析中,我们发现miR-423-5p起到了抑制表达的作用,而TP53则是激活表达的作用。本原创性课题是生殖医学和代谢性疾病发生研究结合的崭新课题,研究成果将对人类胚胎源性心血管病发病机制的阐明作出贡献。
[Abstract]:The first part was based on the proteomic analysis of the umbilical artery of pre-eclampsia based on iTRAQ-2D-LC-MS/MS, which showed that preeclampsia had a significant impact on maternal and fetal health. Adverse conditions in the uterus may lead to cardiovascular disease in adults, but little is known about its molecular mechanism. Therefore, we use quantitative proteomics to study the molecular mechanism of cardiovascular dysfunction in pre-eclampsia. ITRAQ results show that we have identified a total of 1521 proteins, 1496 of which have quantitative information. Further analysis revealed that there were 53 differentially expressed proteins in the umbilical artery, 22 of which were up-regulated and 31 were down-regulated. By using IPA software for further bioinformatics analysis, we found a group of proteins closely related to cardiovascular diseases, such as SLIT3, CD9FN1, DAPK3, CRKL, FBN1C 1, QBP1, CSNK2B1, AKR1B1, TMOD1, LRPA, FBN2, PRKCAA, these proteins are closely related to cardiovascular disease and function. The functional aspect is mainly related to angiogenesis, while the related disease is most closely related to hypertension hypertension. Three proteins, AKR1B1FN1FBN1, were confirmed by western b lot, and the results were in agreement with the results of proteomics. In the upstream regulatory analysis of these cardiovascular function-related proteins, we found that miR-9-5p could inhibit the expression of four proteins, AKRIB 1, FBN 1, FBN 2 and PRKCA. The interaction network of cardiovascular function-related proteins shows that the FN1 protein is in an axial position. The results provide a new perspective for revealing vascular dysfunction in preeclampsia. Part two: proteomics of adult offspring based on animal model of preeclampsia is a very important research field in the world. Epidemiological investigation shows that the adverse environment caused by preeclampsia may lead to different degrees of disorder of fetal vascular function, which increases the risk of cardiovascular disease during the development of offspring, but its molecular mechanism remains to be studied. In the previous study, we analyzed the differential protein expression profiles of umbilical artery in pre-eclampsia. In order to further study the changes of vascular function in pre-eclampsia, we established an animal model of pre-eclampsia. After feeding the offspring to adulthood (1 year), we collected the thoracic aorta for quantitative proteomics. The results of iTRAQ showed that we identified a total of 1825 proteins. Further analysis revealed that 106 proteins were significantly differentially expressed. Among them, 75 proteins were up-regulated and 31 proteins were down-regulated. By analyzing the disease and function of IPA software, we found that 20 of the differentially expressed proteins were related to cardiovascular disease and function. Angiogenesis-angiogenesis is the most closely related cardiovascular function, and related diseases are most closely related to occlusion of artery occlusion and atherosclerosis. In the upstream regulatory analysis of these cardiovascular function-related proteins, we found that miR-423-5p inhibits expression and TP53 activates expression. This original subject is a new research topic of reproductive medicine and metabolic diseases. The research results will contribute to the elucidation of the pathogenesis of human embryonic cardiovascular disease.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R714.244

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