益肾健脾养血通利方治疗多囊卵巢综合征的尿蛋白质组学研究

发布时间：2018-05-08 17:21

本文选题：蛋白质组学益 + 多囊卵巢综合征　；参考：《北京中医药大学》2017年硕士论文

【摘要】：多囊卵巢综合征(polycystic ovarian syndrome,PCOS)是一种复杂、多因性的内分泌紊乱和代谢性疾病。其近期临床表现主要为月经稀发或闭经、慢性无排卵、不孕、多毛及痤疮等,远期并发症为高血压、糖尿病、高血脂、子宫内膜癌等。现代中医学认为PCOS相当于中医疾病中痰湿型不孕或闭经,相对应的中医病因病机为痰浊阻滞胞宫。前期研究结果显示益肾健脾养血通利方(菟丝子、车前子、杜仲、薏苡仁、冬瓜仁等)对PCOS临床症状及生化指标有明显改善作用;但是尚缺乏对其疗效机制的临床研究。蛋白质组学是近年来蛋白质研究的新途径,能够大规模、高通量的显示蛋白表达谱。但由于其操作成本高,对技术水平要求高,临床应用相对较少。本课题首先确认尿液蛋白的提取方法,然后以益肾健脾养血通利方对PCOS患者进行治疗,并运用质谱技术及尿蛋白质组研究策略,比较分析益肾健脾养血通利方治疗前后患者尿蛋白质表达谱及表达产物的差异,在蛋白质代谢水平上对该方改善PCOS的疗效机制进行阐述,为该方在临床的治疗应用提供确切的科学研究基础和实验依据。本试验研究目的是建立PCOS患者、益肾健脾养血通利方治疗PCOS后患者尿液蛋白质谱,筛查差异蛋白。采用乙醇沉淀法提取尿液蛋白,BCA法测蛋白浓度,每一组纳入10例样本,经过还原烷基化、胰蛋白酶切、LC-18SPE柱脱盐等操作,使用液相-质谱/质谱方法对各组尿液蛋白进行鉴定。通过蛋白经质谱鉴定、数据库检索和数据分析,共确认获得2300种蛋白质(Mascot ion score10,peptide FDR0.5%)以及显著差异表达蛋白涉及相关的通路7条。通过对三组样本质谱定量信息的两两T-test,得到差异蛋白(p0.05):给药前组vs给药6个月后组:119个蛋白质(Group1);给药前组vs给药12个月后组:154个蛋白质(Group2);给药后6个月组vs给药12个月后组:46个蛋白质(Group3);由此可见,给药前和给药后的病人蛋白表达是存在显著差异的,说明药物治疗具有显著的改善PCOS的作用。给药6个月和给药12个月并未有许多差异,说明随着药物干预时间的延长治疗效果并未有显著提升。由此可见,益肾健脾养血通利方治疗六个月显著有效,继续治疗,药效维持且并未有提升。目的:前期研究结果证实益肾健脾养血通利方对多囊卵巢综合征具有治疗作用,为进一步研究其作用机制和益肾健脾养血通利方治疗前后患者尿蛋白质表达谱的差异,在尿蛋白质代谢水平上对益肾健脾养血通利方改善PCOS的疗效机制进行阐述。1.探讨不同尿液蛋白提取方法对蛋白种类的影响。2.运用质谱技术及尿蛋白质组学研究方法,比较分析益肾健脾养血通利方治疗前后患者尿蛋白质表达谱差异性。3.在蛋白水平上探讨益肾健脾养血通利方治疗多囊卵巢综合征的通路及功能蛋白。方法:1.采用乙醇、丙酮和TCA沉淀方法提取尿液蛋白,运用SDS-PAGE电泳方法观察尿液蛋白电泳图谱2.采用LC对样本进行分离,MALDI-TOF/TOF质谱对样本进行定性和定量检测结果:1.乙醇沉淀法所得的蛋白回收率和丙酮以及TCA沉淀法相比有显著优势,并且,在24小时内,沉淀时间越久蛋白回收率越高。2.SDS-PAGE电泳发现三个组样品大多数电泳条带相似,但是,也呈现出差别电泳带,电泳谱图清晰、条带丰富、蛋白种类多,适合质谱鉴定。3.通过蛋白经质谱鉴定、数据库检索和数据分析,共确认获得2300种蛋白质,各组间差异蛋白可能参与的功能通路7个。三组样本两两T-test,得到差异蛋白数分别是119、154和46个。结论:1.乙醇沉淀法回收率明显高于丙酮和TCA沉淀法,且沉淀大于1小时蛋白电泳图像清晰。2.给药6个月后,药物干预作用明显,给药12个月,刺激蛋白的变化作用逐渐减弱,有回调趋势。且给药6个月和给药12个月两组病人之间差异不大。3.差异蛋白主要为行使功能的分泌蛋白。因而主要以外泌体形式定位于细胞外;显著差异表达蛋白主要涉及相关的通路7条,尤以PI3K-Akt信号通路最为显著和突出,且此通路和胰岛素抵抗有关,是PCOS产生IR的主要机制。
[Abstract]:Polycystic ovarian syndrome (PCOS) is a complex, multicause endocrine disorder and metabolic disease. Its recent clinical manifestations are mainly menstrual dilute or amenorrhea, chronic anovulatory, infertility, hairy and acne. The long-term complications are hypertension, diabetes, hyperlipidemia, endometrial cancer, etc. PCOS is equivalent to phlegm type and amenorrhea in TCM diseases, and the pathogenesis of TCM is phlegm block. The results of early study showed that Yishen Jianpi Yangxue Tongli (Cuscuta, Plantago, eucommia, coix seed, winter melon kernel) had an obvious improvement on the clinical symptoms and biochemical indexes of PCOS, but it still lacked the mechanism of its curative effect. Clinical research. Proteomics is a new approach to protein research in recent years. It is able to display protein expression profiles on a large scale and high throughput. However, due to its high operating cost, high technical requirements and relatively less clinical application. This topic first confirms the extraction method of urine protein, and then uses Yishen Jianpi Yangxue Tongli prescription for PCOS patients. By using mass spectrometry and urine protein group, the differences of protein expression profiles and expression products were compared and analyzed before and after the treatment of Yishen Jianpi Yangxue Tongli prescription, and the therapeutic mechanism of PCOS was expounded on the level of protein metabolism to provide the exact scientific research for the clinical application of the prescription. Basic and experimental basis. The purpose of this study was to establish PCOS patients, Yishen Jianpi Yangxue Tongli prescription for the treatment of urine protein mass spectrometry after PCOS, screening differential proteins. Using ethanol precipitation method to extract urine protein, BCA method to measure protein concentration, each group was included in 10 samples, after the original alkylation, trypsin cut, LC-18SPE column desalination and so on. The urine proteins were identified by liquid phase mass spectrometry / mass spectrometry. Through mass spectrometry identification, database retrieval and data analysis, 2300 proteins (Mascot ion score10, peptide FDR0.5%) and significant differentially expressed proteins involved 7 pathways. The quantitative information of mass spectrometry of three groups of samples was two. Two T-test, differential protein (P0.05): group vs for 6 months after administration: 119 protein (Group1); vs for 12 months after administration: 154 protein (Group2), and 6 months after the administration of VS to group 12 months after the administration: 46 protein (Group3); therefore, there is a significant difference in the expression of protein before and after the administration of the drug. It shows that drug therapy has a significant effect on the improvement of PCOS. There is no significant difference between the 6 months and 12 months of administration. It shows that the effect of the treatment is not significantly improved with the prolongation of the time of drug intervention. Thus, it is obvious that Yishen Jianpi Yangxue Tongli decoction is effective for six months, followed by continued treatment, the effect is maintained and not promoted. The results of the previous study showed that Yishen Jianpi Yangxue Tongli recipe has a therapeutic effect on polycystic ovary syndrome. It is a further study of its mechanism of action and the difference of urinary protein expression profiles of patients before and after treatment of Yishen Jianpi Yangxue Tongli prescription, and the effect mechanism of Yishen Jianpi Yangxue Tongli prescription on improving PCOS in urine protein metabolism level. The effects of different urine protein extraction methods on protein types were discussed by.1...2. using mass spectrometry and urine proteomics methods were used to compare the differential.3. of urine protein expression profiles before and after the treatment of Yishen Jianpi Yangxue Tongli decoction. The treatment of polycystic ovary syndrome by Yishen Jianpi Yangxue Tongli recipe was discussed. Methods: 1. the urine protein was extracted with ethanol, acetone and TCA, and the urine protein electrophoresis was observed by SDS-PAGE electrophoresis. The samples were separated by LC, and the samples were determined by MALDI-TOF/TOF mass spectrometry: the recovery of protein and the precipitate of acetone and TCA by the 1. ethanol precipitation method. There is a significant advantage compared with the method, and in 24 hours, the longer the precipitation time is, the higher the protein recovery rate is, the more.2.SDS-PAGE electrophoresis found that most of the three groups of electrophoresis strips are similar. However, there are also different electrophoresis bands. The electrophoresis bands are clear, the bands are rich and the protein types are many. It is suitable for mass spectrometric identification of.3. through mass spectrometry identification and database retrieval. And data analysis, a total of 2300 proteins were obtained, and 7 functional pathways may be involved in the difference proteins between each group. Three groups of samples were 22 T-test, and the number of differential proteins was 119154 and 46 respectively. Conclusion: the recovery rate of the 1. ethanol precipitation method was significantly higher than that of acetone and TCA precipitation method, and the precipitation was more than 1 hours and the protein electrophoresis image was clear.2. for 6. After the month, the effect of drug intervention was obvious. After 12 months of administration, the effect of the change of stimulant protein gradually weakened, and there was a trend of callback. And the difference of the difference protein between the 6 months and the two groups of the 12 months of administration was not.3., and the main external secretory type was located outside the cell. 7 related pathways, especially the PI3K-Akt signaling pathway, are most prominent and prominent, and this pathway is related to insulin resistance, and is the main mechanism of PCOS producing IR.

【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.6

【参考文献】