本文選題：蛋白質(zhì)組學(xué)益 + 多囊卵巢綜合征�。� 參考：《北京中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：多囊卵巢綜合征(polycystic ovarian syndrome,PCOS)是一種復(fù)雜、多因性的內(nèi)分泌紊亂和代謝性疾病。其近期臨床表現(xiàn)主要為月經(jīng)稀發(fā)或閉經(jīng)、慢性無(wú)排卵、不孕、多毛及痤瘡等,遠(yuǎn)期并發(fā)癥為高血壓、糖尿病、高血脂、子宮內(nèi)膜癌等�，F(xiàn)代中醫(yī)學(xué)認(rèn)為PCOS相當(dāng)于中醫(yī)疾病中痰濕型不孕或閉經(jīng),相對(duì)應(yīng)的中醫(yī)病因病機(jī)為痰濁阻滯胞宮。前期研究結(jié)果顯示益腎健脾養(yǎng)血通利方(菟絲子、車前子、杜仲、薏苡仁、冬瓜仁等)對(duì)PCOS臨床癥狀及生化指標(biāo)有明顯改善作用;但是尚缺乏對(duì)其療效機(jī)制的臨床研究。蛋白質(zhì)組學(xué)是近年來(lái)蛋白質(zhì)研究的新途徑,能夠大規(guī)模、高通量的顯示蛋白表達(dá)譜。但由于其操作成本高,對(duì)技術(shù)水平要求高,臨床應(yīng)用相對(duì)較少。本課題首先確認(rèn)尿液蛋白的提取方法,然后以益腎健脾養(yǎng)血通利方對(duì)PCOS患者進(jìn)行治療,并運(yùn)用質(zhì)譜技術(shù)及尿蛋白質(zhì)組研究策略,比較分析益腎健脾養(yǎng)血通利方治療前后患者尿蛋白質(zhì)表達(dá)譜及表達(dá)產(chǎn)物的差異,在蛋白質(zhì)代謝水平上對(duì)該方改善PCOS的療效機(jī)制進(jìn)行闡述,為該方在臨床的治療應(yīng)用提供確切的科學(xué)研究基礎(chǔ)和實(shí)驗(yàn)依據(jù)。本試驗(yàn)研究目的是建立PCOS患者、益腎健脾養(yǎng)血通利方治療PCOS后患者尿液蛋白質(zhì)譜,篩查差異蛋白。采用乙醇沉淀法提取尿液蛋白,BCA法測(cè)蛋白濃度,每一組納入10例樣本,經(jīng)過還原烷基化、胰蛋白酶切、LC-18SPE柱脫鹽等操作,使用液相-質(zhì)譜/質(zhì)譜方法對(duì)各組尿液蛋白進(jìn)行鑒定。通過蛋白經(jīng)質(zhì)譜鑒定、數(shù)據(jù)庫(kù)檢索和數(shù)據(jù)分析,共確認(rèn)獲得2300種蛋白質(zhì)(Mascot ion score10,peptide FDR0.5%)以及顯著差異表達(dá)蛋白涉及相關(guān)的通路7條。通過對(duì)三組樣本質(zhì)譜定量信息的兩兩T-test,得到差異蛋白(p0.05):給藥前組vs給藥6個(gè)月后組:119個(gè)蛋白質(zhì)(Group1);給藥前組vs給藥12個(gè)月后組:154個(gè)蛋白質(zhì)(Group2);給藥后6個(gè)月組vs給藥12個(gè)月后組:46個(gè)蛋白質(zhì)(Group3);由此可見,給藥前和給藥后的病人蛋白表達(dá)是存在顯著差異的,說明藥物治療具有顯著的改善PCOS的作用。給藥6個(gè)月和給藥12個(gè)月并未有許多差異,說明隨著藥物干預(yù)時(shí)間的延長(zhǎng)治療效果并未有顯著提升。由此可見,益腎健脾養(yǎng)血通利方治療六個(gè)月顯著有效,繼續(xù)治療,藥效維持且并未有提升。目的:前期研究結(jié)果證實(shí)益腎健脾養(yǎng)血通利方對(duì)多囊卵巢綜合征具有治療作用,為進(jìn)一步研究其作用機(jī)制和益腎健脾養(yǎng)血通利方治療前后患者尿蛋白質(zhì)表達(dá)譜的差異,在尿蛋白質(zhì)代謝水平上對(duì)益腎健脾養(yǎng)血通利方改善PCOS的療效機(jī)制進(jìn)行闡述。1.探討不同尿液蛋白提取方法對(duì)蛋白種類的影響。2.運(yùn)用質(zhì)譜技術(shù)及尿蛋白質(zhì)組學(xué)研究方法,比較分析益腎健脾養(yǎng)血通利方治療前后患者尿蛋白質(zhì)表達(dá)譜差異性。3.在蛋白水平上探討益腎健脾養(yǎng)血通利方治療多囊卵巢綜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,運(yùn)用SDS-PAGE電泳方法觀察尿液蛋白電泳圖譜2.采用LC對(duì)樣本進(jìn)行分離,MALDI-TOF/TOF質(zhì)譜對(duì)樣本進(jìn)行定性和定量檢測(cè)結(jié)果:1.乙醇沉淀法所得的蛋白回收率和丙酮以及TCA沉淀法相比有顯著優(yōu)勢(shì),并且,在24小時(shí)內(nèi),沉淀時(shí)間越久蛋白回收率越高。2.SDS-PAGE電泳發(fā)現(xiàn)三個(gè)組樣品大多數(shù)電泳條帶相似,但是,也呈現(xiàn)出差別電泳帶,電泳譜圖清晰、條帶豐富、蛋白種類多,適合質(zhì)譜鑒定。3.通過蛋白經(jīng)質(zhì)譜鑒定、數(shù)據(jù)庫(kù)檢索和數(shù)據(jù)分析,共確認(rèn)獲得2300種蛋白質(zhì),各組間差異蛋白可能參與的功能通路7個(gè)。三組樣本兩兩T-test,得到差異蛋白數(shù)分別是119、154和46個(gè)。結(jié)論:1.乙醇沉淀法回收率明顯高于丙酮和TCA沉淀法,且沉淀大于1小時(shí)蛋白電泳圖像清晰。2.給藥6個(gè)月后,藥物干預(yù)作用明顯,給藥12個(gè)月,刺激蛋白的變化作用逐漸減弱,有回調(diào)趨勢(shì)。且給藥6個(gè)月和給藥12個(gè)月兩組病人之間差異不大。3.差異蛋白主要為行使功能的分泌蛋白。因而主要以外泌體形式定位于細(xì)胞外;顯著差異表達(dá)蛋白主要涉及相關(guān)的通路7條,尤以PI3K-Akt信號(hào)通路最為顯著和突出,且此通路和胰島素抵抗有關(guān),是PCOS產(chǎn)生IR的主要機(jī)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.

【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R285.6

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