本文選題：蛋白質(zhì)組學(xué) + 生物標(biāo)志物��； 參考：《天津醫(yī)科大學(xué)》2017年博士論文

【摘要】：目的:通過蛋白質(zhì)組學(xué)的同位素標(biāo)記相對和絕對定量(Isobaric tags for relative and absolute quantitation,iTRAQ)技術(shù)探索糖尿病患者與健康人群中血清低豐度蛋白的種類及表達(dá)水平異常,篩查與糖尿病相關(guān)的血清生物學(xué)標(biāo)志物,進(jìn)一步對蛋白質(zhì)組學(xué)篩查出的差異蛋白在人群研究中進(jìn)行驗證;在基礎(chǔ)研究中對差異表達(dá)蛋白影響胰島素信號轉(zhuǎn)導(dǎo)機(jī)制進(jìn)行探索,以期為人群研究的蛋白質(zhì)組學(xué)數(shù)據(jù)庫及糖尿病患者血清蛋白分布和功能研究提供新的數(shù)據(jù),為與糖尿病發(fā)病機(jī)制相關(guān)的蛋白質(zhì)研究探索新的方向。方法:在新診斷2型糖尿病患者中用蛋白質(zhì)組學(xué)的iTRAQ技術(shù)觀察血清中的各種低豐度蛋白表達(dá)水平,與健康人群相比較篩選出表達(dá)水平異常的蛋白,應(yīng)用生物醫(yī)學(xué)軟件對鑒定出的蛋白進(jìn)行分類并分析其細(xì)胞組成成分和參與的生物學(xué)進(jìn)程。從第一部分篩選出的差異蛋白中挑選兩種異常降低蛋白轉(zhuǎn)化生長因子β誘導(dǎo)蛋白(Transforming growth factor-β-induced protein,TGFBIp)及胰島素樣生長因子酸不穩(wěn)定亞基(Insulin-like growth factor acid-labile subunit,IGFLAS)用酶聯(lián)免疫吸附試驗(Enzyme-linked immunosorbent assay,ELISA)在糖尿病患者、非糖尿病的慢性疾病患者、健康人群中進(jìn)行驗證,觀察在不同人群中這兩種蛋白的表達(dá)水平,以驗證蛋白質(zhì)組學(xué)研究結(jié)果。進(jìn)一步觀察TGFBIp及IGFLAS表達(dá)水平與糖尿病患者臨床資料之間的相關(guān)性,繪制受試者工作特征(Receiver operating characteristic,ROC)曲線,判定差異表達(dá)蛋白對診斷糖尿病的效力。采用高胰島素和高葡萄糖誘導(dǎo)人肝癌細(xì)胞(Hep G2)建立胰島素抵抗(Insulin Resistance,IR)細(xì)胞模型,使用IGFALS-si RNA使細(xì)胞中IGFALS表達(dá)沉默,觀察Hep G2葡萄糖消耗量的變化。采用ELISA法檢測IGFALS在各組細(xì)胞中的表達(dá)量。采用蛋白質(zhì)印記(Western Blotting,WB)方法檢測蛋白激酶B(Protein kinases B,PKB,也稱為Akt)、核因子κB(Nuclear factor-κ-gene binding,NF-κB)及磷酸化Akt(p-Akt)、磷酸化NF-κB(p-NF-κB)在IGFALS-si RNA轉(zhuǎn)染前后及IR各組細(xì)胞中蛋白表達(dá)量的變化。結(jié)果:2型糖尿病患者中鑒定到的血清蛋白數(shù)量為222個,與健康組相比差異蛋白72個;其中38個蛋白表達(dá)上調(diào),34個蛋白表達(dá)下調(diào)。在72個差異蛋白中,從細(xì)胞組分匯總分類有17%為胞外蛋白,16%為胞質(zhì)蛋白,10%為膜蛋白,4%為核內(nèi)蛋白。從生物進(jìn)程匯總分類有33%參與調(diào)節(jié)功能,19%參與脂質(zhì)代謝,11%參與代謝過程,8%參與免疫反應(yīng),7%參與穩(wěn)態(tài)調(diào)節(jié),6%參與炎癥反應(yīng)。人群研究發(fā)現(xiàn)TGFBIp在慢性病組(CD組)明顯升高,糖尿病組(DM組)較對照組(CON組)有降低趨勢,但差異無統(tǒng)計學(xué)意義(220.09 ng/ml±46.19 ng/ml比233.07 ng/ml±45.83 ng/ml,P=0.118)。IGFALS在三組中有明顯差異,在DM組值最低(12.21 ng/ml±4.21 ng/ml),其次為CD組(16.93 ng/ml±4.21 ng/ml),在CON組值最高(18.85 ng/ml±5.63 ng/ml),差異有統(tǒng)計學(xué)意義(F=48.90,P0.01)。TGFBIp與各項臨床指標(biāo)無統(tǒng)計學(xué)相關(guān)性,IGFALS與FBG、Hb A1C呈負(fù)相關(guān),r值分別為-0.23,-0.32(P均0.05),與DBP呈正相關(guān),r值為0.35(P0.01)。IGFALS的Cutoff值為15.91 ng/ml,此時靈敏度為65.2%,特異度為84.8%,曲線下面積(Areas under the curve,AUC)為0.802,具有統(tǒng)計學(xué)意義(P0.01)。以高胰島素和高糖分別培養(yǎng)48h和24h后均不會顯著影響細(xì)胞增殖。以胰島素6×10-6 mol/L、3×10-6 mol/L、6×10-7 mol/L的濃度干擾24h時,以葡萄糖44 mmo/L、55 mmol/L、66 mmol/L的濃度干擾12h時對細(xì)胞胰島素敏感性影響較大。沉默表達(dá)IGFALS后細(xì)胞對葡萄糖的利用顯著減少(P0.01)。胰島素誘導(dǎo)的IR細(xì)胞中IGFALS分泌量顯著低于對照組(136.83 ng/ml±7.45 ng/ml,142.27 ng/ml±10.11ng/ml,147.65 ng/ml±8.14 ng/ml比187.99 ng/ml±6.13 ng/ml,P均0.01)。高糖誘導(dǎo)的IR細(xì)胞中IGFALS分泌量顯著低于對照組(131.30 ng/ml±4.33 ng/ml,144.09 ng/ml±7.00 ng/ml比187.99 ng/ml±6.13 ng/ml P均0.01)。IGFALS-home-890組的IGFALS分泌量較對照組顯著降低(114.55 ng/ml±7.50ng/ml比187.99 ng/ml±6.13 ng/ml,P0.01)。WB檢測結(jié)果表明三組細(xì)胞p-Akt水平較對照組顯著降低(1.23±0.12,1.11±0.14,0.99±0.05比1.48±0.11,F=10.832,P0.01),Akt表達(dá)僅在胰島素誘導(dǎo)IR組有差異(1.78±0.19比2.42±0.29,P0.05)。三組中p-NF-κB及NF-κB的表達(dá)量較對照組均有顯著增高(p-NF-κB:0.41±0.05,0.48±0.11,0.39±0.04比0.23±0.03,F=7.706,P=0.01;NF-κB:0.86±0.08,0.90±0.07,0.96±0.07比0.65±0.04,F=12.760,P=0.002)。結(jié)論:在糖尿病人群中能夠應(yīng)用蛋白質(zhì)組學(xué)的iTRAQ技術(shù)篩查差異表達(dá)的低豐度蛋白,為尋找糖尿病患者血清生物標(biāo)志物提供更多的可選擇性。糖尿病患者血清中IGFALS較健康對照組顯著降低,與患者的Hb A1c、FBG呈負(fù)相關(guān),IGFALS作為診斷糖尿病的生物學(xué)標(biāo)志物特異性較好,敏感性欠佳。高糖及高胰島素誘導(dǎo)的IR細(xì)胞中,IGFALS的水平顯著下降。沉默表達(dá)IGFALS后,胰島素信號轉(zhuǎn)導(dǎo)通路中Akt的磷酸化水平降低,炎癥信號轉(zhuǎn)導(dǎo)通路中NF-κB及其磷酸化水平升高。IGFALS可能通過作用于胰島素及炎癥信號轉(zhuǎn)導(dǎo)通路影響胰島素抵抗。
[Abstract]:Objective: To explore the types and expression levels of low abundance proteins in diabetic patients and healthy people by relative and absolute quantification (Isobaric tags for relative and absolute quantitation, iTRAQ), and to screen the serum biomarkers related to diabetes and further to the protein. The differential protein screened by the group is verified in the population study. In the basic study, the differential expression protein affects the mechanism of insulin signal transduction, in order to provide new data for the proteomics database and the study of the distribution and function of serum protein in diabetic patients, and to be related to the pathogenesis of diabetes. The new direction of protein research is explored. Methods: in the newly diagnosed type 2 diabetic patients, the protein expression level in the serum is observed by the proteomic iTRAQ technique. The proteins with abnormal expression level are screened out compared with the healthy people, and the proteins are classified and analyzed by the biomedical software. Components and biological processes involved. Select two abnormally reduced protein transforming growth factor beta induced proteins (Transforming growth factor- beta -induced protein, TGFBIp) and insulin like growth factor acid unstable subunits (Insulin-like growth factor acid-labile subunit, IGFLAS) from the differential proteins selected from the first part. An enzyme linked immunosorbent assay (Enzyme-linked immunosorbent assay, ELISA) was used to verify the expression level of these two proteins in the patients with diabetes, non diabetic chronic diseases, and in a healthy population to verify the results of proteomics research. Further observation of the expression level of TGFBIp and IGFLAS and diabetes mellitus The correlation between the clinical data and the Receiver operating characteristic (ROC) curve was drawn to determine the effect of differential expression protein on the diagnosis of diabetes. The insulin resistance (Insulin Resistance, IR) cell model was established by using high insulin and Hyperglucose induced human hepatoma cells (Hep G2), and IGFALS-si RNA was used. The expression of IGFALS expression in Hep G2 was observed. ELISA method was used to detect the expression of IGFALS in each cell. Protein kinase B (Protein kinases B) was used to detect the protein kinase B (Protein kinases B). P-Akt), the changes in the protein expression of NF- kappa B (p-NF- kappa B) in IGFALS-si RNA transfection and IR groups. Results: the number of serum proteins identified in type 2 diabetic patients was 222, and 72 of the difference proteins were compared with the healthy group, of which 38 proteins were up-regulated and 34 proteins were down regulated. In 72 differential proteins, the cells were from the cells. There were 17% extracellular proteins, 16% cytoplasmic proteins, 10% membrane proteins, 10% membrane proteins and 4% intranuclear proteins. 33% participated in the regulation function, 19% participated in lipid metabolism, 11% participated in the metabolic process, 8% participated in the immune response, 7% participated in the immune response, 7% was involved in the homeostasis regulation, and 6% was involved in the inflammatory reaction. The population study found that TGFBIp was in the chronic disease group (group CD) Significantly higher, the diabetes group (DM group) had a lower trend than the control group (group CON), but there was no significant difference (220.09 ng/ml + 46.19 ng/ml ratio 233.07 ng/ml + 45.83 ng/ml, P=0.118).IGFALS in the three groups, the lowest in the DM group (12.21 ng/ml + 4.21 ng/ ml), and the second group (16.93, 4.21 + 4.21), and the highest (18.8 5 ng/ml + 5.63 ng/ml), the difference was statistically significant (F=48.90, P0.01).TGFBIp had no statistical correlation with various clinical indexes, IGFALS was negatively correlated with FBG, Hb A1C, R value was -0.23, -0.32 (0.05) was positively correlated with the value of 0.35 (15.91), at this time the sensitivity was 65.2%, the specificity was 84.8%, under the curve. The area (Areas under the curve, AUC) was 0.802, and was statistically significant (P0.01). Both 48h and 24h were not significantly affected by high insulin and high glucose, respectively. The concentration of insulin 6 x 10-6 mol/L, 3 x 10-6 mol/L, 6 x 10-7 mol/L interfered 24h, with the concentration of grape sugar 44, 55, and 66 The insulin sensitivity was greatly affected. The use of IGFALS cells after silent expression of IGFALS decreased significantly (P0.01). The secretion of IGFALS in insulin induced IR cells was significantly lower than that in the control group (136.83 ng/ml + 7.45 ng/ml, 142.27 ng/ml + 10.11ng/ml, 147.65 ng/ml + 8.14 ng/ml compared to 187.99 ng/ml + 6.13 ng/ml, 0.01). The secretion of IGFALS in the cell was significantly lower than that in the control group (131.30 ng/ml + 4.33 ng/ml, 144.09 ng/ml + 7 ng/ml ratio, 187.99 ng/ml + 6.13 ng/ml P 0.01). The secretion of IGFALS in the.IGFALS-home-890 group was significantly lower than that in the control group (114.55 ng/ml + 7.50ng/ml ratio 187.99 + 6.13). The expression of Akt was significantly lower (1.23 + 0.12,1.11 + 0.14,0.99 + 0.05 than 1.48 + 0.11, F=10.832, P0.01). The expression of Akt was significantly different in the IR induced IR group (1.78 + 0.19 versus 2.42 + 0.29, P0.05). The p-NF- kappa B and NF- kappa B were significantly higher than those of the control group (p-NF- kappa 0.41 + 0.04 + 0.23 + 0.23 + 0.23 + 0.03. Kappa B:0.86 + 0.08,0.90 + 0.07,0.96 + 0.65 + 0.04, 0.65 + 0.04, F=12.760, P=0.002). Conclusion: a proteomic iTRAQ technique can be used to screen differentially expressed low abundance proteins in the diabetic population, providing more choice for finding serum biomarkers in diabetic patients. The serum IGFALS in diabetic patients is more than that of the healthy control group. The decrease is negatively related to the patient's Hb A1c, FBG, and IGFALS as a biomarker for the diagnosis of diabetes is better and less sensitive. The level of IGFALS in the IR cells induced by high glucose and high insulin significantly decreases. After silent expression of IGFALS, the level of phosphorylation of Akt in the pathway of insulin signal transduction is reduced, and the signal transduction pathway of the inflammatory signal transduction pathway is reduced. The increase of NF- kappa B and its phosphorylation level may affect insulin resistance through the action of insulin and inflammatory signal transduction pathway in.IGFALS.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R446.6;R587.1

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