基于iTRAQ技术的糖尿病血清蛋白标志物筛选及机制研究

发布时间：2018-05-19 18:32

本文选题：蛋白质组学 + 生物标志物　；参考：《天津医科大学》2017年博士论文

【摘要】：目的:通过蛋白质组学的同位素标记相对和绝对定量(Isobaric tags for relative and absolute quantitation,iTRAQ)技术探索糖尿病患者与健康人群中血清低丰度蛋白的种类及表达水平异常,筛查与糖尿病相关的血清生物学标志物,进一步对蛋白质组学筛查出的差异蛋白在人群研究中进行验证;在基础研究中对差异表达蛋白影响胰岛素信号转导机制进行探索,以期为人群研究的蛋白质组学数据库及糖尿病患者血清蛋白分布和功能研究提供新的数据,为与糖尿病发病机制相关的蛋白质研究探索新的方向。方法:在新诊断2型糖尿病患者中用蛋白质组学的iTRAQ技术观察血清中的各种低丰度蛋白表达水平,与健康人群相比较筛选出表达水平异常的蛋白,应用生物医学软件对鉴定出的蛋白进行分类并分析其细胞组成成分和参与的生物学进程。从第一部分筛选出的差异蛋白中挑选两种异常降低蛋白转化生长因子β诱导蛋白(Transforming growth factor-β-induced protein,TGFBIp)及胰岛素样生长因子酸不稳定亚基(Insulin-like growth factor acid-labile subunit,IGFLAS)用酶联免疫吸附试验(Enzyme-linked immunosorbent assay,ELISA)在糖尿病患者、非糖尿病的慢性疾病患者、健康人群中进行验证,观察在不同人群中这两种蛋白的表达水平,以验证蛋白质组学研究结果。进一步观察TGFBIp及IGFLAS表达水平与糖尿病患者临床资料之间的相关性,绘制受试者工作特征(Receiver operating characteristic,ROC)曲线,判定差异表达蛋白对诊断糖尿病的效力。采用高胰岛素和高葡萄糖诱导人肝癌细胞(Hep G2)建立胰岛素抵抗(Insulin Resistance,IR)细胞模型,使用IGFALS-si RNA使细胞中IGFALS表达沉默,观察Hep G2葡萄糖消耗量的变化。采用ELISA法检测IGFALS在各组细胞中的表达量。采用蛋白质印记(Western Blotting,WB)方法检测蛋白激酶B(Protein kinases B,PKB,也称为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转染前后及IR各组细胞中蛋白表达量的变化。结果:2型糖尿病患者中鉴定到的血清蛋白数量为222个,与健康组相比差异蛋白72个;其中38个蛋白表达上调,34个蛋白表达下调。在72个差异蛋白中,从细胞组分汇总分类有17%为胞外蛋白,16%为胞质蛋白,10%为膜蛋白,4%为核内蛋白。从生物进程汇总分类有33%参与调节功能,19%参与脂质代谢,11%参与代谢过程,8%参与免疫反应,7%参与稳态调节,6%参与炎症反应。人群研究发现TGFBIp在慢性病组(CD组)明显升高,糖尿病组(DM组)较对照组(CON组)有降低趋势,但差异无统计学意义(220.09 ng/ml±46.19 ng/ml比233.07 ng/ml±45.83 ng/ml,P=0.118)。IGFALS在三组中有明显差异,在DM组值最低(12.21 ng/ml±4.21 ng/ml),其次为CD组(16.93 ng/ml±4.21 ng/ml),在CON组值最高(18.85 ng/ml±5.63 ng/ml),差异有统计学意义(F=48.90,P0.01)。TGFBIp与各项临床指标无统计学相关性,IGFALS与FBG、Hb A1C呈负相关,r值分别为-0.23,-0.32(P均0.05),与DBP呈正相关,r值为0.35(P0.01)。IGFALS的Cutoff值为15.91 ng/ml,此时灵敏度为65.2%,特异度为84.8%,曲线下面积(Areas under the curve,AUC)为0.802,具有统计学意义(P0.01)。以高胰岛素和高糖分别培养48h和24h后均不会显著影响细胞增殖。以胰岛素6×10-6 mol/L、3×10-6 mol/L、6×10-7 mol/L的浓度干扰24h时,以葡萄糖44 mmo/L、55 mmol/L、66 mmol/L的浓度干扰12h时对细胞胰岛素敏感性影响较大。沉默表达IGFALS后细胞对葡萄糖的利用显著减少(P0.01)。胰岛素诱导的IR细胞中IGFALS分泌量显著低于对照组(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)。高糖诱导的IR细胞中IGFALS分泌量显著低于对照组(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组的IGFALS分泌量较对照组显著降低(114.55 ng/ml±7.50ng/ml比187.99 ng/ml±6.13 ng/ml,P0.01)。WB检测结果表明三组细胞p-Akt水平较对照组显著降低(1.23±0.12,1.11±0.14,0.99±0.05比1.48±0.11,F=10.832,P0.01),Akt表达仅在胰岛素诱导IR组有差异(1.78±0.19比2.42±0.29,P0.05)。三组中p-NF-κB及NF-κB的表达量较对照组均有显著增高(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)。结论:在糖尿病人群中能够应用蛋白质组学的iTRAQ技术筛查差异表达的低丰度蛋白,为寻找糖尿病患者血清生物标志物提供更多的可选择性。糖尿病患者血清中IGFALS较健康对照组显著降低,与患者的Hb A1c、FBG呈负相关,IGFALS作为诊断糖尿病的生物学标志物特异性较好,敏感性欠佳。高糖及高胰岛素诱导的IR细胞中,IGFALS的水平显著下降。沉默表达IGFALS后,胰岛素信号转导通路中Akt的磷酸化水平降低,炎症信号转导通路中NF-κB及其磷酸化水平升高。IGFALS可能通过作用于胰岛素及炎症信号转导通路影响胰岛素抵抗。
[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.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R446.6;R587.1

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