ITRAQ技术筛选系统性红斑狼疮合并股骨头坏死的血清差异蛋白质组学变化
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摘要
背景:系统性红斑狼疮合并股骨头坏死是由于长期大剂量应用皮质类固醇糖皮质激素造成股骨头活性成分死亡的病理过程,但其早期诊治缺乏特异的生物标志物。目的:应用同位素相对标记与绝对定量技术(i TRAQ)筛选系统性红斑狼疮合并激素性股骨头坏死的血清标志物。方法:分别收集系统性红斑狼疮合并激素性股骨头坏死患者血清8例(实验组)、系统性红斑狼疮使用激素后无股骨头坏死的患者血清(对照组)10例,采用i TRAQ联合二维液相色谱-串联质谱技术(2D-LC-MS/MS)对差异蛋白质进行筛选与鉴定。结果与结论:①共鉴定出10种差异蛋白质,与对照组相比有统计学差异,其中实验组中铜篮蛋白、免疫球蛋白Ig G、免疫球蛋白κ链、补体C2等4种蛋白表达上调;内皮细胞蛋白C受体、人源性激肽释放酶结合蛋白、色素上皮衍生因子、四连接素、胰岛素样生长因子2、甲状腺素运载蛋白等6种蛋白表达下调;②KEGG通路分析提示以上10个蛋白与Wnt信号通路、补体与凝血级联反应、系统性红斑狼疮这3条信号通路密切相关;③结果说明,通过蛋白质组学技术成功得到了可靠的激素性股骨头坏死血清差异蛋白质,为激素性股骨头坏死的诊断及其发病机制提供了理论依据。
BACKGROUND: Systemic lupus erythematosus(SLE) combined with osteonecrosis of the femoral head(ONFH) is a result of long-term application of steroids that induces death of active ingredients in femoral head. But there is a lack of specific biomarker for early diagnosis. OBJECTIVE: To screen the systemic markers of SLE with steroid-induced ONFH using isobaric tags for relative and absolute quantitation(iTRAQ) technology. METHODS: Serum of eight patients with SLE and steroid-induced ONFH was collected and used as the experimental group, and serum of 10 patients with SLE but without steroid-induced ONFH served as the control group. The differentially expressed proteins were screened and identified by iTRAQ technique and two-dimensional liquid chromatography-tandem mass spectrometry. RESULTS AND CONCLUSION: Totally 10 kinds of differentially expressed proteins were identified. There was significant difference between two groups. In the experimental group, the expression levels of copper basket protein, immunoglobulin IgG, immunoglobulin κ chain and complement C2 were significantly up-regulated. The expression levels of endothelial cell protein C receptor, human kallikrein-binding protein, pigment epithelium-derived factor, tetracycline, insulin-like growth factor 2, and thyroxine carrier protein were down-regulated. KEEG pathway analysis indicated that 10 kinds of proteins were closely related to Wnt signaling pathway, coagulation and complement cascades reaction signaling pathway and SLE signaling pathway. In summary, the differentially expressed proteins are obtained successfully in steroid-induced ONFH by proteomics, which provides theoretical basis for the diagnosis and pathogenesis of steroid-induced ONFH.
BACKGROUND: Systemic lupus erythematosus(SLE) combined with osteonecrosis of the femoral head(ONFH) is a result of long-term application of steroids that induces death of active ingredients in femoral head. But there is a lack of specific biomarker for early diagnosis. OBJECTIVE: To screen the systemic markers of SLE with steroid-induced ONFH using isobaric tags for relative and absolute quantitation(iTRAQ) technology. METHODS: Serum of eight patients with SLE and steroid-induced ONFH was collected and used as the experimental group, and serum of 10 patients with SLE but without steroid-induced ONFH served as the control group. The differentially expressed proteins were screened and identified by iTRAQ technique and two-dimensional liquid chromatography-tandem mass spectrometry. RESULTS AND CONCLUSION: Totally 10 kinds of differentially expressed proteins were identified. There was significant difference between two groups. In the experimental group, the expression levels of copper basket protein, immunoglobulin IgG, immunoglobulin κ chain and complement C2 were significantly up-regulated. The expression levels of endothelial cell protein C receptor, human kallikrein-binding protein, pigment epithelium-derived factor, tetracycline, insulin-like growth factor 2, and thyroxine carrier protein were down-regulated. KEEG pathway analysis indicated that 10 kinds of proteins were closely related to Wnt signaling pathway, coagulation and complement cascades reaction signaling pathway and SLE signaling pathway. In summary, the differentially expressed proteins are obtained successfully in steroid-induced ONFH by proteomics, which provides theoretical basis for the diagnosis and pathogenesis of steroid-induced ONFH.
引文
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[14]孔园园.血管内皮细胞蛋白C受体的研究进展[J].华东六省一市生物化学与分子生物学会2010年学术交流会, 2010.
[15]何梦梅,于靖.激肽释放酶结合蛋白KBP在炎症中的作用机制及研究进展[J].医学综述, 2015,21(18):3268-3271.
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[24]孙少松,郑欣,姚圣城,等.色素上皮衍生因子对高浓度地塞米松刺激下成骨细胞迁移、分化的影响[J].中国矫形外科杂志, 2016,24(24):2280-2285.
[25]贾岩波,刘万林,任逸众.激素性股骨头坏死发病机制中细胞凋亡的研究进展[J].中国组织工程研究,2012,16(13):2444-2450.
[26]Salajegheh A.Insulin Like Growth Factor(IGF)//Angiogenesis in Health, Disease and Malignancy. Springer International Publishing, 2016.
[27]胡玲萍,吴雪华.老年男性2型糖尿病并发骨质疏松相关危险因素研究现状[J].中国临床保健杂志, 2014,17(1):107-109.
[28]隋春华,陈奕,余娇,等. 2型糖尿病患者胰岛素样生长因子-2与骨密度的关系[J].中国临床医学,2011,18(2):196-197.
[29]赵玉岩,郭磊,田蕾,等.骨性关节炎软骨细胞中胰岛素样生长因子Ⅱ表达的研究[J].中国老年学,2009, 29(2):195-197.
[30]Wewer UM, Albrechtsen R.A Potential Role for Tetranectin in Mineralization during Osteogenesis. J Cell Biol. 1994;127(6 Pt 1):1767-1775.
[31]茹德文,颜玉峰,李炳,等.四连接素基因敲除小鼠表现出脊柱后凸畸形和骨质疏松症的特征[J].复旦学报(医学版), 2016, 43(2):159-163.
[32]孙家祥,李艳,刘利洪,等.系统性红斑狼疮患者外周血T淋巴细胞亚群、免疫球蛋白及补体变化分析[J].临床和实验医学杂志,2013,12(9):654-655.
[33]程健华,赵修竹.B因子——补体第二激活途径中的一个关键分子[J].基础医学与临床,1987,7(3):3.
[34]邹刚,卫海荣.类风湿性关节炎患者血清免疫球蛋白和补体水平的检测与分析[J].中国实用医刊,2006,33(11):82-83.
[35]Ando Y.Transthyretin-its function and pathogenesis.Rinsho Byori. 2006;54(5):497-502.
[36]蒋洪彦,王世鑫,李雪华,等.严重急性呼吸综合征康复人员并发股骨头缺血性坏死血清相关蛋白研究[J].中华预防医学杂志,2008,42(7):522-526.
[2]Zhao L,Wu X,Wu H,et al.Symptomatic knee osteonecrosis in patients with systemic lupus erythematosus:a case-control study. Rheumatol Int. 2016;36(8):1105-1111.
[3]Padovan M,Castellino G,Bortoluzzi A,et al.Factors and comorbidities associated with central nervous system involvement in systemic lupus erythematosus:a retrospective cross-sectional case-control study from a single center.Rheumatol Int. 2012;32(1):129-135.
[4]Weinstein RS.Glucocorticoid-induced osteoporosis and osteonecrosis. Endocrinol Metab Clin North Am.2012;41(3):595–611.
[5]陈宇.股骨头坏死的病因构成及发病特征分析[J].中国伤残医学,2015,24(20):167-168.
[6]叶奕亨,陈凯,金可可,等.塌陷前期股骨头坏死的保头手术治疗的进展[J].中国骨伤, 2017, 30(3):287-292.
[7]张鹤山,李子荣.股骨头坏死诊断与治疗的专家建议[J].中华骨科杂志,2007,27(2):146-148.
[8]杨述华.骨坏死学[M].北京:人民军医出版社,2002:148.
[9]Zhang H,Zhang L,Wang J,et al.Proteomic analysis of bone tissues of patients with osteonecrosis of the femoral head.Omics A Journal of Integrative Biology.2009;13(6):453.
[10]谢秀枝,王欣,刘丽华,等.iTRAQ技术及其在蛋白质组学中的应用[J].中国生物化学与分子生物学报,2011, 27(7):616-621.
[11]汪亮,刘耀升,刘蜀彬.糖皮质激素诱导股骨头坏死发病机制的研究进展[J].中华损伤与修复杂志:电子版, 2015,10(5):60-66.
[12]Garcia FL, Ramalli EL, Picado CH.Coagulation disorders in patients with femoral head osteonecrosis. Acta Ortop Bras.2013;21(1):43-45.
[13]李蕾,王培昌.蛋白C抗凝系统及生物学作用最新研究进展[C]//2012北京检验医学年会暨中青年检验医学论坛. 2012.
[14]孔园园.血管内皮细胞蛋白C受体的研究进展[J].华东六省一市生物化学与分子生物学会2010年学术交流会, 2010.
[15]何梦梅,于靖.激肽释放酶结合蛋白KBP在炎症中的作用机制及研究进展[J].医学综述, 2015,21(18):3268-3271.
[16]郝廷,刘万林,苏秀兰.血管内皮生长因子与激素性股骨头坏死关系的研究进展[J].临床医学工程,2009, 16(1):85-87.
[17]邢永梅,刁勇.人源性激肽释放酶结合蛋白与内皮细胞功能[J].生命科学, 2012,24(1):29-35.
[18]毕雪,汲广东.铜蓝蛋白的结构功能以及演化[J].鲁东大学学报(自然科学版),2015,31(4):318-324.
[19]Chen YS,Pelekanos RA,Ellis RL,et al.Small Molecule Mesengenic Induction of Human Induced Pluripotent Stem Cells to Generate Mesenchymal Stem/Stromal Cells. Stem Cells Translational Medicine. 2012,1(2):83.
[20]Kinzebach S, Bieback K.Expansion of Mesenchymal Stem/Stromal cells under xenogenic-free culture conditions[M]//Mesenchymal Stem Cells-Basics and Clinical Application I. Springer Berlin Heidelberg, 2012:33.
[21]祝兆波,魏波.脂质促进成骨细胞凋亡及作用机制的研究进展[J].中国矫形外科杂志,2015,21(1):52-54.
[22]Craword SE,Fitchev P,Veliceasa D,et al.The many facets of PEDF in drug discovery and disease:a diamond in the rough or split personality disorder?.Expert Opinion on Drug Discovery. 2013; 8(7):769-792.
[23]宋娜.肩峰下滑囊间充质干细胞多能性的探讨及PEDF蛋白在成骨分化过程中的作用[D].长春:吉林大学,2013.
[24]孙少松,郑欣,姚圣城,等.色素上皮衍生因子对高浓度地塞米松刺激下成骨细胞迁移、分化的影响[J].中国矫形外科杂志, 2016,24(24):2280-2285.
[25]贾岩波,刘万林,任逸众.激素性股骨头坏死发病机制中细胞凋亡的研究进展[J].中国组织工程研究,2012,16(13):2444-2450.
[26]Salajegheh A.Insulin Like Growth Factor(IGF)//Angiogenesis in Health, Disease and Malignancy. Springer International Publishing, 2016.
[27]胡玲萍,吴雪华.老年男性2型糖尿病并发骨质疏松相关危险因素研究现状[J].中国临床保健杂志, 2014,17(1):107-109.
[28]隋春华,陈奕,余娇,等. 2型糖尿病患者胰岛素样生长因子-2与骨密度的关系[J].中国临床医学,2011,18(2):196-197.
[29]赵玉岩,郭磊,田蕾,等.骨性关节炎软骨细胞中胰岛素样生长因子Ⅱ表达的研究[J].中国老年学,2009, 29(2):195-197.
[30]Wewer UM, Albrechtsen R.A Potential Role for Tetranectin in Mineralization during Osteogenesis. J Cell Biol. 1994;127(6 Pt 1):1767-1775.
[31]茹德文,颜玉峰,李炳,等.四连接素基因敲除小鼠表现出脊柱后凸畸形和骨质疏松症的特征[J].复旦学报(医学版), 2016, 43(2):159-163.
[32]孙家祥,李艳,刘利洪,等.系统性红斑狼疮患者外周血T淋巴细胞亚群、免疫球蛋白及补体变化分析[J].临床和实验医学杂志,2013,12(9):654-655.
[33]程健华,赵修竹.B因子——补体第二激活途径中的一个关键分子[J].基础医学与临床,1987,7(3):3.
[34]邹刚,卫海荣.类风湿性关节炎患者血清免疫球蛋白和补体水平的检测与分析[J].中国实用医刊,2006,33(11):82-83.
[35]Ando Y.Transthyretin-its function and pathogenesis.Rinsho Byori. 2006;54(5):497-502.
[36]蒋洪彦,王世鑫,李雪华,等.严重急性呼吸综合征康复人员并发股骨头缺血性坏死血清相关蛋白研究[J].中华预防医学杂志,2008,42(7):522-526.