早期激素性股骨头坏死差异蛋白质组学分析及相关蛋白表达验证
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摘要
随着激素在临床上广泛应用,激素性股骨头缺血性坏死(steroid-induced avascular necrosis of femoral head,SANFH)已居非创伤性股骨头坏死的首位。目前SANFH的发病机制尚不明确,众多发病学说往往仅从单一的分子或基因水平上对SANFH发病机理进行研究,无法整体、动态、多层次地了解SANFH发生过程中相关调控基因和相关功能基因蛋白产物的种类、数量及峰值变化规律。随着后基因组时代的来临,蛋白质组学研究的兴起,为我们研究SANFH的发病机制提供了新的思路及技术手段。
目的:(1)通过反复大剂量注射地塞米松联合增加股骨头负重的方法建立兔早期SANFH模型,为后续比较蛋白质组学研究提供较为理想的动物模型。(2)优化兔股骨头蛋白质组双向电泳(2-DE)技术。(3)创建早期SANFH不同时点(注射激素后3w、6w、9w、12w )与正常股骨头的蛋白质表达图谱,经图像分析及质谱鉴定后筛选出差异蛋白并观察其变化规律。(4)对差异表达蛋白Annexin A1进行表达验证,以证实2-DE结果的可靠性。
方法:(1)健康成年新西兰大白兔48只,随机分为模型组和对照组。模型组臀肌注射地塞米松针(10mg/kg),每次间隔48h,通过迫使兔子站立进食水增加股骨头负重,对照组相同时点注射等量生理盐水。分别于给药后3w,6w,9w,12w处死兔子,每次模型组和对照组各6只。观察动物一般状态、股骨头外观、病理学改变(包括骨髓腔内脂肪细胞、骨小梁及骨细胞)及骨细胞Caspase-3活性变化情况。(2)用健康成年新西兰大白兔股骨头作为样本,改进2-DE程序中的几个关键环节,对样品制备(骨组织破碎、细胞裂解、除盐)、等电聚焦程序、固相胶条的选择和凝胶染色方法进行一系列比较和优化。(3)获取SANFH早期病理变化过程的不同时间点(注射激素后3w、6w、9w、12w)及正常股骨头坏死标本,利用2-DE技术创建早期SANFH不同时点与正常股骨头的蛋白表达图谱,运用基质辅助激光解析飞行时间质谱(MALDI-TOF-MS)对差异点进行鉴定。(4)应用免疫印迹(Western Blot)技术检测Annexin A1在各组的表达水平,并与2-DE结果进行比较。
结果:(1)模型组给药3w后可见骨髓腔内脂肪细胞体积增大,数量增多,并且随给药时间的延长脂肪细胞肥大更为明显,甚至发生融合,骨髓干细胞相对减少,脂肪细胞面积百分比大于对照组(P<0.05);模型组6w后空骨陷凹率大于对照组(P <0.05);模型组9w后可见骨小梁稀疏变细、结构紊乱、断裂,骨小梁面积百分比小于对照组(P<0.05);电镜下骨细胞形态发生异常改变;模型组给药6w后骨细胞Caspase-3活性高于对照组(P <0.05)。(2)采用组织匀浆法破碎骨组织,改良细胞裂解液进行蛋白质提取,丙酮沉淀法除盐,选择24cmPH3-10NL的固相胶条上样,调整后的等电聚焦(IEF)程序进行电泳,胶体考马斯亮兰染色法进行凝胶染色可以得到较为理想的蛋白表达图谱。(3)获得分辨率和重复性较好的早期SANFH不同时间点及正常股骨头的蛋白表达图谱。通过比较后发现24个差异蛋白点(差异大于2倍),经质谱成功鉴定出20个,其中7种蛋白质在早期SANFH中呈现表达上升,另外13种蛋白呈现表达下降。(4)Western blot显示早期SANFH中Annexin A1的表达水平低于对照组,其表达趋势与2-DE结果相一致。
结论:(1)反复大剂量注射地塞米松联合增加股骨头负重的方法可以诱导出兔早期SANFH模型。(2)建立并优化了兔股骨头蛋白质2-DE技术,得到了比较理想的2-DE图谱,为进一步开展疾病差异蛋白质组学研究提供了良好的技术条件。(3)经过优化后的2-DE技术,获得了早期SANFH不同时点及正常股骨头蛋白质表达图谱。发现了20种差异表达的蛋白,丰富了疾病相关的差异蛋白质组学的数据,初步筛选出潜在的与疾病早期发病机制相关的重要蛋白质。(4)Western blot结果与2-DE结果相一致,验证了之前实验获得Annexin A1表达变化趋势的正确性,Annexin A1可以成为进一步研究疾病早期发病机制的候选蛋白分子。
With the widespread clinical application of hormone, SANFH has become the most common disease in non-traumatic femoral head necrosis. Currently, the pathogenesis of SANFH remains unclear, many theories can only explain part of the pathogenesis from single molecular or genetic level, not whole, dynamic, multi-level to observe the type, quantity, peak variation of the SANFH related regulatory genes and associated gene protein product.With the post-genome era coming and the rise of proteomics, proteomics can provide new ideas and techniques to study the pathogenesis of SANFH.
Objective: (1)To create a rabbit model of early SANFH by given a high doses of dexamethasone and increase the loading of femoral head. (2)To optimize femoral head dimensional gel electrophoresis (2-DE) technology. (3) To create protein expression profiles of early SANFH(3w, 6w, 9w, 12w after the injection of hormones)and normal femoral head, identify the difference proteins between early SANFH and normal femoral head by image analysis and mass spectrometry and observe their changing patterns. (4) To verify the expression of Annexin A1 by Western blot.
Methods: (1)The 48 rabbits were randomly divided into the model groups and control group. Rabbits in the model group were injected dexamethasone (10mg/kg) and forced to hold upright position 1 hour everyday, while rabbits in the control group were injected the same amount of saline. Animals were sacrificed at 3w,6w,9w,12w after injection, using optical microscope and transmission electron microscopy to observe the histopathological changes and measuring the Caspase-3 activity in osteocyte.(2)Femoral head from normal rabbits were used for samples preparation, optimizating the technique by improving the 2-DE process in some steps, including sample preparation, isoelectric focusing procedures, IPG strip selection and gel staining.(3) Preparing samples of early SANFH(3w, 6w, 9w, 12w)and normal femoral head, using MALDI-TOF-MS to identify the difference protein after creating 2-DE images. (4) Using Western Blot to detect Annexin A1 expression levels in each group.
Results: (1) Histopathological changes could be observed under the light microscope at 3 weeks after administration, including fat cell hypertrophy and relatively decreasing of mesenchymal stem cells, the percentage of fat cell area is higher (P<0.05). The percentage of empty osteocyte lacunae in the model group was significantly higher than in the control group at 6 weeks after administration (P<0.05). Trabecular bone becomes thinner even collapse in model group at 9 weeks after administration. Under the electron microscope, morphological changes in bone cells were happened. At 6 weeks after administration, osteocyte Caspase-3 activity in the model group was higher than the control group (P<0.05). (2)Ideal images can be obtain after optimizing the 2-DE technique.(3) 20 different proteins were identified,7 proteins up-regulate in the early SANFH,meanwhile 13 proteins down-regulate. (4)Result in Western Blot showing that the expression of Annexin A1 in early SANFH was lower than the control group, the expression trends were consistent with 2-DE.
Conclusions: (1) Using high-dose dexamethasone combined with increasing loading of the femoral head can creat a rabbit model of early SANFH. (2) The optimization of the femoral head protein 2-DE technology are important to the further research on SANFH. (3)20 differentially expressed protein were found, they may become the potential biomarks to SANFH. (4)The result is consistent with the 2-DE, the trend of Annexin A1 expression down-regulate in SANFH was verified by Western Blot,Annexin A1 may be one of importance proteins in early SANFH.
目的:(1)通过反复大剂量注射地塞米松联合增加股骨头负重的方法建立兔早期SANFH模型,为后续比较蛋白质组学研究提供较为理想的动物模型。(2)优化兔股骨头蛋白质组双向电泳(2-DE)技术。(3)创建早期SANFH不同时点(注射激素后3w、6w、9w、12w )与正常股骨头的蛋白质表达图谱,经图像分析及质谱鉴定后筛选出差异蛋白并观察其变化规律。(4)对差异表达蛋白Annexin A1进行表达验证,以证实2-DE结果的可靠性。
方法:(1)健康成年新西兰大白兔48只,随机分为模型组和对照组。模型组臀肌注射地塞米松针(10mg/kg),每次间隔48h,通过迫使兔子站立进食水增加股骨头负重,对照组相同时点注射等量生理盐水。分别于给药后3w,6w,9w,12w处死兔子,每次模型组和对照组各6只。观察动物一般状态、股骨头外观、病理学改变(包括骨髓腔内脂肪细胞、骨小梁及骨细胞)及骨细胞Caspase-3活性变化情况。(2)用健康成年新西兰大白兔股骨头作为样本,改进2-DE程序中的几个关键环节,对样品制备(骨组织破碎、细胞裂解、除盐)、等电聚焦程序、固相胶条的选择和凝胶染色方法进行一系列比较和优化。(3)获取SANFH早期病理变化过程的不同时间点(注射激素后3w、6w、9w、12w)及正常股骨头坏死标本,利用2-DE技术创建早期SANFH不同时点与正常股骨头的蛋白表达图谱,运用基质辅助激光解析飞行时间质谱(MALDI-TOF-MS)对差异点进行鉴定。(4)应用免疫印迹(Western Blot)技术检测Annexin A1在各组的表达水平,并与2-DE结果进行比较。
结果:(1)模型组给药3w后可见骨髓腔内脂肪细胞体积增大,数量增多,并且随给药时间的延长脂肪细胞肥大更为明显,甚至发生融合,骨髓干细胞相对减少,脂肪细胞面积百分比大于对照组(P<0.05);模型组6w后空骨陷凹率大于对照组(P <0.05);模型组9w后可见骨小梁稀疏变细、结构紊乱、断裂,骨小梁面积百分比小于对照组(P<0.05);电镜下骨细胞形态发生异常改变;模型组给药6w后骨细胞Caspase-3活性高于对照组(P <0.05)。(2)采用组织匀浆法破碎骨组织,改良细胞裂解液进行蛋白质提取,丙酮沉淀法除盐,选择24cmPH3-10NL的固相胶条上样,调整后的等电聚焦(IEF)程序进行电泳,胶体考马斯亮兰染色法进行凝胶染色可以得到较为理想的蛋白表达图谱。(3)获得分辨率和重复性较好的早期SANFH不同时间点及正常股骨头的蛋白表达图谱。通过比较后发现24个差异蛋白点(差异大于2倍),经质谱成功鉴定出20个,其中7种蛋白质在早期SANFH中呈现表达上升,另外13种蛋白呈现表达下降。(4)Western blot显示早期SANFH中Annexin A1的表达水平低于对照组,其表达趋势与2-DE结果相一致。
结论:(1)反复大剂量注射地塞米松联合增加股骨头负重的方法可以诱导出兔早期SANFH模型。(2)建立并优化了兔股骨头蛋白质2-DE技术,得到了比较理想的2-DE图谱,为进一步开展疾病差异蛋白质组学研究提供了良好的技术条件。(3)经过优化后的2-DE技术,获得了早期SANFH不同时点及正常股骨头蛋白质表达图谱。发现了20种差异表达的蛋白,丰富了疾病相关的差异蛋白质组学的数据,初步筛选出潜在的与疾病早期发病机制相关的重要蛋白质。(4)Western blot结果与2-DE结果相一致,验证了之前实验获得Annexin A1表达变化趋势的正确性,Annexin A1可以成为进一步研究疾病早期发病机制的候选蛋白分子。
With the widespread clinical application of hormone, SANFH has become the most common disease in non-traumatic femoral head necrosis. Currently, the pathogenesis of SANFH remains unclear, many theories can only explain part of the pathogenesis from single molecular or genetic level, not whole, dynamic, multi-level to observe the type, quantity, peak variation of the SANFH related regulatory genes and associated gene protein product.With the post-genome era coming and the rise of proteomics, proteomics can provide new ideas and techniques to study the pathogenesis of SANFH.
Objective: (1)To create a rabbit model of early SANFH by given a high doses of dexamethasone and increase the loading of femoral head. (2)To optimize femoral head dimensional gel electrophoresis (2-DE) technology. (3) To create protein expression profiles of early SANFH(3w, 6w, 9w, 12w after the injection of hormones)and normal femoral head, identify the difference proteins between early SANFH and normal femoral head by image analysis and mass spectrometry and observe their changing patterns. (4) To verify the expression of Annexin A1 by Western blot.
Methods: (1)The 48 rabbits were randomly divided into the model groups and control group. Rabbits in the model group were injected dexamethasone (10mg/kg) and forced to hold upright position 1 hour everyday, while rabbits in the control group were injected the same amount of saline. Animals were sacrificed at 3w,6w,9w,12w after injection, using optical microscope and transmission electron microscopy to observe the histopathological changes and measuring the Caspase-3 activity in osteocyte.(2)Femoral head from normal rabbits were used for samples preparation, optimizating the technique by improving the 2-DE process in some steps, including sample preparation, isoelectric focusing procedures, IPG strip selection and gel staining.(3) Preparing samples of early SANFH(3w, 6w, 9w, 12w)and normal femoral head, using MALDI-TOF-MS to identify the difference protein after creating 2-DE images. (4) Using Western Blot to detect Annexin A1 expression levels in each group.
Results: (1) Histopathological changes could be observed under the light microscope at 3 weeks after administration, including fat cell hypertrophy and relatively decreasing of mesenchymal stem cells, the percentage of fat cell area is higher (P<0.05). The percentage of empty osteocyte lacunae in the model group was significantly higher than in the control group at 6 weeks after administration (P<0.05). Trabecular bone becomes thinner even collapse in model group at 9 weeks after administration. Under the electron microscope, morphological changes in bone cells were happened. At 6 weeks after administration, osteocyte Caspase-3 activity in the model group was higher than the control group (P<0.05). (2)Ideal images can be obtain after optimizing the 2-DE technique.(3) 20 different proteins were identified,7 proteins up-regulate in the early SANFH,meanwhile 13 proteins down-regulate. (4)Result in Western Blot showing that the expression of Annexin A1 in early SANFH was lower than the control group, the expression trends were consistent with 2-DE.
Conclusions: (1) Using high-dose dexamethasone combined with increasing loading of the femoral head can creat a rabbit model of early SANFH. (2) The optimization of the femoral head protein 2-DE technology are important to the further research on SANFH. (3)20 differentially expressed protein were found, they may become the potential biomarks to SANFH. (4)The result is consistent with the 2-DE, the trend of Annexin A1 expression down-regulate in SANFH was verified by Western Blot,Annexin A1 may be one of importance proteins in early SANFH.
引文
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11张义福,刘建,孟国林等.兔激素性股骨头坏死病程中肝脂肪酶改变的意义.第四军医大学学报, 2006, 27(8): 737-740.
12吴云刚,童培建,肖鲁伟等.激素性股骨头坏死介入治疗过程中VEGF表达的实验研究.中国中医骨伤科杂志,2002,1O(4):30-34.
13熊明月,王坤正,党晓谦.早期激素性股骨头坏死骨细胞凋亡的实验研究.中国修复重建外科杂志, 2007, 21(3): 262-265.
14 Baltimore D. Our genome unveiled. Nature, 2001, 409(6822): 814-816.
15 Leite-BrowningML,McCawley LJ,Choi OH,et al.Interactions ofα2- HS-glycoprotein(fetuin) with MMP-3 and murine squamous cell carcinoma cells.Int J Oncol,2002,21(5):965-971.
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17 Ix JH, Wassel CL, Bauer DC, Fetuin-A and BMD in older persons: the Health Aging and Body Composition (Health ABC) study. J Bone Miner Res, 2009,24(3):514-21.
18 Reynolds JL, Skepper JN, McNair R.Multifunctional Roles for Serum Protein Fetuin-A in Inhibition of Human Vascular Smooth Muscle Cell Calcification. Am Soc Nephrol, 2005,16(10):2920-30.
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37 Haut Donahue TL, Genetos DC, Jacobs CR, et al.AnnexinV disruption impairs mechanically induced calcium signaling in osteoblastic cells. Bone ,2004,35:656-663.
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11张义福,刘建,孟国林等.兔激素性股骨头坏死病程中肝脂肪酶改变的意义.第四军医大学学报, 2006, 27(8): 737-740.
12吴云刚,童培建,肖鲁伟等.激素性股骨头坏死介入治疗过程中VEGF表达的实验研究.中国中医骨伤科杂志,2002,1O(4):30-34.
13熊明月,王坤正,党晓谦.早期激素性股骨头坏死骨细胞凋亡的实验研究.中国修复重建外科杂志, 2007, 21(3): 262-265.
14 Baltimore D. Our genome unveiled. Nature, 2001, 409(6822): 814-816.
15 Leite-BrowningML,McCawley LJ,Choi OH,et al.Interactions ofα2- HS-glycoprotein(fetuin) with MMP-3 and murine squamous cell carcinoma cells.Int J Oncol,2002,21(5):965-971.
16 Jahnen-Dechent W, Schinke T, Trindl A,et al.Cloning and targeted deletion of the mouse fetuin gene. J Biol Chem,1997,272(50):31496-31503.
17 Ix JH, Wassel CL, Bauer DC, Fetuin-A and BMD in older persons: the Health Aging and Body Composition (Health ABC) study. J Bone Miner Res, 2009,24(3):514-21.
18 Reynolds JL, Skepper JN, McNair R.Multifunctional Roles for Serum Protein Fetuin-A in Inhibition of Human Vascular Smooth Muscle Cell Calcification. Am Soc Nephrol, 2005,16(10):2920-30.
19刘元林,于晓妉,刘晓丹,等.间充质干细胞定向成骨细胞分化的蛋白质组学分析.第四军医大学学报,2006,27(17):1151-1154.
20 Fraser JH, Helfrich MH,et al.Hydrogen peroxide,but no tsuperoxide, stimulates bone resorption in mouse calvariae.Bone,1996,19(3): 223-226.
21 Cosgriff SW.Thromboembolic complications associated with ACTH and cortisone therapy. Am Med Assoc,1951,147(10):924-926.
22 Hamilton JA, Lingelbach S, Partridge NC,et al. Regulation of plasminogen activator production by bone-resorbing hormones in normal and malignant osteoblasts.Endocrinology,1985,116(6):2186-2191.
23朱盛修.股骨头缺血性坏死诊疗学.湖南科学技术出版社,1999,81-91.
24王新生,许振华,陈风苞,等.激素性股骨头缺血性坏死发病机理的实验研究.中华骨科杂志,1995,15(3):168-170.
25 Glueck GJ,Freberg R, Tracy T, et al.Thrombophilia and hypofibrinolysis: Pathophysiologies of osteonecrosis. Clin Orthop,1997,334:43-56.
26 Oxlund H,Mosekilde L,Ouoft G,et al.Reduced concentration of collagen reducible cross links in human trabecullar bone with respect to age and osteoporosis.Bone,1996,19:479-484.
27 Garnero P,Sornay-rendu E,Hapuy MC,et al.Increased bone turnover in late post-menopausal women is a major determinant of osteoporosis.J Bone Miner Res,1996,11:337-349.
28 Masaki T,Tokuda M,Ohnishi M,et al.Enhanced expression of protein kinase substrate Annexin in human hepatocellular carcinoma.Hepatology,1996,24 (1):72-81.
29宋海燕.Annexin家族与肿瘤转移.国外医学分子生物学分册,2003,25(增刊):111-114.
30刘懿,凌诒萍,钟慈声.Annexin依赖磷脂结合蛋白在细胞分泌中的作用.生理科学进展,1997,28:367-369.
31崔杰峰,刘银坤,代智,等.转移潜能不同人肝癌细胞系差异蛋白Annexin A1的功能解析.生物化学与生物物理进展,2005,32(12):1141-1149.
32 Damazo AS, Moradi BN, Oliani SM, et al. Role of Annexin 1 gene expression in mouse craniofacial bone development. Birth Defects Res A Clin Mol Teratol,2007,79(7):524-532.
33 Clark, A. R. Anti?inflammatory functions of glucocorticoid?induced genes. Mol. Cell. Endocrinol,2007,275(1-2):79-97.
34 Ai-Xia Zhang,Wei-Hua Yu,Bao-Feng Ma,at al. Proteomic identification ofdifferently expressed proteins responsible for osteoblast differentiation from human mesenchymal stem cells. Mol Cell Biochem,2007, 304(1-2): 167-179.
35 Wang W, Kirsch T.Retinoic acid stimulates Annexinmediated growth plate chondrocyte mineralization. Cell Biology,2002,157(6):1061-1070.
36 Kirsch T, Harrison G, Golub EE, et al.The roles of Annexins and types II and X collagen in matrix vesicle-mediated mineralization of growth plate cartilage. J Biol Chem,2000,275(45):35577-35583.
37 Haut Donahue TL, Genetos DC, Jacobs CR, et al.AnnexinV disruption impairs mechanically induced calcium signaling in osteoblastic cells. Bone ,2004,35:656-663.
1 Clark, A. R. Anti?inflammatory functions of glucocorticoid?induced genes. Mol. Cell. Endocrinol,2007,275 (1-2):79-97.
2 Bensalem N, Ventura AP, Vallée B,et al. Down-regulation of the anti-inflammatory protein Annexin A1 in cystic fibrosis knock-out mice and patients. Mol Cell Proteomics,2005, 4(10):1591-601.
3 Tabe Y, Jin L, Contractor R,et al. Novel role of HDAC inhibitors in AML1/ETO AML cells: activation of apoptosis and phagocytosis through induction of Annexin A1. Cell Death Differ,2007,14(8):1443-56.
4 McNeil AK, Rescher U, Gerke V,et al. Requirement for Annexin A1 in plasma membrane repair. J Biol Chem,2006, 281(46):35202-7.
5 Masaki T,Tokuda M,Ohnishi M,et al.Enhanced expression of protein kinase substrate Annexin in human hepatocellular carcinoma.Hepatology,1996,24 (1):72-81.
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