不同分期骨关节炎关节液中相关降解酶的表达差异
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摘要
背景:目前对骨关节患者滑膜、关节软骨中相关降解酶的研究较多,对关节液中相关因子的检测亦有报道,但有关不同病变分期相关因子表达情况的报道较少,且其与病变程度相关性研究报道亦相对较少。目的:检测不同分期骨关节炎患者关节液中细胞外调节蛋白激酶、基质金属蛋白酶13和人类含Ⅰ型血小板结合蛋白基序的解聚蛋白样金属蛋白酶4/5(a disintegrin and metalloproteinase with thrombospondin-like motifs,ADAMTS4/5)的表达。方法:选择2016年10月至2017年12月在石河子大学医学院第一附属医院骨科行关节镜手术的骨关节炎患者94例,根据临床症状与K-L X射线分级分为早期组(n=27)、中期组(n=32)、晚期组(n=35);选择行截肢的健康患者、单纯膝外伤行关节镜检查者及自愿参与试验的门诊体检者10例,作为对照组。采集4组受试者关节液,采用荧光定量PCR法和ELISA法检测细胞外调节蛋白激酶、基质金属蛋白酶13和ADAMTS4/5的表达情况。试验经石河子大学医学院第一附属医院医学伦理委员会批准,批准号:2017-052-01。结果与结论:①荧光定量PCR检测:早、中、晚期组的各基因表达均高于对照组(P <0.001),早、中、晚期组的细胞外调节蛋白激酶、基质金属蛋白酶13基因表达呈逐渐上升趋势(P <0.01),早期组ADAMTS4/5基因表达高于中、晚期组(P <0.01);②ELISA检测:早、中、晚期组的关节液中各因子表达均高于对照组(P <0.001),早、中、晚期组的细胞外调节蛋白激酶、基质金属蛋白酶13表达呈逐渐上升趋势(P <0.01),早期组ADAMTS4/5表达高于中、晚期组(P <0.01);③相关性分析:细胞外调节蛋白激酶与基质金属蛋白酶13表达水平呈正相关关系(P <0.01),ADAMTS4与ADAMTS5表达呈正相关关系(P <0.01),ADAMTS4、ADAMTS5与细胞外调节蛋白激酶、基质金属蛋白酶13表达水平呈负相关关系(P<0.05);④结果表明:细胞外调节蛋白激酶、基质金属蛋白酶13及ADAMTS4/5的表达与膝骨性关节炎临床症状较为密切,研究这些因子在骨性关节炎不同分期中的分子机制,有望为临床诊断及预后判定提供一定的依据。
BACKGROUND: At present, there are many studies on the degrading enzymes in synovial membrane and articular cartilage in patients with osteoarthritis. The related factors in joint fluid have also been reported, but the expression of related factors in different stages of osteoarthritis is little reported. There are few reports on the correlation between these factors and the disease severity.OBJECTIVE: To detect the expression of extracellular regulated protein kinase, matrix metalloproteinase 13 and a disintegrin and metalloproteinase with thrombospondin-like motifs 4/5 in patients with different stages of osteoarthritis.METHODS: Ninety-four patients with osteoarthritis undergoing arthroscopic surgery at the Department of Orthopedics, Department of Orthopedics, the First Affiliated Hospital of the Medical College, Shihezi University, from October 2016 to December 2017 were included.According to clinical symptoms and K-L X-ray classification, the patients were divided into early-(n=27), medium-(n=32), and late-stage(n=35) groups. Ten healthy patients who underwent amputation, knee trauma undergoing arthroscopy examination and outpatients who volunteered to participate in the experiment were as control group. The synovial fluids of each group were collected to detect the expression levels of extracellular regulated protein kinase, matrix metalloproteinase 13 and a disintegrin and metalloproteinase with thrombospondin-like motifs 4/5 by qRT-PCR and ELISA. The study was approved by the Ethics Committee of the First Affiliated Hospital of the Medical College,Shihezi University, approval number: 2017-052-01.RESULTS AND CONCLUSION:(1) The mRNA expression levels of each gene detected by qRT-PCR in the early-, middle-and late-stage groups were significantly higher than those in the control group(P < 0.001). The mRNA expression of extracellular regulated protein kinase and matrix metalloproteinase 13 in the early-, middle-and late-stage groups was on a rise(P < 0.01). The mRNA expression of a disintegrin and metalloproteinase with thrombospondin-like motifs 4/5 in the early-stage group was significantly higher than that in the middle-and latestage groups(P < 0.01).(2) The protein expression levels of each gene detected by ELISA in the early-, middle-and late-stage groups were significantly higher than those in the control group(P < 0.001). The protein expression of extracellular regulated protein kinase and matrix metalloproteinase 13 in the early-, middle-and late-stage groups was on a rise(P < 0.01). The protein expression of a disintegrin and metalloproteinase with thrombospondin-like motifs 4/5 in the early-stage group was significantly higher than that in the middle-and late-stage groups(P < 0.01).(3) Correlation analysis showed that there was a positive correlation between extracellular regulated protein kinase and matrix metalloproteinase 13 expression(P < 0.01), a disintegrin and metalloproteinase with thrombospondin-like motifs 4 and 5 expression levels were positively correlated(P < 0.01). The expression levels of a disintegrin and metalloproteinase with thrombospondin-like motifs 4/5 were negatively correlated with the expression levels of extracellular regulated protein kinase and matrix metalloproteinase 13(P < 0.05).(4)These results show that the expressions of extracellular regulated protein kinase, matrix metalloproteinase 13 and a disintegrin and metalloproteinase with thrombospondin-like motifs 4/5 are closely related to the clinical symptoms of knee osteoarthritis. The molecular mechanism of these factors in different stages of osteoarthritis is expected to provide reference for the clinical diagnosis and prognosis.
BACKGROUND: At present, there are many studies on the degrading enzymes in synovial membrane and articular cartilage in patients with osteoarthritis. The related factors in joint fluid have also been reported, but the expression of related factors in different stages of osteoarthritis is little reported. There are few reports on the correlation between these factors and the disease severity.OBJECTIVE: To detect the expression of extracellular regulated protein kinase, matrix metalloproteinase 13 and a disintegrin and metalloproteinase with thrombospondin-like motifs 4/5 in patients with different stages of osteoarthritis.METHODS: Ninety-four patients with osteoarthritis undergoing arthroscopic surgery at the Department of Orthopedics, Department of Orthopedics, the First Affiliated Hospital of the Medical College, Shihezi University, from October 2016 to December 2017 were included.According to clinical symptoms and K-L X-ray classification, the patients were divided into early-(n=27), medium-(n=32), and late-stage(n=35) groups. Ten healthy patients who underwent amputation, knee trauma undergoing arthroscopy examination and outpatients who volunteered to participate in the experiment were as control group. The synovial fluids of each group were collected to detect the expression levels of extracellular regulated protein kinase, matrix metalloproteinase 13 and a disintegrin and metalloproteinase with thrombospondin-like motifs 4/5 by qRT-PCR and ELISA. The study was approved by the Ethics Committee of the First Affiliated Hospital of the Medical College,Shihezi University, approval number: 2017-052-01.RESULTS AND CONCLUSION:(1) The mRNA expression levels of each gene detected by qRT-PCR in the early-, middle-and late-stage groups were significantly higher than those in the control group(P < 0.001). The mRNA expression of extracellular regulated protein kinase and matrix metalloproteinase 13 in the early-, middle-and late-stage groups was on a rise(P < 0.01). The mRNA expression of a disintegrin and metalloproteinase with thrombospondin-like motifs 4/5 in the early-stage group was significantly higher than that in the middle-and latestage groups(P < 0.01).(2) The protein expression levels of each gene detected by ELISA in the early-, middle-and late-stage groups were significantly higher than those in the control group(P < 0.001). The protein expression of extracellular regulated protein kinase and matrix metalloproteinase 13 in the early-, middle-and late-stage groups was on a rise(P < 0.01). The protein expression of a disintegrin and metalloproteinase with thrombospondin-like motifs 4/5 in the early-stage group was significantly higher than that in the middle-and late-stage groups(P < 0.01).(3) Correlation analysis showed that there was a positive correlation between extracellular regulated protein kinase and matrix metalloproteinase 13 expression(P < 0.01), a disintegrin and metalloproteinase with thrombospondin-like motifs 4 and 5 expression levels were positively correlated(P < 0.01). The expression levels of a disintegrin and metalloproteinase with thrombospondin-like motifs 4/5 were negatively correlated with the expression levels of extracellular regulated protein kinase and matrix metalloproteinase 13(P < 0.05).(4)These results show that the expressions of extracellular regulated protein kinase, matrix metalloproteinase 13 and a disintegrin and metalloproteinase with thrombospondin-like motifs 4/5 are closely related to the clinical symptoms of knee osteoarthritis. The molecular mechanism of these factors in different stages of osteoarthritis is expected to provide reference for the clinical diagnosis and prognosis.
引文
[1]高世超,殷海波,刘宏潇,等.MAPK信号通路在骨关节炎发病机制中的研究进展[J].中国骨伤,2014,27(5):441-444.
[2]Yang CY,Chanalaris A,Troeberg L.ADAMTS and ADAM metalloproteinases in osteoarthritis-looking beyond the'usual suspects'.Osteoarthritis Cartilage. 2017;25(7):1000-1009.
[3]赵昌盛,钟群杰,林剑浩.中国膝关节骨关节炎流行病学调查现状[J].广东医学,2016,37(13):2050-2052.
[4]Tang X,Wang S,Zhan S,et al.The Prevalence of Symptomatic Knee Osteoarthritis in China:Results From the China Health and Retirement Longitudinal Study.Arthritis Rheumatol. 2016;68(3):648-653.
[5]Carr AJ,Robertsson O,Graves S,et al.Knee replacement. Lancet. 2012;379(9823):1331-1340.
[6]Pivec R,Johnson AJ,Mears SC,et al.Hip arthroplasty.Lancet. 2012;380(9855):1768-1777.
[7]Bijlsma JW,Berenbaum F,Lafeber FP.Osteoarthritis:an update with relevance for clinical practice.Lancet.2011;377(9783):2115-2126.
[8]Glyn-Jones S,Palmer AJ,Agricola R,et al.Osteoarthritis. Lancet.2015;386(9991):376-387.
[9]Verma P,Dalal K.ADAMTS-4 and ADAMTS-5:key enzymes in osteoarthritis.J Cell Biochem. 2011;112(12):3507-3514.
[10]王利,王宪,孔炜.新型金属蛋白酶ADAMTS家族的研究进展[J].生理科学进展,2008,39(1):49-52.
[11]Watanabe H.Aggrecan and its chondroitin sulfate in cartilage.Seikagaku.2008;80(1):28-32.
[12]中华医学会骨科学分会.骨关节炎诊治指南(2007年版)[J].中国矫形外科杂志,2014,22(3):287-288.
[13]Martel-Pelletier J,Boileau C,Pelletier JP,et al.Cartilage in normal and osteoarthritis conditions.Best Pract Res Clin Rheumatol. 2008;22(2):351-384.
[14]Braun HJ,Gold GE.Diagnosis of osteoarthritis:imaging.Bone. 2012;51(2):278-288.
[15]曾明珠,段戡,袁长深,等.不同分期骨关节炎滑膜细胞中COX-2 mRNA表达的差异[J].中国组织工程研究, 2014,18(7):1003-1008.
[16]王维山,史晨辉,何仁豪,等. MicroRNAs、u PA及MMP-3在骨关节炎患者滑膜组织中的表达及相关性[J].中国老年学杂志, 2014,34(4):871-873.
[17]胡毅,任云萍,张勇,等.持续被动运动条件下骨关节炎软骨细胞Erk活性及增殖[J].中国组织工程研究,2016,20(42):6265-6270.
[18]邓小兰,晋贞超,王健,等.ERK途径可能参与抗坏血酸诱导的神经干细胞向多巴胺能细胞分化[J].第三军医大学学报,2013,35(22):2430-2434.
[19]KELLGREN JH,LAWRENCE JS.Radiological assessment of osteo-arthrosis.Ann Rheum Dis. 1957;16(4):494-502.
[20]Joshi S,Platanias LC.Mnk kinase pathway:Cellular functions and biological outcomes.World J Biol Chem.2014;5(3):321-333.
[21]Fecher LA,Amaravadi RK,Flaherty KT.The MAPK pathway in melanoma.Curr Opin Oncol. 2008;20(2):183-189.
[22]王涛,殷红,廖江龙,等.骨关节炎与MAPK信号通路关系的研究概况[J].中国民族民间医药,2017,26(19):27-29,33.
[23]陈泽华,李楠.ERK1/2信号通路与骨关节炎软骨细胞活性及其治疗的相关性研究[J].风湿病与关节炎,2015,4(1):63-65,71.
[24]Prasadam I,Crawford R,Xiao Y.Aggravation of ADAMTS and matrix metalloproteinase production and role of ERK1/2 pathway in the interaction of osteoarthritic subchondral bone osteoblasts and articular cartilage chondrocytes--possible pathogenic role in osteoarthritis. J Rheumatol. 2012;39(3):621-634.
[25]付长龙,林洁,赵忠胜,等.电针抑制Ras/Raf/MEK1/2/ERK1/2信号通路介导骨关节炎软骨超微结构退变[J].中国组织工程研究, 2017,21(32):5134-5139.
[26]黄媛霞,徐海斌,郭春.IL-1β和MMP-13在兔骨关节炎模型软骨和滑液中的表达[J].西安交通大学学报(医学版), 2017,38(4):507-511,528.
[27]Fukui N,Miyamoto Y,Nakajima M,et al.Zonal gene expression of chondrocytes in osteoarthritic cartilage.Arthritis Rheum. 2008;58(12):3843-3853.
[28]魏凡华,张玉颖,于修平.基质金属蛋白酶ADAMTS的研究进展[J].中国细胞生物学学报,2014,36(8):1186-1192.
[29]王维山,史晨辉,李长俊,等.OA患者关节液u PA和MMP-3,9,13,14的表达水平与关节功能的相关性研究[J].中国骨质疏松杂志, 2014,20(6):602-605,610.
[30]Dreier R,Grassel S,Fuchs S,et al.Pro-MMP-9 is a specific macrophage product and is activated by osteoarthritic chondrocytes via MMP-3 or a MT1-MMP/MMP-13 cascade.Exp Cell Res. 2004;297(2):303-312.
[31]Jiang TJ. Detection of levels of serum and knee synovial fluid MMP-9and TNF-αin patients with osteoarthritis and its clinical significance.Medical Journal of National Defending Forces in Southwest China.2009;19(4):380-382.
[32]兰平文,刘跃洪,刘树平,等.不同病变阶段OA软骨细胞中TGF-β信号通路相关分子的表达规律[J].四川医学,2017,38(10):1128-1132.
[33]祁雷,姚运峰,李子煜,等.大鼠骨关节炎模型的建立及软骨组织中MMP-13和ADAMTS-5的表达[J].局解手术学杂志, 2018,27(3):157-163.
[34]Malfait AM,Liu RQ,Ijiri K,et al.Inhibition of ADAM-TS4 and ADAM-TS5prevents aggrecan degradation in osteoarthritic cartilage.J Biol Chem.2002;277(25):22201-22208.
[35]Song RH,Tortorella MD,Malfait AM,et al.Aggrecan degradation in human articular cartilage explants is mediated by both ADAMTS-4 and ADAMTS-5.Arthritis Rheum.2007;56(2):575-585.
[36]Tortorella MD,Malfait AM,Deccico C,et al.The role of ADAM-TS4(aggrecanase-1)and ADAM-TS5(aggrecanase-2)in a model of cartilage degradation.Osteoarthritis Cartilage. 2001;9(6):539-552.
[37]Stanton H,Rogerson FM,East CJ,et al.ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro. Nature. 2005;434(7033):648-652.
[38]Gendron C,Kashiwagi M,Lim NH,et al.Proteolytic activities of human ADAMTS-5:comparative studies with ADAMTS-4.J Biol Chem.2007;282(25):18294-18306.
[39]李保驰,王维山,董金波,等.MMP-3、MMP-13在骨性关节炎患者滑膜中的表达及意义[J].中国骨质疏松杂志, 2014,20(6):593-596,697.
[40]陈临新.膝关节骨关节炎的诊疗[J].中国全科医学, 2009,12(4):297-298.
[2]Yang CY,Chanalaris A,Troeberg L.ADAMTS and ADAM metalloproteinases in osteoarthritis-looking beyond the'usual suspects'.Osteoarthritis Cartilage. 2017;25(7):1000-1009.
[3]赵昌盛,钟群杰,林剑浩.中国膝关节骨关节炎流行病学调查现状[J].广东医学,2016,37(13):2050-2052.
[4]Tang X,Wang S,Zhan S,et al.The Prevalence of Symptomatic Knee Osteoarthritis in China:Results From the China Health and Retirement Longitudinal Study.Arthritis Rheumatol. 2016;68(3):648-653.
[5]Carr AJ,Robertsson O,Graves S,et al.Knee replacement. Lancet. 2012;379(9823):1331-1340.
[6]Pivec R,Johnson AJ,Mears SC,et al.Hip arthroplasty.Lancet. 2012;380(9855):1768-1777.
[7]Bijlsma JW,Berenbaum F,Lafeber FP.Osteoarthritis:an update with relevance for clinical practice.Lancet.2011;377(9783):2115-2126.
[8]Glyn-Jones S,Palmer AJ,Agricola R,et al.Osteoarthritis. Lancet.2015;386(9991):376-387.
[9]Verma P,Dalal K.ADAMTS-4 and ADAMTS-5:key enzymes in osteoarthritis.J Cell Biochem. 2011;112(12):3507-3514.
[10]王利,王宪,孔炜.新型金属蛋白酶ADAMTS家族的研究进展[J].生理科学进展,2008,39(1):49-52.
[11]Watanabe H.Aggrecan and its chondroitin sulfate in cartilage.Seikagaku.2008;80(1):28-32.
[12]中华医学会骨科学分会.骨关节炎诊治指南(2007年版)[J].中国矫形外科杂志,2014,22(3):287-288.
[13]Martel-Pelletier J,Boileau C,Pelletier JP,et al.Cartilage in normal and osteoarthritis conditions.Best Pract Res Clin Rheumatol. 2008;22(2):351-384.
[14]Braun HJ,Gold GE.Diagnosis of osteoarthritis:imaging.Bone. 2012;51(2):278-288.
[15]曾明珠,段戡,袁长深,等.不同分期骨关节炎滑膜细胞中COX-2 mRNA表达的差异[J].中国组织工程研究, 2014,18(7):1003-1008.
[16]王维山,史晨辉,何仁豪,等. MicroRNAs、u PA及MMP-3在骨关节炎患者滑膜组织中的表达及相关性[J].中国老年学杂志, 2014,34(4):871-873.
[17]胡毅,任云萍,张勇,等.持续被动运动条件下骨关节炎软骨细胞Erk活性及增殖[J].中国组织工程研究,2016,20(42):6265-6270.
[18]邓小兰,晋贞超,王健,等.ERK途径可能参与抗坏血酸诱导的神经干细胞向多巴胺能细胞分化[J].第三军医大学学报,2013,35(22):2430-2434.
[19]KELLGREN JH,LAWRENCE JS.Radiological assessment of osteo-arthrosis.Ann Rheum Dis. 1957;16(4):494-502.
[20]Joshi S,Platanias LC.Mnk kinase pathway:Cellular functions and biological outcomes.World J Biol Chem.2014;5(3):321-333.
[21]Fecher LA,Amaravadi RK,Flaherty KT.The MAPK pathway in melanoma.Curr Opin Oncol. 2008;20(2):183-189.
[22]王涛,殷红,廖江龙,等.骨关节炎与MAPK信号通路关系的研究概况[J].中国民族民间医药,2017,26(19):27-29,33.
[23]陈泽华,李楠.ERK1/2信号通路与骨关节炎软骨细胞活性及其治疗的相关性研究[J].风湿病与关节炎,2015,4(1):63-65,71.
[24]Prasadam I,Crawford R,Xiao Y.Aggravation of ADAMTS and matrix metalloproteinase production and role of ERK1/2 pathway in the interaction of osteoarthritic subchondral bone osteoblasts and articular cartilage chondrocytes--possible pathogenic role in osteoarthritis. J Rheumatol. 2012;39(3):621-634.
[25]付长龙,林洁,赵忠胜,等.电针抑制Ras/Raf/MEK1/2/ERK1/2信号通路介导骨关节炎软骨超微结构退变[J].中国组织工程研究, 2017,21(32):5134-5139.
[26]黄媛霞,徐海斌,郭春.IL-1β和MMP-13在兔骨关节炎模型软骨和滑液中的表达[J].西安交通大学学报(医学版), 2017,38(4):507-511,528.
[27]Fukui N,Miyamoto Y,Nakajima M,et al.Zonal gene expression of chondrocytes in osteoarthritic cartilage.Arthritis Rheum. 2008;58(12):3843-3853.
[28]魏凡华,张玉颖,于修平.基质金属蛋白酶ADAMTS的研究进展[J].中国细胞生物学学报,2014,36(8):1186-1192.
[29]王维山,史晨辉,李长俊,等.OA患者关节液u PA和MMP-3,9,13,14的表达水平与关节功能的相关性研究[J].中国骨质疏松杂志, 2014,20(6):602-605,610.
[30]Dreier R,Grassel S,Fuchs S,et al.Pro-MMP-9 is a specific macrophage product and is activated by osteoarthritic chondrocytes via MMP-3 or a MT1-MMP/MMP-13 cascade.Exp Cell Res. 2004;297(2):303-312.
[31]Jiang TJ. Detection of levels of serum and knee synovial fluid MMP-9and TNF-αin patients with osteoarthritis and its clinical significance.Medical Journal of National Defending Forces in Southwest China.2009;19(4):380-382.
[32]兰平文,刘跃洪,刘树平,等.不同病变阶段OA软骨细胞中TGF-β信号通路相关分子的表达规律[J].四川医学,2017,38(10):1128-1132.
[33]祁雷,姚运峰,李子煜,等.大鼠骨关节炎模型的建立及软骨组织中MMP-13和ADAMTS-5的表达[J].局解手术学杂志, 2018,27(3):157-163.
[34]Malfait AM,Liu RQ,Ijiri K,et al.Inhibition of ADAM-TS4 and ADAM-TS5prevents aggrecan degradation in osteoarthritic cartilage.J Biol Chem.2002;277(25):22201-22208.
[35]Song RH,Tortorella MD,Malfait AM,et al.Aggrecan degradation in human articular cartilage explants is mediated by both ADAMTS-4 and ADAMTS-5.Arthritis Rheum.2007;56(2):575-585.
[36]Tortorella MD,Malfait AM,Deccico C,et al.The role of ADAM-TS4(aggrecanase-1)and ADAM-TS5(aggrecanase-2)in a model of cartilage degradation.Osteoarthritis Cartilage. 2001;9(6):539-552.
[37]Stanton H,Rogerson FM,East CJ,et al.ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro. Nature. 2005;434(7033):648-652.
[38]Gendron C,Kashiwagi M,Lim NH,et al.Proteolytic activities of human ADAMTS-5:comparative studies with ADAMTS-4.J Biol Chem.2007;282(25):18294-18306.
[39]李保驰,王维山,董金波,等.MMP-3、MMP-13在骨性关节炎患者滑膜中的表达及意义[J].中国骨质疏松杂志, 2014,20(6):593-596,697.
[40]陈临新.膝关节骨关节炎的诊疗[J].中国全科医学, 2009,12(4):297-298.