骨桥蛋白-CD44配体受体反应对人膝骨关节炎软骨细胞透明质酸表达的影响  

Effect of ligand-receptor interaction of osteopontin-CD44 on the expression of hyaluronic acid in human knee osteoarthritic chondrocytes in vitro

作　　者：袁宇豪 刘擎[1,2] 张方杰 罗伟[1] YUAN Yuhao;LIU Qing;ZHANG Fangjie;LUOWei(Department of Orthopedics,Xiangya Hospital,Central South University,Changsha 410008;Department of Spine Surgery,Second Xiangya Hospital,Central South University,Changsha 410011;Department of Emergency,Xiangya Hospital,Central South University,Changsha 410008,China)

机构地区：[1]中南大学湘雅医院骨科,长沙410008 [2]中南大学湘雅二医院脊柱外科,长沙410011 [3]中南大学湘雅医院急诊科,长沙410008

出　　处：《中南大学学报（医学版）》2020年第9期1015-1023,共9页Journal of Central South University ：Medical Science

摘　　要：目的:通过干预体外培养人膝骨关节炎(osteoarthritis,OA)软骨细胞中的骨桥蛋白(osteopontin,OPN)与CD44配体受体反应,观察其对软骨细胞透明质酸(hyaluronic acid,HA)表达的影响。方法:分别从膝OA患者和恶性肿瘤患者的膝关节软骨组织中获取OA软骨细胞和正常软骨细胞,对正常软骨细胞和OA软骨细胞进行检测,然后对OA软骨细胞进行干预分析。将OA软骨细胞分为如下4组:阴性对照组,用完全培基正常培养,不加任何干预试剂;OPN干预组,以人重组OPN(recombinant human OPN,rhOPN)干预24 h;CD44阻断组,预先采用CD44受体特异性拮抗剂干预软骨细胞1 h以阻断OPN-CD44结合后,再加入rhOPN干预23 h;CD44同型组,预先采用CD44干预软骨细胞1 h后,加入rhOPN干预23 h。此外,在OPN-CD44轴线的研究中,OA软骨细胞又分为OA阴性对照组、沉默OPN(si-OPN)干预组、rhOPN干预组和rhOPN+CD44抗体组(rhOPN+CD44 Ab组)。采用蛋白质印迹法、real-time PCR和ELISA分别对OPN,CD44,透明质酸合酶(hyaluronate synthase,HAS)和HA的表达水平进行检测。结果:OA软骨细胞中OPN,CD44和HAS1的蛋白质表达水平及HA的分泌水平均高于正常软骨细胞;与OPN干预组相比,CD44阻断组HAS1,HAS2,HAS3和HA的表达水平均降低,差异均有统计学意义(均P<0.05);与OPN干预组相比,CD44同型组HAS1,HAS2,HAS3和HA的表达水平差异均无统计学意义(均P>0.05)。对OPN-CD44轴线的研究结果显示:与OA阴性对照组相比,rhOPN干预组CD44的表达降低,但HAS1的蛋白质和m RNA水平却显著上调,差异均有统计学意义(均P<0.05);与OA阴性对照组相比,si-OPN干预组CD44的表达上调,但HAS1的蛋白质和mRNA水平却受到抑制,差异均有统计学意义(均P<0.05);与OA阴性对照组相比,rhOPN+CD44 Ab组HAS1的蛋白质和mRNA水平也受到显著抑制,差异均有统计学意义(均P<0.05)。结论:OA软骨细胞内OPN对HAS1的表达具有促进作用,且OPN是通过与CD44配体受体反应刺激软骨细胞HAObjective:To investigate the effect of ligand-receptor interaction of osteopontin(OPN)-CD44 on the expression of hyaluronic acid(HA)in the cultured human knee osteoarthritis(OA)chondrocytes via interfering the reaction between OPN and CD44 ligand-receptor.Methods:The OA chondrocytes and normal chondrocytes were obtained from knee joint cartilage tissues in the patients with knee OA and malignant tumor respectively.The normal chondrocytes and OA chondrocytes were detected and analyzed,and then the intervention analysis of OA chondrocytes was carried out.The OA chondrocytes were divided into 4 groups:a negative control group,which was cultured with complete medium without any molecular intervention reagent;an OPN intervention group,which was cultured with recombinant human OPN(rh OPN)for 24 hours;a CD44 blocking group,which were pretreated with CD44 receptor specific antagonist for 1 hour to block the binding of OPNCD44,and then treated with rh OPN for 23 hours;a CD44 homotype group,which was pretreated with CD44 for 1 hour and then treated with rh OPN for 23 hours.In addition,the study for OPN-CD44 axis was also divided into 4 groups:an OA-negative control group(OA-NC group),a si-OPN intervention group,a rh OPN intervention group,and a rh OPN+CD44 antibody(Ab)group.Western blotting,real-time PCR,and enzyme linked immunosorbent assay(ELISA)were used to detect the protein and m RNA expression levels of OPN,CD44,hyaluronate synthase(HAS),and HA,respectively.Results:The protein expression levels of OPN,CD44,and HAS1 and the secretion levels of HA in the OA chondrocytes were higher than those in the normal chondrocytes.Compared with the OPN intervention group,the expression levels of HAS1,HAS2,HAS3 and HA in the CD44 blocking group were lower than those in OPN intervention group(all P<0.05);but there was no significant difference in the expression levels of HAS1,HAS2,HAS3 and HA between the CD44 homotype group and the OPN intervention group(all P>0.05).The results of OPN-CD44 axis study showed that:compared with the OA

关 键 词：骨桥蛋白 CD44 软骨细胞 透明质酸 透明质酸合酶 膝骨关节炎 

分 类 号：R684.3[医药卫生—骨科学]