阿仑膦酸钠体外对软骨细胞的影响及对兔前交叉韧带切断后关节软骨和软骨下骨的作用  

IN VITRO EFFECT OF ALENDRONATE ON CHONDROCYTES AND ARTICULAR CARTILAGE AND SUBCHONDRAL BONE IN RABBIT ANTERIOR CRUCIATE LIGAMENT TRANSECTION MODEL

作　　者：胡宏宇[1] 张柳[1] 李彬[1] 张尹娜[1] 刘晓宁[1] 田发明[1] 程潭[1] 王哲彦[1] 

机构地区：[1]华北煤炭医学院附属医院骨科,河北唐山063000

出　　处：《中国修复重建外科杂志》2009年第12期1474-1481,共8页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：河北省科技领军人才创新基金项目(06547008D-8)~~

摘　　要：目的探讨阿仑膦酸钠(alendronate,ALN)对受IL-1β干预后软骨细胞的影响,以及对前交叉韧带切断术(anterior cruciate ligament transection,ACLT)后兔骨性关节炎模型关节软骨和软骨下骨的保护作用。方法取3月龄雄性日本大耳白兔关节软骨采用酶消化法分离软骨细胞,并进行体外培养。取第3代软骨细胞随机分为3组:ALN组(A1组)及IL-1β组(B1组),采用含10ng/mL人重组IL-1β的DMEM完全培养液培养2d后,分别更换含或不含1×10-6mol/L ALN的DMEM完全培养液培养3d;空白组(C1组)细胞以DMEM完全培养液培养5d。取各组细胞行ColⅡ及MMP-13免疫细胞化学染色观察及实时RT-PCR检测。另取24只3月龄雄性日本大耳白兔,随机分为3组(n=8)。ACLT+ALN组(A2组)及ACLT组(B2组)制备ACLT模型,假手术组(C2组)以同法显露前交叉韧带后缝合切口。术后4d A2组以10μg/(kg·d)皮下注射浓度为25μg/mL的ALN,B2组和C2组予等量生理盐水。8周后处死动物行膝关节大体观察,取股骨内侧髁行组织学观察及ColⅡ及MMP-13免疫组织化学染色观察,对胫骨进行软骨下骨组织形态学分析。结果C1组软骨细胞胞浆中见ColⅡ免疫细胞化学染色表达呈强阳性,A1组呈阳性表达,B1组少见阳性显色。A1组积分吸光度(IA)值高于B1组(P<0.05),与C1组差异无统计学(P>0.05)。C1组软骨细胞胞浆中未见MMP-13免疫细胞化学染色阳性表达,A1组呈阳性表达,B1组呈强阳性表达。A1组IA值低于B1组(P<0.05),但高于C1组(P<0.05)。实时RT-PCR检测示A1组ColⅡmRNA表达高于B1组(P<0.05),与C1组差异无统计学意义(P>0.05);MMP-13mRNA表达低于B1组(P<0.05),但高于C1组(P<0.05)。体内实验8周后C2组膝关节面无溃疡,A2组膝关节面有少量溃疡,B2组关节面溃疡较多并有全层溃疡。A2组Mankin评分低于B2组(P<0.05),但高于C2组(P<0.05)。免疫组织化学染色见C2组关节软骨中ColⅡ蛋白表达呈强阳性,A2组呈阳性表达,B2组少见阳性显色;A2组IA值�Objective To examine the effects of alendronate （ALN） on IL-1β-stimulated chondrocyte of rabbit in vitro and on cartilage and subchondral bone in rabbit osteoarthritis （OA） induced by anterior cruciate ligament transection （ACLT）. Methods The chondrocytes from articular surface of healthy 3-month-old Japanese White rabbits were obtained by the method of enzyme digestion and cultured in vitro. The third generation chondrocytes were assigned into three groups：the chondrocytes were cultured in DMEM medium with 10 ng/mL IL-1β for 2 days,subsequently with （ALN group,group A1） or without （IL-1β group,group B1） 1 × 10-6 mol/L ALN for 3 days; the chondrocytes in vacant group （group C1） were cultured in DMEM medium for 5 days. The expression of Col II and MMP-13 were analyzed by immunocytochemical staining observation and real time RT-PCR test. Another twenty-four 3-month-old male Japanese White rabbits were randomized into three groups （n=8 per group）. The OA model was made by ACLT in ACLT＋ALN group （group A2） and ACLT group （group B2）; the joint cave was sutured after exposure of ACL in sham group （group C2）. After 4 days,the rabbits of group A2 received the subcutaneous injection of ALN at a dosage of 10 μg/（kg·d） for 8 weeks. Rabbits of group B2 and C2 received equal normal saline treatment. After 8 weeks,the rabbits were executed. The macro-pathologic changes of right knee joints were observed,so were the histological changes of femoral condyles. Expression levels of Col II and MMP-13 were detected by immunohistochemical staining. The bone histomorphometry analysis was applied to subchondral bone of proximal tibia. Results In vitro,the Col II immunocytochemical staining showed intensely positive staining in group C1,and the intensity of staining was slightly decreased in group A1,but the intensity of Col II immunocytochemical staining was extremely lower in the group B1. The integrated absorbance （IA） value for Col II in group A1 was significantly higher than

关 键 词：骨性关节炎 阿仑膦酸钠 ColⅡ MMP-13 兔 

分 类 号：R684.3[医药卫生—骨科学] R681.3[医药卫生—外科学]