阿仑膦酸钠对IL-1β体外诱导培养的大鼠膝关节软骨细胞影响的实验研究  

EFFECT OF ALENDRONATE ON INTERLEUKIN-1β INDUCED CHONDROCYTES OF RAT IN VITRO

作　　者：王哲彦[1] 王文雅[1] 张柳[2] 田发明[2] 程潭[2] 杨林[3] 

机构地区：[1]华北煤炭医学院病理教研室,河北唐山063000 [2]华北煤炭医学院附属医院骨外科 [3]成都大学医护学院药理教研室

出　　处：《中国修复重建外科杂志》2011年第1期50-55,共6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：目的二膦酸盐类药物可抑制骨重塑,通过观察阿仑膦酸钠(alendronate,ALN)对IL-1β体外诱导培养的大鼠膝关节软骨细胞的影响,探讨ALN治疗骨性关节炎(osteoarthritis,OA)的可行性。方法取15只1月龄SPF级SD大鼠(雌雄不限,体重100～150g)膝关节软骨,体外培养软骨细胞并传至第3代。倒置相差显微镜下观察细胞形态并进行鉴定。将第3代软骨细胞分为3组:空白对照组(A组)软骨细胞用常规DMEM完全培养液培养5d;IL-1β诱导组(B组)软骨细胞以10ng/mL重组人IL-1β培养2d后更换为常规DMEM完全培养液培养3d;IL-1β诱导后加ALN培养组(C组)软骨细胞以10ng/mL重组人IL-1β培养2d后更换为浓度为1×10-6mol/L的ALN培养3d。培养后取各组细胞进行免疫细胞化学染色及实时PCR检测,观察细胞中Ⅱ型胶原(collagen typeⅡ,ColⅡ)、基质金属蛋白酶13(matrix metalloproteinase13,MMP-13)和β-连环蛋白(β-catenin)的表达水平。结果甲苯胺蓝染色示培养的细胞呈异染性,证实为软骨细胞。免疫细胞化学染色显示:A、B、C组ColⅡ表达积分吸光度(IA)值分别为15.3770±0.5718、5.4632±0.4504、10.2907±0.4992,C组高于B组,但低于A组,差异有统计学意义(P<0.05);MMP-13表达IA值分别为2.7775±0.1996、6.9981±0.3297、3.0686±0.2056,C组明显低于B组(P<0.05),但与A组比较差异无统计学意义(P>0.05);β-catenin表达IA值分别为4.3903±0.5519、11.7999±0.3487、6.6117±0.3818,C组低于B组,但高于A组,差异均有统计学意义(P<0.05)。实时PCR检测示,C组ColⅡmRNA表达高于B组,MMP-13及β-catenin的mRNA表达低于B组,比较差异均有统计学意义(P<0.05);ColⅡ和β-catenin mRNA表达高于A组,MMP-13mRNA表达低于A组,比较差异均有统计学意义(P<0.05)。结论 ALN可能通过抑制IL-1β诱导的大鼠膝关节软骨细胞中ColⅡ的降解并下调MMP-13及β-catenin因子的表达,对软骨细胞具有一定的保护作用。Objective To investigate the feasibility of alendronate（ALN） in treating osteoarthritis（OA） by observing the effects of ALN on interleukin 1β（IL-1β） induced chondrocytes of rat in vitro.Methods The chondrocytes of knee articular surface from 15 SD rats（1-month-old,female or male,weighing 100-150 g） were cultured.The chondrocytes were observed by inverted phase contrast microscope and identified by toluidine blue staining and HE staining.The third passage chondrocytes were divided into 3 groups：the chondrocytes were cultured with DMEM for 5 days in group A,with 10 ng/mL IL-1β for 2 days and with DMEM for 3 days in group B,and with 10 ng/mL IL-1β for 2 days and with 1 × 10-6 mol/L ALN for 3 days in group C.Immunocytochemistry and real-time PCR were performed to determine the expression levels of collagen type II（Col II）,matrix metalloproteinase 13（MMP-13）,and β-catenin.Results Toluidine blue staining proved that the cultured cells were chondrocytes.The integrated absorbency（IA） value of Col II in group C（10.290 7± 0.499 2） was lower than that in group A（15.377 0± 0.571 8） and higher than that in group B（5.463 2± 0.450 4）,showing significant differences（P 0.05）.The IA value of MMP-13 in group C（3.068 6± 0.205 6） was significantly lower than that in group B（6.998 1± 0.329 7,P 0.05）,but there was no significant differenc when compared with group A（2.777 5± 0.199 6,P 0.05）.The IA value of β-catenin in group C（6.611 7± 0.381 8） was lower than that in group B（11.799 9± 0.348 7） and higher than that in group A（4.390 3± 0.551 9）,showing significant differences（P 0.05）.The mRNA expression of Col II in group C was significantly higher than those in groups A and B（P 0.05）,the mRNA expression of MMP-13 in group C was significantly lower than that in group B（P 0.05） but there was no significant difference when compared with group A（P 0.05）.The mRNA expression of β-catenin in group C was significantly lower than that in group B

关 键 词：骨性关节炎 阿仑膦酸钠 软骨细胞 Ⅱ型胶原 基质金属蛋白酶13 β-连环蛋白大鼠 

分 类 号：R965[医药卫生—药理学]