核心蛋白聚糖抑制人软骨细胞MMP13基因表达的体外研究

目的体外原代细胞培养人正常关节软骨细胞,观察不同浓度的外源性核心蛋白聚糖(decorin,DCN)对软骨细胞基质金属蛋白酶13(matrix metalloproteases 13,MMP13)表达的影响。方法采用胰蛋白酶和Ⅱ型胶原酶结合消化法分离培养人透明软骨细胞,观察体外培养的软骨细胞形态,阿尔新蓝染色和Ⅱ型胶原免疫组化鉴定透明软骨细胞,从基因水平(实时定量PCR)检测不同浓度的外源性DCN(对照组:0,A组:2.5μg/ml,B组:25μg/ml,C组:250μg/ml)作用下透明软骨细胞对MMP13、X型胶原(Collagen X)表达的影响。结果①正常透明软骨细胞培养呈多角型,扁平状,换液培养5 d时,细胞呈多边形、星形等,排列紧密呈"铺石板"样。②本法分离培养的原代透明软骨细胞的阿尔新蓝染色和Ⅱ型胶原免疫组化均为阳性。③随着外源性DCN浓度(0～250μg/ml)的不断增大,Collagen X的表达明显降低(P<0.01),且成剂量依赖关系(A:0.78±0.03,B:0.71±0.04,C:0.63±0.01),MMP13的表达则在DCN高浓度组(250μg/ml)时明显降低(0.85±0.02,P<0.01)。结论外源性DCN抑制透明软骨细胞的Collagen X的表达且成剂量依赖关系,高浓度(250μg/ml)对MMP13的表达起明显抑制作用。表明外源性DCN可能通过抑制透明软骨细胞肥大化抑制软骨细胞MMP13的表达。

Decorin inhibits MMP13 gene expression in human chondrocytes:an in vitro study

Objective To observe the effect of different exogenous decorin(DCN) levels on matrix metalloproteases 13(MMP13) gene expression in normal human articular chondrocytes primarily cultured in vitro.Methods Human hyaline chondrocytes were isolated and cultured with trypsin and type 2 collagenase in combination with digestion to observe their morphology.The cells were identified with Alcian blue staining and type 2 collagen immunohistochemistry.Effect of different exogenous DCN levels(0,2.5,25 and 250 μg/ml) on expression of MMP13 and collagen X in hyaline chondrocytes was detected by RT-PCR.Results The cultured normal hyaline chondrocytes were polygonal and flat in shape,and turned into polygonal and star in shape in 5 d after they were cultured with another medium and closely arranged as a "stone shop".Alcian blue staining and type 2 collagen immunohistochemistry were positive for primarily cultured hyaline chondrocytes.The expression of collagen X was significantly decreased(0.78±0.03,0.71±0.04,0.63±0.01) with the increasing exogenous DCN level(0 to 250 μg/ml) in a dose-dependent manner(P<0.01),while the MMP13 expression significantly decreased(0.85±0.02) at the highest DCN level(250 μg/ml,P<0.01).Conclusion Exogenous DCN inhibits expression of collagen X in hyaline chondrocytes in a dose-dependent manner and high DCN level(250 μg/ml) plays a significant role in inhibiting the expression of MMP13,which indicates that exogenous DCN inhibits the MMP13 expression in hyaline chondrocytes by suppressing their hypertrophy.