Caron J P, Tardif G, Martel-Pelletier J, DiBattista J A, Geng C, Pelletier J P
Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing 48824, USA.
Am J Vet Res. 1996 Nov;57(11):1631-4.
To determine whether matrix metalloprotease 13 (MMP-13; collagenase 3) is produced by equine chondrocytes and to investigate modulation of its expression by recombinant human interleukin 1 beta (rhIL-1 beta) and corticosteroids.
Equine chondrocytes in monolayer culture were stimulated with rhIL-1 beta. Total RNA was extracted, purified, and reverse transcribed into DNA. Using appropriate primers, a putative MMP-13 fragment was amplified by polymerase chain reaction, and cloned into a bacterial vector. The resultant fragment was purified and sequenced, then was used to prepare a digoxigenin-labeled cRNA probe. Monolayer cultures of first-passage chondrocytes were treated with rhIL-1 beta in the presence or absence of dexamethasone (10(-6)M) or methylprednisolone acetate (10(-9)M to 10(-5)M), in addition to positive and negative controls. Cellular RNA was extracted and resolved on agarose gels and subjected to northern blot analysis, using the equine MMP-13 probe.
Reverse transcriptase-polymerase chain reaction enabled isolation of a 0.6-kb fragment of equine MMP-13 cDNA that had 93% homology with the human MMP-13 cDNA sequence. rhIL-1 significantly stimulated MMP-13 expression in the chondrocytes. Methylprednisolone acetate inhibited the stimulatory effects of rhIL-1 in dose-dependent manner that was statistically significant at 10(-5)M.
Novel information was gained on the existence of MMP-13 and its expression in equine chondrocytes, which suggests a possible role for this enzyme in matrix degradation in horses with arthritis.
确定马软骨细胞是否产生基质金属蛋白酶13（MMP - 13；胶原酶3），并研究重组人白细胞介素1β（rhIL - 1β）和皮质类固醇对其表达的调节作用。
用rhIL - 1β刺激单层培养的马软骨细胞。提取、纯化总RNA并逆转录成DNA。使用合适的引物，通过聚合酶链反应扩增出一个假定的MMP - 13片段，并克隆到细菌载体中。纯化并测序所得片段，然后用于制备地高辛标记的cRNA探针。除了阳性和阴性对照外，将第一代软骨细胞的单层培养物在有或没有地塞米松（10⁻⁶M）或醋酸甲泼尼龙（10⁻⁹M至10⁻⁵M）存在的情况下用rhIL - 1β处理。提取细胞RNA，在琼脂糖凝胶上进行分离，并使用马MMP - 13探针进行Northern印迹分析。
逆转录酶 - 聚合酶链反应能够分离出一个0.6 kb的马MMP - 13 cDNA片段，该片段与人MMP - 13 cDNA序列具有93%的同源性。rhIL - 1显著刺激软骨细胞中MMP - 13的表达。醋酸甲泼尼龙以剂量依赖性方式抑制rhIL - 1的刺激作用，在10⁻⁵M时具有统计学意义。
获得了关于MMP - 13在马软骨细胞中的存在及其表达的确切信息，这表明该酶在患有关节炎的马的基质降解中可能发挥作用。
