Balkman C E, Nixon A J
Comparative Orthopaedics Laboratory, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.
Am J Vet Res. 1998 Jan;59(1):30-6.
To clone and determine molecular structure of equine stromelysin 1 (matrix metalloproteinase 3) and examine stromelysin expression in articular cartilage. SAMPLES AND PROCEDURE: Total RNA was harvested from equine arthritic cartilage specimens and was used for reverse transcription and polymerase chain reaction amplification to develop overlapping complementary DNA (cDNA) clones. Four cDNA sequences were ligated into plasmid (pGEM3Z) constructs and subcloned into bacterial expression vectors, and sequence was determined by automated dye terminator sequencing. Stromelysin mRNA expression was assessed in normal and arthritic cartilage and synovium by northern blotting. Interleukin 1 (IL-1) regulation of stromelysin transcriptional activity in articular chondrocytes cultured in the presence of 0, 20, and 50 ng of IL-1 alpha/ml was assessed by northern blotting of total RNA isolated from the cell layer and probed with 32P-labeled stromelysin cDNA.
4 overlapping clones provided the full-length cDNA sequence of equine stromelysin, including portions of untranslated 5' and 3' regions, and the entire translated portion coding for the stromelysin prepropeptide. The coding region of 1,431 base pairs was well conserved between species, with 86, 83, and 78% sequence homology to that of human, rabbit, and mouse stromelysin, respectively. Predicted amino-acid (AA) sequence data indicated highly conserved features. Comparison of the equine AA sequence revealed 89, 88, and 84% homology to the AA structure in human, rabbit, and mouse stromelysin, respectively. Minimal stromelysin mRNA expression was evident in normal cartilage and synovium, and increased expression was evident in arthritic cartilage. Marked dose-dependent up-regulation of stromelysin transcriptional activity was evident in chondrocyte cultures exposed to 20 and 50 ng of IL-1/ml.
Stromelysin DNA sequence in horses is similar to that in people and rodents. Constitutive stromelysin message amounts in normal cartilage and synovium are low, but considerably increased in arthritic cartilage and in chondrocytes exposed to IL-1.
克隆并确定马基质溶解素1（基质金属蛋白酶3）的分子结构，并检测其在关节软骨中的表达。样本与方法：从马关节炎软骨标本中提取总RNA，用于逆转录和聚合酶链反应扩增，以构建重叠互补DNA（cDNA）克隆。将4个cDNA序列连接到质粒（pGEM3Z）构建体中，并亚克隆到细菌表达载体中，通过自动染料终止测序法确定序列。通过Northern印迹法评估正常和关节炎软骨及滑膜中基质溶解素mRNA的表达。通过对从细胞层分离的总RNA进行Northern印迹分析，并用32P标记的基质溶解素cDNA进行探针杂交，评估在含有0、20和50 ng白细胞介素1α/ml的条件下培养的关节软骨细胞中白细胞介素1（IL-1）对基质溶解素转录活性的调节作用。
4个重叠克隆提供了马基质溶解素的全长cDNA序列，包括部分5'和3'非翻译区，以及编码基质溶解素前原肽的整个翻译区。1431个碱基对的编码区在物种间高度保守，与人、兔和小鼠基质溶解素的序列同源性分别为86%、83%和78%。预测的氨基酸（AA）序列数据显示出高度保守的特征。马AA序列与人类、兔和小鼠基质溶解素的AA结构的同源性分别为89%、88%和84%。在正常软骨和滑膜中基质溶解素mRNA表达极低，而在关节炎软骨中表达增加。在暴露于20和50 ng IL-1/ml的软骨细胞培养物中，基质溶解素转录活性明显呈剂量依赖性上调。
马的基质溶解素DNA序列与人及啮齿动物的相似。正常软骨和滑膜中基质溶解素的基础表达量较低，但在关节炎软骨和暴露于IL-1的软骨细胞中显著增加。
