Fortier L A, Nixon A J, Mohammed H O, Lust G
Comparative Orthopaedics Laboratory, College of Veterinary Medicine, Cornell University, Ithaca, NY 14583, USA.
Am J Vet Res. 1997 Jan;58(1):66-70.
To determine the effects of transforming growth factor-beta 1 (TGF-beta 1) on the synthesis of DNA, collagen, and proteoglycans (PG) by equine chondrocytes.
Articular cartilage obtained from multiple joints of a 4-month-old foal.
Chondrocytes were isolated by collagenase digestion, cultured in monolayer, trypsinized, and implanted at a cellular density of 10 x 10(6) chondrocytes/ml in a three-dimensional fibrin matrix. Chondrocytes in culture were supplemented with TGF-beta 1 at concentrations of 0, 1, 5, or 10 ng/ml in serum-free medium or medium containing fetal bovine serum (FBS). Total PG accumulation, [35S]-labeled PG synthesis, PG monomer hydrodynamic size, type II collagen production, total DNA content, and [3H]thymidine incorporation into DNA were determined at 7 and 14 days of culture.
Chondrocytes maintained a rounded phenotype, dedifferentiating slightly to a more fibroblastic appearance only in medium containing FBS and 10 ng of TGF-beta 1/ml. Type II collagen immunoreaction on day 14 was decreased in the pericellular matrix in cultures containing FBS and 1, 5, and 10 ng of TGF-beta 1/ml, and in all serum-free culture conditions compared to FBS and 0 ng of TGF-beta 1/ml. Total proteoglycan accumulation and [35S]-labeled proteoglycan synthesis in cultures on days 7 and 14 were increased by the addition of exogenous TGF-beta 1 in serum-free conditions and decreased by TGF-beta 1 in FBS-supplemented conditions. Calculation of the partition coefficients for PG indicated that there was synthesis of low molecular weight PG in serum-free conditions and larger sized proteoglycans in FBS-supplemented conditions. Proteoglycan molecular size was unchanged by the addition of TGF-beta 1. Total DNA content of chondrocytes increased with the addition of TGF-beta 1 in FBS-supplemented conditions and decreased in serum-free conditions.
In a solid three-dimensional fibrin matrix, the effects of TGF-beta 1 on chondrocyte biological activity depend on the culture duration and on the presence of FBS in the medium. Stimulatory effects of TGF-beta 1 were most pronounced in serum-free culture conditions with high concentration of TGF-beta 1 (5 and 10 ng/ml) on day 7 and with low concentration of TGF-beta 1 (1 ng/ml) on day 14.
TGF-beta 1 may not be a suitable growth factor for enhancement of equine articular grafting in sites exposed to serum.
确定转化生长因子β1（TGF-β1）对马软骨细胞DNA、胶原蛋白和蛋白聚糖（PG）合成的影响。
从一匹4月龄马驹的多个关节获取的关节软骨。
通过胶原酶消化分离软骨细胞，单层培养，胰蛋白酶处理后，以10×10⁶个软骨细胞/毫升的细胞密度植入三维纤维蛋白基质中。在无血清培养基或含胎牛血清（FBS）的培养基中，分别添加浓度为0、1、5或10纳克/毫升的TGF-β1培养软骨细胞。在培养7天和14天时，测定总PG积累量、[³⁵S]标记的PG合成量、PG单体流体动力学大小、II型胶原蛋白产量、总DNA含量以及[³H]胸腺嘧啶核苷掺入DNA的量。
软骨细胞维持圆形表型，仅在含FBS和10纳克TGF-β1/毫升的培养基中稍有去分化，呈现更成纤维细胞样外观。与含FBS和0纳克TGF-β1/毫升的培养基相比，在含FBS且添加1、5和10纳克TGF-β1/毫升的培养基中以及所有无血清培养条件下，培养14天时细胞周围基质中的II型胶原蛋白免疫反应降低。在无血清条件下添加外源性TGF-β1可增加培养7天和14天时的总蛋白聚糖积累量和[³⁵S]标记的蛋白聚糖合成量，而在添加FBS的条件下，TGF-β1则使其降低。PG分配系数的计算表明，在无血清条件下合成低分子量PG，在添加FBS的条件下合成较大尺寸的蛋白聚糖。添加TGF-β1后蛋白聚糖分子大小未改变。在添加FBS的条件下，添加TGF-β1可使软骨细胞的总DNA含量增加，在无血清条件下则降低。
在固态三维纤维蛋白基质中，TGF-β1对软骨细胞生物学活性的影响取决于培养时间和培养基中FBS的存在。在无血清培养条件下，高浓度TGF-β1（5和10纳克/毫升）在第7天以及低浓度TGF-β1（1纳克/毫升）在第14天的刺激作用最为明显。
TGF-β1可能不是促进血清暴露部位马关节移植的合适生长因子。
