Stewart Allison A, Byron Christopher R, Pondenis Holly C, Stewart Matthew C
Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802, USA.
Am J Vet Res. 2008 Aug;69(8):1013-21. doi: 10.2460/ajvr.69.8.1013.
To determine whether expansion of equine mesenchymal stem cells (MSCs) by use of fibroblast growth factor-2 (FGF-2) prior to supplementation with dexamethasone during the chondrogenic pellet culture phase would increase chondrocytic matrix markers without stimulating a hypertrophic chondrocytic phenotype.
MSCs obtained from 5 young horses.
First-passage equine monolayer MSCs were supplemented with medium containing FGF-2 (0 or 100 ng/mL). Confluent MSCs were transferred to pellet cultures and maintained in chondrogenic medium containing 0 or 10(7)M dexamethasone. Pellets were collected after 1, 7, and 14 days and analyzed for collagen type II protein content; total glycosaminoglycan content; total DNA content; alkaline phosphatase (ALP) activity; and mRNA of aggrecan, collagen type II, ALP, and elongation factor-1alpha.
Treatment with FGF-2, dexamethasone, or both increased pellet collagen type II content, total glycosaminoglycan content, and mRNA expression of aggrecan. The DNA content of the MSC control pellets decreased over time. Treatment with FGF-2, dexamethasone, or both prevented the loss in pellet DNA content over time. Pellet ALP activity and mRNA were increased in MSCs treated with dexamethasone and FGF-2-dexamethasone. After pellet protein data were standardized on the basis of DNA content, only ALP activity of MSCs treated with FGF-2-dexamethasone remained significantly increased.
Dexamethasone and FGF-2 enhanced chondrogenic differentiation of MSCs, primarily through an increase in MSC numbers. Treatment with dexamethasone stimulated ALP activity and ALP mRNA, consistent with the progression of cartilage toward bone. This may be important for MSC-based repair of articular cartilage.
确定在软骨形成微团培养阶段，先使用成纤维细胞生长因子2（FGF-2）扩增马间充质干细胞（MSCs），然后添加地塞米松，是否会增加软骨细胞基质标志物，同时不刺激肥大软骨细胞表型。
从5匹幼马获取的MSCs。
第一代马单层MSCs用含FGF-2（0或100 ng/mL）的培养基进行补充。汇合的MSCs转移至微团培养物中，并在含0或10(-7)M地塞米松的软骨形成培养基中维持培养。在1、7和14天后收集微团，并分析II型胶原蛋白含量、总糖胺聚糖含量、总DNA含量、碱性磷酸酶（ALP）活性以及聚集蛋白聚糖、II型胶原蛋白、ALP和延伸因子-1α的mRNA。
用FGF-2、地塞米松或两者处理均增加了微团II型胶原蛋白含量、总糖胺聚糖含量以及聚集蛋白聚糖的mRNA表达。MSC对照微团的DNA含量随时间下降。用FGF-2、地塞米松或两者处理可防止微团DNA含量随时间的损失。用FGF-2 -地塞米松处理的MSCs中微团ALP活性和mRNA增加。在基于DNA含量对微团蛋白数据进行标准化后，仅用FGF-2 -地塞米松处理的MSCs的ALP活性仍显著增加。
地塞米松和FGF-2增强了MSCs的软骨形成分化，主要是通过增加MSC数量实现的。用地塞米松处理刺激了ALP活性和ALP mRNA，这与软骨向骨的进展一致。这对于基于MSC的关节软骨修复可能很重要。
