Garza-Veloz Idalia, Romero-Diaz Viktor J, Martinez-Fierro Margarita L, Marino-Martinez Ivan A, Gonzalez-Rodriguez Manuel, Martinez-Rodriguez Herminia G, Espinoza-Juarez Marcela A, Bernal-Garza Dante A, Ortiz-Lopez Rocio, Rojas-Martinez Augusto
Arthritis Res Ther. 2013 Jul 30;15(4):R80. doi: 10.1186/ar4260.
Adipose-derived stem cells (ASCs) have the potential to differentiate into cartilage under stimulation with some reported growth and transcriptional factors, which may constitute an alternative for cartilage replacement approaches. In this study, we analyzed the in vitro chondrogenesis of ASCs transduced with adenoviral vectors encoding insulin-like growth factor-1 (IGF-1), transforming growth factor beta-1 (TGF-β1), fibroblast growth factor-2 (FGF-2), and sex-determining region Y-box 9 (SOX9) either alone or in combinations.
Aggregate cultures of characterized ovine ASCs were transduced with 100 multiplicity of infections of Ad.IGF-1, Ad.TGF-β1, Ad.FGF-2, and Ad.SOX9 alone or in combination. These were harvested at various time points for detection of cartilage-specific genes expression by quantitative real-time PCR or after 14 and 28 days for histologic and biochemical analyses detecting proteoglycans, collagens (II, I and X), and total sulfated glycosaminoglycan and collagen content, respectively.
Expression analyses showed that co-expression of IGF-1 and FGF-2 resulted in higher significant expression levels of aggrecan, biglycan, cartilage matrix, proteoglycan, and collagen II (all P ≤0.001 at 28 days). Aggregates co-transduced with Ad.IGF-1/Ad.FGF-2 showed a selective expression of proteoglycans and collagen II, with limited expression of collagens I and × demonstrated by histological analyses, and had significantly greater glycosaminoglycan and collagen production than the positive control (P ≤0.001). Western blot analyses for this combination also demonstrated increased expression of collagen II, while expression of collagens I and × was undetectable and limited, respectively.
Combined overexpression of IGF-1/FGF-2 within ASCs enhances their chondrogenic differentiation inducing the expression of chondrogenic markers, suggesting that this combination is more beneficial than the other factors tested for the development of cell-based therapies for cartilage repair.
脂肪来源干细胞（ASC）在一些已报道的生长和转录因子刺激下具有分化为软骨的潜力，这可能为软骨替代方法提供一种替代方案。在本研究中，我们分析了用编码胰岛素样生长因子-1（IGF-1）、转化生长因子β-1（TGF-β1）、成纤维细胞生长因子-2（FGF-2）和性别决定区Y框9（SOX9）的腺病毒载体单独或联合转导的ASC的体外软骨形成。
用100个感染复数的Ad.IGF-1、Ad.TGF-β1、Ad.FGF-2和Ad.SOX9单独或联合转导已鉴定的绵羊ASC的聚集体培养物。在不同时间点收获这些培养物，通过定量实时PCR检测软骨特异性基因表达，或在14天和28天后进行组织学和生化分析，分别检测蛋白聚糖、胶原蛋白（II、I和X）以及总硫酸化糖胺聚糖和胶原蛋白含量。
表达分析表明，IGF-1和FGF-2的共表达导致聚集蛋白聚糖、双糖链蛋白聚糖、软骨基质、蛋白聚糖和胶原蛋白II的表达水平显著更高（在28天时均P≤0.001）。用Ad.IGF-1/Ad.FGF-2共转导的聚集体显示蛋白聚糖和胶原蛋白II的选择性表达，组织学分析显示胶原蛋白I和X的表达有限，并且其糖胺聚糖和胶原蛋白产量显著高于阳性对照（P≤0.001）。该组合的蛋白质印迹分析也显示胶原蛋白II的表达增加，而胶原蛋白I和X的表达分别未检测到和有限。
ASC内IGF-1/FGF-2的联合过表达增强了它们的软骨形成分化，诱导软骨形成标志物的表达，表明这种组合比测试的其他因子更有利于基于细胞的软骨修复治疗的开发。
