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目的:探讨转化生长因子β1(TGF-β1)联合软骨源性形态发生蛋白-2(CDMP-2)体外诱导犬成肌细胞向软骨细胞表型定向分化的可行性。方法:取成年比格犬大腿股直肌肌肉,以机械分解法与二步酶消化法分离获得成肌细胞,取第4代成肌细胞,以2.0×104细胞/cm2的密度接种于24孔培养板,使用含CDMP-2和(或)TGF-β1的完全培养液诱导分化成肌细胞,观察细胞生长情况。诱导14 d后获取细胞,进行HE染色、甲苯胺蓝染色,观察细胞形态及胞内高硫酸化的蛋白聚糖变化。免疫细胞化学染色检测II型胶原蛋白着色情况。RT-PCR检测I型、II型胶原蛋白、aggrecan及Sox9 mRNA表达。阿尔新蓝法定量检测细胞糖胺聚糖(GAG)含量。结果:3个加入细胞因子的实验组成肌细胞形态由长梭形向多角形、类圆形转变;HE染色、甲苯胺蓝染色和软骨特异性I型和Ⅱ型胶原免疫组化法检测及RT-PCR检测中,实验组均有不同程度的阳性表达;TGF-β1与CDMP-2联合应用组糖胺聚糖含量明显高于其他三组(P<0.05)。结论:CDMP-2和TGF-β1可以单独诱导高密度单层培养的犬成肌细胞向软骨细胞分化,表达软骨细胞特异性细胞表型,并且CDMP-2和TGF-β1有协同作用。
Objective: To investigate the feasibility of transforming growth factor β1 (TGF-β1) and cartilage-derived morphogenetic protein-2 (CDMP-2) induced directional differentiation of canine myoblasts into chondrocytes in vitro. METHODS: The muscle of adult rectus beagle thigh muscle was obtained by mechanical disintegration and two-step enzymatic digestion. The myoblasts of the fourth generation were seeded into the 24-well at the density of 2.0 × 104 cells / cm2 Plates were cultured and differentiated into myoblasts using complete medium containing CDMP-2 and / or TGF-β1 to observe cell growth. After induction for 14 days, the cells were harvested and stained with hematoxylin and eosin (HE). Toluidine blue staining was performed to observe the changes of cell morphology and intracellular hyper-sulfated proteoglycans. Immunocytochemical staining for collagen type II staining. The mRNA expression of type I, type II collagen, aggrecan and Sox9 was detected by RT-PCR. Alzheimer’s method was used to quantitatively detect the content of glycosaminoglycan (GAG). RESULTS: The morphology of myocytes in the three experimental groups with cytokines changed from long spindle to polygonal and round-like, HE staining, toluidine blue staining and cartilage-specific type I and type II collagen immunohistochemistry and RT -PCR test, the experimental group had different degrees of positive expression; the combination of TGF-β1 and CDMP-2 glycosaminoglycan content was significantly higher than the other three groups (P <0.05). CONCLUSION: CDMP-2 and TGF-β1 can induce the differentiation of chondrocytes into high-density monolayer cultured canine chondrocytes to express chondrocyte-specific cell phenotype, and CDMP-2 and TGF-β1 have synergistic effects.