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目的 探讨生长因子(bFGF,TGF -β1)诱导下,培养时间对兔间充质干细胞(MSCs)体外软骨形成的影响。方法 取兔 MSCs 梯度离心,分离培养。取 P3、P5、P7、P10 代细胞,在 bFGF、TGF- β1 各20 ng/ml诱导下培养。观察细胞生长形态变化,用甲苯胺蓝染色观察细胞蛋白多糖的分泌。RT- PCR半定量检测Ⅱ型胶原mRNA的相对表达量,取关节软骨细胞作阳性对照。结果 各代细胞形态无明显差异。MSCs甲苯胺蓝染色呈异染性,RT- PCR检测Ⅱ型胶原阳性表达,各代细胞灰度值,P3 0 .16±0 .05,P5 0 .37±0. 03,P7 0 .88±0 .08,P10 0. 47±0. 05,软骨细胞 2 08±0 13。P7代细胞灰度值达最大(P<0 .01),软骨细胞是 MSCs的 3 倍以上。结论 生长因子能维持 MSCs细胞活性,促进生长繁殖。一定培养时间内,软骨生成能力渐增强,但随时间进一步延长,软骨分化减弱。
Objective To investigate the effect of culture time on the in vitro chondrogenesis of mesenchymal stem cells (MSCs) induced by growth factor (bFGF, TGF-β1). Methods Rabbit MSCs gradient centrifugation, separation and culture. P3, P5, P7 and P10 on behalf of the cells, bFGF, TGF-β1 20 ng / ml each induced culture. Morphological changes of cells were observed, and the secretion of cell proteoglycan was observed by toluidine blue staining. RT-PCR semiquantitative detection of type II collagen mRNA relative expression, take articular chondrocytes as a positive control. Results The morphological changes of all generations showed no significant difference. MSCs were stained with toluidine blue, and the expression of type Ⅱ collagen was detected by RT-PCR. The gray value of each cell line was P3 0 .16 ± 0 .05, P5 0 .37 ± 0. 03, P7 0 .88 ± 0 .08, P10 0. 47 ± 0.05, chondrocytes 208 ± 0 13. P7 generation cell gray value reached the maximum (P <0. 01), chondrocytes is more than 3 times MSCs. Conclusion Growth factors can maintain the activity of MSCs and promote the growth and reproduction. Within a certain incubation time, the chondrogenic ability gradually increased, but further prolonged with time, weakened cartilage differentiation.