兔骨髓间质干细胞成软骨分化的诱导及体内研究

Study on cartilage differentiation of rabbit marrow messenchymal stem cell in vitro and in vivo

目的研究骨形态发生蛋白-2(BMP-2)对兔骨髓间质干细胞(MSCs)的定向诱导作用,探讨MSCs的成软骨能力。方法分离并培养兔MSCs,BMP-2诱导MSCs向软骨细胞分化,观察其形态学改变及碱性磷酸酶(ALP)活性。将诱导后的MSCs接种于PGA无纺网体外培养2周,将MSCs-PGA无纺网复合体植入裸鼠背部皮下,2个月后取材,进行组织学观察。结果经BMP-2诱导后,MSCs的形态由长梭形向多角形和三角形转化,群体倍增时间约为3.0 d。诱导5、10 d后,ALP活性为0.282±0.015、0.502±0.012,明显高于对照组(0.265±0.010、0.315±0.021)(P<0.01),ALP染色阳性。MSCs-PGA无纺网复合体植入裸鼠后,组织学可见软骨陷窝,陷窝内可见核蓝染的成软骨细胞,证实MSCs具有体内成软骨能力。结论经BMP-2诱导的MSCs向软骨细胞分化和增殖,在裸鼠体内可形成软骨组织。

Objective To study the cartilage differentiation of rabbit marrow messenchymal stern cells （MSCs） orientationally induced by bone morphogenetic protein-2 （BMP-2）. Methods The MSCs were isolated and from rabbit thighbone marrow cultured. Growth kinetic was studied by growth curve. The MSCs had been cultured in the BMP-2 medium for 10 days,and then the alkaline phosphatase（ALP） activity was observed. The induced MSCs/PGA was implanted in the nude mice subcutaneously. The histological analysis of the graft was performed in 2 months after implantation. Results The MSCs were successfully cultured and induced by the BMP-2. The growth curve showed that the population doubling time was 3.0 days. The ALP activities were 0.282 ±0.015,0.502±0.012 in 5 d and 10 d respectively in BMP-2 group and 0.265±0.010,significantly higher than that in control group 0.315±0.021 （P 〈 0.01）. The cells displayed fibroblast-like morphology. Histological evaluation showed the cartilage formation was successfully achieved in 2 months in vivo. Conclusion The BMP-2 can induce MSCs to differentiate and proliferate chondral cells, and MSCs have the capability to generate cartilage in nude mice.