神经生长因子对大鼠骨髓基质细胞成骨向分化的影响

Effects of nerve growth factor on the differentiation of rat bone marrow stroma cells into osteoblasts

目的:观察神经生长因子(nerve growth factor,NGF)对大鼠骨髓基质细胞(BMSCs)向成骨细胞分化的影响。方法:5周龄雄性SD大鼠,贴壁法培养股骨、胫骨BMSCs。实验分为空白对照组、NGF组、K252a+NGF组和K252a组。在加入NGF前,按分组加入酪氨酸激酶A(TrkA)受体特异性抑制剂K252a孵育30min。通过MTT法检测24h、48h和72h细胞增殖情况,PNPP法检测成骨诱导3d、5d和7d细胞碱性磷酸酶(alkalinephosphatase,ALP)活性,茜素红S钙染法观察成骨诱导14d矿化结节量。结果:同一观察时间点,4组之间BMSCs的增殖差异无统计学意义。成骨诱导3d、5d,NGF组ALP水平显著高于空白对照组(P<0.5);与NGF组相比,K252a+NGF组ALP水平显著降低(P<0.05);7d时,它们之间差异无统计学意义。成骨诱导14d,NGF组钙结节数量和面积显著高于空白对照组;阻断TrkA受体后,K252a+NGF组钙结节数量和面积明显降低。结论:NGF可能通过TrkA受体,在一定程度上促进大鼠骨髓基质细胞向成骨细胞分化、增强其矿化能力,且此促进作用可以被TrkA受体抑制剂K252a阻断;阻断TrkA受体与否,NGF均不能促进大鼠骨髓基质细胞增殖。

Objective： To investigate the effects of Nerve growth factor （NGF） on the differentiation of rat bone marrow stromal cells（BMSCs） into osteoblasts. Methods： BMSCs were obtained from the femur and tibia bones of 5-week old male SD rats and cultured by attachment method in vitro. They were divided into 4 groups： Blank control group, NGF group, K252a＋NGF group and K252a group. Before NGF, the K252a, TrkA inhibitor, was added into cultures for 30 min following the divided groups. The proliferation of BMSCs was evaluated through MTT assay after 24h, 48h and 72h, respectively. The activity of alkaline phosphatase （ALP） was studied by PNPP after osteogenic-inducing for 3d, 5d and 7d. Mineralized nodules were observed by Alizarin Red S calcium stain after 14-day osteogenic induction. Results＂ At the same observation time point, there was no statistical difference in the proliferation of BMSCs between 4 groups. After osteogenic-inducing for 3d and 5d, the ALP level of NGF group was higher than the Blank control groups; the ALP level of K252a＋NGF groups obviously reduced when comparing with NGF groups（P〈0.05）. At 7d, there was no significant difference between them. After 14-day osteogenic induction, the count and area of the calcium nodule in NGF group was significantly higher than that in Blank control group. After blocking TrkA, the count and area of the nodule in K252a＋NGF group reduced significantly. Conclusions： It was showed that NGF could promote the differentiation and the mineralization of BMSCs in a certain degree and this promotion could be inhibited by TrkA inhibitor K252a. Blocks TrkA or not, NGF couldn＇t facilitate the proliferation of BMSCs.