Regulatory Effects Of Nerve Growth Factor On The Proliferation And Differentiation Of Goats Bone Marrow Mesenchymal Stem Cells Into Osteoblasts

Objective The goats bone marrow mesenchymal stem cells (BMSCs) were cultured and induced to osteoblasts in vitro. To investigate the effects of nerve growth factor(NGF) on the proliferation and differentiation of BMSCs into osteoblasts after adding the NGF, and confirm an experiment that NGF can facilitate osteogenesis in distraction osteogenesis. We hope that the empirical method can establish a stable cell model in the further research. It will make the promotion of new bone formation be possible.Methode The goats BMSCs were isolated and purified by density gradient centrifugation combined with attachment culture method in vitro. Morphological changes of the goats BMSCs were observed. In order to eliminate the foreign cells, the marker protein on the BMSCs surface were identificated by flow cytometry. The third passage of BMSCs were randomly divided into four groups,A (the blank control group): the BMSCs were cultured using L-DMEM supplemented with 10% fetal bovine serum; B (the osteoblast control group): the BMSCs were cultured in mineralization media containing 10% fetal bovine serum, 50mg/L vitamin C, 10 mmol/L beta-sodium glycerophosphate and 10-8mol/L dexamethasone; C (the NGF group): put 100ng/ml NGF into the L-DMEM supplemented with 10% fetal bovine serum; D (the experimental group): put 100ng/ml NGF into the mineralization media. Three, five, seven days later, the proliferation of BMSCs was detected by MTT method. The activities of alkaline phosphatase (ALP) and osteocalcin (OC) were examined at 14 days later. 21 days later, the application of von Kossa stain method is used to detect the calcium nodules, and compared the effects of osteogenic differatiation with every groups.Results 1. The density gradient centrifugation combined with attachment culture method is an convenient way to isolated and purified the goats BMSCs in vitro. The CD90 and CD105 was strong positive while the CD34 and CD45 were negative expressed by flow cytometry.2. MTT results suggested that, in the A and C group, the BMSCs proliferated similar, and there was no obvious difference in the B and D group (P>0.05). But compared with the B group, the BMSCs in the D group proliferated degraded(p<0.05).3. Detection of bone formation performance: To the activities of ALP and OC, there are no obviously difference between A and C groups(p>0.05), but the BMSCs in the B and D groups were significantly stronger than in the A and C groups, and in the D groups, the BMSCs is expressed a higher activity compared with the B group(p<0.05). The staining of Von Kossa method was positive and the black calcium nodules were appeared in the B group. The number and the area of the calcium nodules is stronger in the D group.Conclusion The density gradient centrifugation combined with attachment culture method is an convenient way to isolated and purified the goats BMSCs in vitro. We can procure a higher purity in the third passage. NGF could not accelerate the proliferation of goats BMSCs. But when we used with mineralization media, it can clearly promote the differentiation of goats BMSCs into osteoblasts.