摘要
目的 建立鸡胚额骨成骨细胞的培养方法,研究依普拉芬对其成骨细胞增殖和分化功能的影响。方法 采用混合胶原酶和胰酶、两次消化的方法,获取成骨细胞,经培养传代,取状态良好的第3代细胞在96孔板上进行增殖率和碱性磷酸酶活性的测定。结果 消化培养的细胞具有明显的成骨细胞的特点:属于成纤维目细胞型、碱性磷酸酶黑染、基质前体红染,长期培养能形成矿化结节。应用依普拉芬后发现10-4mol/L IP可抑制成骨细胞增殖,而10-8moL/L对改善成骨细胞的增殖和分化有显著功效(P<0.01),小于10-10mol/L浓度的IP则对成骨细胞与对照组无差异。结论 酶混合消化可以得到较纯的成骨细胞,应用依普拉芬研究表明,适宜浓度的IP可促进鸡胚额骨成骨细胞增殖和分化,提高其碱性磷酸酶活性,增强成骨细胞矿化,促使骨形成。
Objective To study the effect of different concentrations of ipriflavone on the proliferation and differentiation osteoblastic cells, based on the method of isokting the osteobkstic cells from calvariae of embryonic chicken. Methods The osteoblastic cells were harvested by sequential enzyme digestion(0.2% collagenase and 0.25% trypsin) from bone chips isolated from calvariae of embryonic chicken. A second or third passage culture was used, which was seeded in 96-well plates at the density of 1.5 ×104, and cultured with 10-4-10-10M ipriflavone for 72 h.Then MTT and PNPP methods were used to assay the proliferation rate and activity of AKP of osteobkstic cells. Results The cells were characteristic of osteoblast; they were of fibroblastic type, showed black deposits of AKP, red deposits of promatrix by cytochemical staining and formed mineralized nodes. With addition of ipri-fkvone, 10-4M ipriflavone inhibited the osteoblastic function, but the 10-8M iprifkvone significantly improved the proliferation and differentiation of osteoblasts, other concentration of ipriflavones had no obvious effect on the os-teoblastic cells. Conclusion Pure chicken osteobkstic cells can be obtained by sequential digestion method. An appropriate concentration of iprifkvone promotes the proliferation and differentiation of chicken osteoblastic cell.
出处
《中国骨质疏松杂志》
CAS
CSCD
2004年第2期236-238,177,共4页
Chinese Journal of Osteoporosis
基金
国家自然科学基金(3977056)
江苏省自然科学基金(Q990023)
