牛骨胶原肽对成骨细胞分化的影响

摘要

Objective To investigate the effect of 3 mg/ml bovine collagen peptides ( BCP ) on the differentiation of human osteoblasts ( HOB) and mouse MC3T3-E1 pre-osteoblasts.Methods The effect of BCP on the expression of runt-related transcription factor 2 ( Runx2) in MC3T3-E1 cells was detected using Western blotting.HOBs were isolated and cultured with BCP. The concentration of alkaline phosphatase ( ALP) was detected using colorimetric p-nitrophenyl phosphate assay.The concentration of osteocalcin ( OC) was detected using ELISA.After Alizarin red staining and decolorization with 5%perchloric acid, the effect of BCP on the mineralization in HOBs was detected using spectrophotometric method.Results The result of Western blotting revealed that the expression of Runx2 in BCP-treated MC3T3 cells at the 14th day was higher than that in control cells (0.178 ± 0.201 vs.0.146 ±0.582 ) .There was an increasing trend, but no significance was observed ( P>0.05 ) .The results of ALP staining showed that the ALP activity in the BCP-treated cells ( 33859 ±8221.7 ) was much higher than that in the control cells (19900 ±2796, P<0.05) at the 10th day, indicating that BCP could promote the ALP activity in MC3T3-E1 cells at the early stage.The OC activity in BCP-treated cells (0.137 ±0.014) was much higher than that in the control cells (0.086 ±0.023, P<0.05) at the 14th day, indicating that BCP could promote the OC activity in MC3T3-E1 cells at late stage.Calcium deposit test showed that BCP significantly increased mineralization in HOB cells at the 14th day.After decolorization with 5%perchloric acid, the absorbance at 490 nm in BCP-treated cells (0.579 ±0.093) was much higher than that in the control cells (0.193 ±0.021, P<0.01) at the 14th day, indicating that BCP could promote the mineralization in HOB cells.Conclusion BCP plays a positive role in osteoblast differentiation and the mineralized bone matrix formation.Taking all the experiments together, our study indicates a molecular mechanism for the potential prevention and treatment of osteoarthritis and osteoporosis.%目的：研究3 mg／ml牛骨胶原肽（ Bovine collagen peptides， BCP）对人成骨细胞（ Human osteoblast， HOB），小鼠前成骨细胞系MC3T3分化的影响。方法 Western blot检测MC3T3细胞中BCP对Runx2表达的影响；分离培养HOB，利用对硝基苯磷酸比色法检测3 mg／ml BCP对碱性磷酸酶（ Alkaline phosphatase，ALP）含量的影响；骨钙素（ osteocalcin，OC） ELISA法测定BCP对OC含量的影响；茜素红矿化染色检测BCP对HOB矿化，然后用5％高氯酸进行脱色，用吸光度法检测BCP对人成骨细胞矿化程度。结果 Western blot检测表明，14 d后BCP处理的MC3T3细胞中Runx2蛋白表达水平（0．178±0．201）与CN组（0．146±0．582，P＞0．05）比较，有增高趋势。 ALP染色结果表明，BCP混合物处理组的ALP的染色面积（33859±8221）在第10d与CN组（19900±2796）相比，显著增加（ P＜0．05），表明BCP混合物能促进人成骨细胞早期的ALP表达量。BCP混合物处理组的OC吸光度值（0．137±0．014）在第14d高于CN组（0．086±0．023， P＜0．05），表明BCP混合物能促进人成骨细胞晚期阶段的OC含量。茜素红矿化染色结果表明，3mg／ml BCP能显著促进HOB的矿化骨基质的形成。当使用5％的高氯酸脱色后，BCP处理组在490 nm处测吸光度值（0．579±0．093）显著高于CN组（0．193±0．021，P＜0．01），表明BCP混合物能促进人成骨细胞的矿化。结论 BCP在成骨细胞分化和矿化骨基质的形成中发挥了积极作用。综合上述结果，本文为BCP混合物在骨关节炎和骨质疏松症潜在的预防和治疗提供了分子机理。