CAJ | 학술논문

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。
목적：연구3 mg／ml우골효원태（ Bovine collagen peptides， BCP）대인성골세포（ Human osteoblast， HOB），소서전성골세포계MC3T3분화적영향。방법 Western blot검측MC3T3세포중BCP대Runx2표체적영향；분리배양HOB，이용대초기분린산비색법검측3 mg／ml BCP대감성린산매（ Alkaline phosphatase，ALP）함량적영향；골개소（ osteocalcin，OC） ELISA법측정BCP대OC함량적영향；천소홍광화염색검측BCP대HOB광화，연후용5％고록산진행탈색，용흡광도법검측BCP대인성골세포광화정도。결과 Western blot검측표명，14 d후BCP처리적MC3T3세포중Runx2단백표체수평（0．178±0．201）여CN조（0．146±0．582，P＞0．05）비교，유증고추세。 ALP염색결과표명，BCP혼합물처리조적ALP적염색면적（33859±8221）재제10d여CN조（19900±2796）상비，현저증가（ P＜0．05），표명BCP혼합물능촉진인성골세포조기적ALP표체량。BCP혼합물처리조적OC흡광도치（0．137±0．014）재제14d고우CN조（0．086±0．023， P＜0．05），표명BCP혼합물능촉진인성골세포만기계단적OC함량。천소홍광화염색결과표명，3mg／ml BCP능현저촉진HOB적광화골기질적형성。당사용5％적고록산탈색후，BCP처리조재490 nm처측흡광도치（0．579±0．093）현저고우CN조（0．193±0．021，P＜0．01），표명BCP혼합물능촉진인성골세포적광화。결론 BCP재성골세포분화화광화골기질적형성중발휘료적겁작용。종합상술결과，본문위BCP혼합물재골관절염화골질소송증잠재적예방화치료제공료분자궤리。
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.