Liu JunLi, Zhang Bing, Song ShuJun, Ma Ming, Si ShaoYan, Wang YiHu, Xu BingXin, Feng Kai, Wu JiGong, Guo YanChuan
Department of Pathology and Experimental Medicine, 306 Hospital of PLA, Beijing, People's Republic of China.
Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Science, Beijing, People's Republic of China.
PLoS One. 2014 Jun 13;9(6):e99920. doi: 10.1371/journal.pone.0099920. eCollection 2014.
Collagen peptides (CP) compounds, as bone health supplements, are known to play a role in the treatment of osteoporosis. However, the molecular mechanisms of this process remain unclear. This study aimed to investigate the effects of bovine CP compounds on the proliferation and differentiation of MC3T3-E1 cells.
Mouse pre-osteoblast cell line MC3T3-E1 subclone 4 cells were treated with bovine CP compounds. Cell proliferation was analyzed by MTT assays and the cell cycle was evaluated by flow cytometry scanning. Furthermore, MC3T3-E1 cell differentiation was analyzed at the RNA level by real-time PCR and at the protein level by western blot analysis for runt-related transcription factor 2 (Runx2), a colorimetric p-nitrophenyl phosphate assay for alkaline phosphatase (ALP), and ELISA for osteocalcin (OC). Finally, alizarin red staining for mineralization was measured using Image Software Pro Plus 6.0.
Cell proliferation was very efficient after treatment with different concentrations of bovine CP compounds, and the best concentration was 3 mg/mL. Bovine CP compounds significantly increased the percentage of MC3T3-E1 cells in G2/S phase. Runx2 expression, ALP activity, and OC production were significantly increased after treatment with bovine CP compounds for 7 or 14 days. Quantitative analyses with alizarin red staining showed significantly increased mineralization of MC3T3-E1 cells after treatment with bovine CP compounds for 14 or 21 days.
Bovine CP compounds increased osteoblast proliferation, and played positive roles in osteoblast differentiation and mineralized bone matrix formation. Taking all the experiments together, our study indicates a molecular mechanism for the potential treatment of osteoarthritis and osteoporosis.
胶原蛋白肽（CP）化合物作为骨骼健康补充剂，已知在骨质疏松症治疗中发挥作用。然而，这一过程的分子机制仍不清楚。本研究旨在探讨牛源CP化合物对MC3T3-E1细胞增殖和分化的影响。
用牛源CP化合物处理小鼠前成骨细胞系MC3T3-E1亚克隆4细胞。通过MTT法分析细胞增殖情况，并用流式细胞术扫描评估细胞周期。此外，通过实时PCR在RNA水平分析MC3T3-E1细胞分化情况，通过蛋白质印迹分析与矮小相关转录因子2（Runx2）在蛋白质水平分析细胞分化情况，用比色法对碱性磷酸酶（ALP）进行对硝基苯磷酸酯测定，并用酶联免疫吸附测定法（ELISA）检测骨钙素（OC）。最后，使用Image Software Pro Plus 6.0测量茜素红染色的矿化情况。
用不同浓度的牛源CP化合物处理后细胞增殖非常有效，最佳浓度为3 mg/mL。牛源CP化合物显著增加了处于G2/S期的MC3T3-E1细胞的百分比。用牛源CP化合物处理7天或14天后，Runx2表达、ALP活性和OC产生均显著增加。茜素红染色的定量分析表明，用牛源CP化合物处理14天或21天后，MC3T3-E1细胞的矿化显著增加。
牛源CP化合物增加了成骨细胞增殖，并在成骨细胞分化和矿化骨基质形成中发挥了积极作用。综合所有实验，我们的研究揭示了潜在治疗骨关节炎和骨质疏松症的分子机制。
