不同浓度牡蛎蛋白肽对大鼠骨髓间充质干细胞增殖和分化能力的影响

Effects of oyster protein peptide with different concentrations on the proliferation and differentiation of bone marrow mesenchymal stem cells

目的:探讨不同浓度牡蛎蛋白肽对骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成骨分化的影响。方法:选择具备稳定传代能力的第3代大鼠BMSCs,采用MTT试剂盒,检测细胞在0、10、20、50 mg/L不同牡蛎蛋白肽浓度干预培养24 h、3 d、7 d后的细胞活力。然后将BMSCs分为普通培养基组(DMEM)和成骨诱导培养基组(ODM),根据牡蛎蛋白肽的不同浓度,将实验组分为4组,依次为0、10、20、50 mg/L,利用碱性磷酸酶(ALP)染色法和ALP半定量检测法,研究不同浓度牡蛎蛋白肽对BMSCs培养10 d早期成骨分化的影响。应用茜素红染色法,半定量检测不同浓度牡蛎蛋白肽对BMSCs培养21 d后成骨矿化的影响,应用RT-PCR检测成骨相关基因的表达情况。采用SPSS 20.0软件包对数据进行统计学分析。结果:MTT结果表明,经不同浓度药物干预处理后,细胞活力旺盛,无明显异常;但牡蛎蛋白肽药物浓度为50 mg/L时,细胞死亡数有上升趋势。ALP半定量检测结果显示,DMEM和ODM组诱导分化第10天时,20 mg/L浓度组的ALP活性显著高于对照组(P<0.05),50 mg/L浓度组的ALP活性与对照组无显著差异。茜素红半定量检测结果显示,ODM组中20 mg/L实验组的A值显著高于对照组(P<0.05)。与对照组及50 mg/L组相比,10、20 mg/L组RUNX-2和OCN的表达显著上升(P<0.05)。结论:高于50 mg/L浓度的牡蛎蛋白肽对BMSCs的成骨分化表现出明显抑制作用,而低浓度牡蛎蛋白肽,尤其是20 mg/L的牡蛎蛋白肽对BMSCs成骨分化及细胞生长有促进作用。

PURPOSE: To investigate the effect of different concentrations of oyster protein peptide on osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs). METHODS: BMSCs of the third generation from rats with stable subculture ability were selected, and the cell viability was detected by MTT kit at different concentrations of 0,10, 20 and 50 mg/L oyster protein peptide for 24 hours, 3 days and 7 days. BMSCs were divided into two groups: cultured with DMEM or ODM. According to the different concentrations of oyster protein peptide, the experimental groups were divided into four subgroups, which were 0, 10, 20 and 50 mg/L, respectively. The effects of different concentrations of oyster protein peptide on the early osteogenic differentiation of BMSCs were evaluated by ALP staining and ALP semiquantitative detection. Alizarin red staining was used to detect the effect of different concentrations of oyster protein peptide on the osteogenesis and mineralization of BMSCs after 21 days of treatment. RT-PCR was used to defect the expression of bone formation-related genes. SPSS 20.0 software package was used to analyze the data. RESULTS: The results of MTT showed that cell activity was vigorous and there was no obvious abnormality after intervention with different concentrations of drugs, but the number of cell death increased when the concentration of oyster protein peptide was 50 mg/L. The results of ALP semi-quantitative analysis showed that ALP activity of DMEM and ODM group was significantly higher than that of the control group(P<0.05) on the 10 th day of differentiation induction, but there was no significant difference between 50 mg/L group and the control group. Alizarin red semi-quantitative analysis showed that the A value of 20 mg/L in ODM group was significantly higher than that of the control group(P<0.05). The expression of RUNX-2 and OCN in 10 mg/L and 20 mg/L groups were significantly higher than that in control goup and 50 mg/L group(P<0.05).CONCLUSIONS: Oyster protein peptide with a concentration higher than 50 mg/L can significantly inhibit osteogenic differentiation of BMSCs, while oyster protein peptide with a low concentration, especially oyster protein peptide with a concentration of 20 mg/L, can promote osteogenic differentiation and cell growth of BMSCs.