鱼鳞胶对MC3T3-E1细胞成骨性能的影响

The Effect of Fish Scale Glue on the Osteogenic Performance of MC3T3-E1 Cells

摘要: 研究目的：用含有鱼鳞胶的培养基培养小鼠胚胎成骨细胞(MC3T3-E1细胞)，探究鱼鳞胶对MC3T3-E1细胞成骨性能的影响，为临床应用鱼鳞胶治疗骨质疏松症提供理论基础。研究方法：1. 用鱼鳞胶分别以5 mg/mL、10 mg/mL、20 mg/mL的浓度培养MC3T3-E1细胞，同时设置空白对照组和阳性对照组。在培养的第14 d，通过ALP染色检测碱性磷酸酶(ALP)的活性，imageJ软件计算反映蓝紫色沉淀单位面积浓度的平均光密度(AOD)值，并进行组间比较。2. 上述浓度诱导MC3T3-E1细胞，在培养的第21 d，通过茜素红染色(ARS)评估钙结节形成情况。3. 设置空白对照组，处理组，含20 mg/mL鱼鳞胶的处理组，培养MC3T3-E1细胞48 h后，通过逆转录–实时荧光定量PCR (RT-qPCR)分别检测上述三组MC3T3-E1细胞的碱性磷酸酶(ALP)，Runx相关转录因子2 (Runx2)，骨钙素(OCN) mRNA相对表达量的变化。研究结果：1. ALP染色实验结果显示，不同浓度鱼鳞胶干预MC3T3-E1细胞14 d，与对照组相比，10 mg/mL鱼鳞胶组，20 mg/mL鱼鳞胶组和阳性对照组的AOD均比空白对照组增加(P < 0.05)。2. 不同浓度的鱼鳞胶体外培养MC3T3-E1细胞21 d时，茜素红染色结果显示，鱼鳞胶组与空白对照组相比，各组均形成钙化结节，且存在剂量依赖性，即鱼鳞胶浓度越大，结节越明显。3. RT-qPCR结果表明，体外培养MC3T3-E1细胞48 h，处理组和含鱼鳞胶的处理组ALP、OCN mRNA表达量均上调，与空白对照组相比有统计学意义(P < 0.01)，且两组间均有统计学差异(P < 0.05)。处理组和含鱼鳞胶的处理组Runx2 mRNA表达量较空白对照组上调(P < 0.05)，两组间没有统计学差异(P > 0.05)。结论及意义：鱼鳞胶能够显著促进MC3T3-E1细胞的成骨分化成熟、矿化，从而加快骨基质矿化过程，促进骨形成。这些体外实验数据证实了鱼鳞胶对骨质疏松症具有潜在的治疗作用，为全面评价其在临床上防治骨质疏松症的药效作用提供实验参考依据。

Abstract: Research aim: To culture mouse embryonic osteoblasts (MC3T3-E1 cells) in a medium containing fish scale glue, and investigate the effect of fish scale glue on the osteogenic performance of MC3T3-E1 cells, providing a theoretical basis for the clinical application of fish scale glue in the treatment of osteoporosis. Methods: 1. MC3T3-E1 cells were cultured with fish scale glue at concen-trations of 5 mg/mL, 10 mg/mL, and 20 mg/mL, respectively, while a blank control group and a positive control group were set up. On the 14th day of culture, the activity of alkaline phosphatase (ALP) was detected by ALP staining, and the average optical density (AOD) value reflecting the con-centration per unit area of blue-violet precipitates was calculated by imageJ software and compared between groups. 2. MC3T3-E1 cells were induced at the above concentrations. On the 21st day of culture, the formation of calcium nodules was evaluated by alizarin red staining (ARS). 3. Set up a blank control group, a treatment group and a treatment group containing 20 mg/mL fish scale glue. After culturing MC3T3-E1 cells for 48 hours, detect the changes in the relative expression of alka-line phosphatase (ALP), Runx related transcription factor 2 (Runx2), and osteocalcin (OCN) mRNA in the above three groups of MC3T3-E1 cells by reverse transcription real-time fluorescence quan-titative PCR (RT-qPCR). Results: The results of ALP staining experiment showed that the AOD of MC3T3-E1 cells in the 10 mg/mL fish scale glue group, the 20 mg/mL fish scale glue group, and the positive control group increased compared to the blank control group after 14 days of intervention with different concentrations of fish scale glue (P < 0.05). When MC3T3-E1 cells were cultured in vitro with different concentrations of fish scale colloid for 21 days, the results of alizarin red staining showed that compared with the blank control group, calcified nodules were formed in each group, and there was a dose dependent relationship. That is, the greater the concentration of fish scale colloid, the more obvious the nodules were. The results of RT-qPCR showed that the expression of ALP and OCN mRNA in MC3T3-E1 cells cultured in vitro for 48 hours was upregulated in both the treatment group and the treatment group containing fish scale gum, which was statistically signifi-cant compared to the blank control group (P < 0.01), and there was a significant difference between the two groups (P < 0.05). The expression of Runx2 mRNA in the treatment group and the treat-ment group containing fish scale glue was upregulated compared to the blank control group (P < 0.05), and there was no statistical difference between the two groups (P > 0.05). Conclusion and sig-nificance: Fish scale glue can significantly promote the osteogenic differentiation, maturation, and mineralization of MC3T3-E1 cells, thereby accelerating the mineralization process of bone matrix and promoting bone formation. These in vitro experimental data confirm the potential therapeutic effect of fish scale glue on osteoporosis, providing experimental reference for comprehensive evalu-ation of its clinical efficacy in preventing and treating osteoporosis.