| 摘要: |
| 目的 考察晚期糖基化终产物（AGE）对小鼠胚胎成骨细胞系MC3T3-E1细胞增殖和分化的影响及其作用机制。方法 用不同质量浓度（100、200、300 mg/L）AGE作用于MC3T3-E1细胞，采用CCK-8法检测细胞增殖活性，采用流式细胞术检测细胞凋亡率，采用碱性磷酸酶（ALP）染色检测细胞成骨能力，采用qPCR检测成骨相关基因（骨钙素、ALP和Runx2）及Yes相关蛋白（YAP）和β-联蛋白的mRNA表达，采用蛋白质印迹法检测YAP和β-联蛋白的蛋白质表达，采用免疫荧光法观察YAP和β-联蛋白的细胞核内含量及细胞骨架蛋白纤丝状肌动蛋白（F-actin）的表达。结果 MC3T3-E1细胞在200、300 mg/L AGE处理后增殖活性降低、凋亡率增加（P均＜0.05），100 mg/L AGE对细胞增殖和凋亡无明显影响，故选取100 mg/L AGE进行实验。在成骨诱导培养条件下，与对照组相比，MC3T3-E1细胞经100 mg/L AGE处理后ALP染色较浅，骨钙素、ALP和Runx2的mRNA表达均较低（P均＜0.05）。在常规培养条件下，与对照组相比，MC3T3-E1细胞经100 mg/L AGE处理后F-actin形态和分布发生明显改变；YAP的mRNA和蛋白质表达均无明显变化，但其细胞核内含量减少；β-联蛋白的mRNA和蛋白质表达均降低（P均＜0.05），但其细胞核内含量无明显变化。结论 AGE能抑制MC3T3-E1细胞的增殖活性，诱导细胞凋亡，降低成骨分化能力，F-actin、YAP和β-联蛋白参与其调控过程。 |
| 关键词: 晚期糖基化终产物 MC3T3-E1细胞 成骨分化 纤丝状肌动蛋白 Yes相关蛋白 β-联蛋白 |
| DOI：10.16781/j.CN31-2187/R.20201381 |
| 投稿时间：2020-11-18 |
| 基金项目:国家自然科学基金面上项目（81470772），重庆市科卫联合医学科研重点项目（2018ZDXM020） |
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| Advanced glycation end products inhibit osteogenic differentiation of mouse embryonic osteoblasts by regulating F-actin/YAP |
| WU Pei-lian1,2,HU Yun1,3,LIU Dong-rong1,2,ZHENG Lei-lei1,3* |
(1. Stomatological Hospital of Chongqing Medical University, Chongqing 401147, China;
2. Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences, Chongqing 401147, China;
3. Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education, Chongqing 401147, China
*Corresponding author) |
| Abstract: |
| Objective To explore the effects of advanced glycation end product (AGE) on proliferation and differentiation of mouse embryonic osteoblast line MC3T3-E1 cells and its mechanisms.Methods MC3T3-E1 cells were treated with different mass concentrations of AGE (100, 200, and 300 mg/L). Cell proliferation activity and apoptosis rate were detected by cell counting kit 8 and flow cytometry, respectively. Osteogenic ability was detected by alkaline phosphatase (ALP) staining, and the mRNA expression of osteogenesis-related genes (osteocalcin, ALP, and Runx2), Yes-associated protein (YAP), and β-catenin was detected by quantitative polymerase chain reaction. The protein expression of YAP and β-catenin was detected by Western blotting, and the nuclear contents of YAP and β-catenin and the expression of cytoskeleton protein filamentous actin (F-actin) were observed by immunofluorescence assay.Results The proliferation activity of MC3T3-E1 cells was significantly decreased and the apoptosis rates were significantly increased after treatment with 200 or 300 mg/L AGE (all P < 0.05), and 100 mg/L AGE had no significant effect on cell proliferation or apoptosis and was selected for the experiment. Under osteogenic induction culture condition, compared with the control group, the ALP staining was shallow in MC3T3-E1 cells after treatment with 100 mg/L AGE, and the mRNA expression levels of osteocalcin, ALP and Runx2 were significantly lower (all P < 0.05). Under conventional culture condition, compared with the control group, the morphology and distribution of F-actin were significantly changed in MC3T3-E1 cells after treatment with 100 mg/L AGE; there were no significant changes in the mRNA or protein expression of YAP, but the nuclear contents were decreased; and the mRNA and protein expression levels of β-catenin were significantly decreased (both P < 0.05), but the nuclear contents had no significant change.Conclusion AGE can inhibit proliferation activity, induce apoptosis, and inhibit osteogenic differentiation ability of MC3T3-E1 cells. F-actin, YAP, and β-catenin participate in the regulation process. |
| Key words: advanced glycation end products MC3T3-E1 cells osteogenic differentiation filamentous actin Yes-associated protein β-catenin |
