摘要
目的:探讨后肾间充质细胞(MMC)及其上清液和其活性成分VEGF-C所作用受体VEGFR2/3对成骨细胞成骨的影响。方法:将MMC与成骨细胞系MC3T3-E1混合培养,检测成骨标志物碱性磷酸酶(ALP)活性与染色情况;茜素红染色法观察矿化结节形成情况。制备MMC上清液,将成骨细胞分为成骨诱导对照组和MMC上清液组,CCK-8法检测细胞增殖活性;检测ALP活性、染色情况与矿化结节形成情况;蛋白质印记法检测成骨相关蛋白Runx2、OCN、OPN的表达水平。加入VEGFR2和VEGFR3的特异性抑制剂Ki8751和MAZ51后,通过检测上述指标观察其促成骨作用有无减弱。结果:混合培养使成骨细胞ALP活性升高、染色增强,细胞表面矿化结节数量增多、体积增大。加入MMC上清液后,成骨细胞增殖活性升高,ALP活性升高、染色增强,Runx2、OCN、OPN表达升高,矿化结节数量增多、体积增大。抑制VEGFR2/3后,MMC上清液的促成骨作用减弱,ALP活性下降、染色减弱,Runx2、OCN、OPN的表达下降,细胞表面矿化结节数量减少、体积减小。结论:MMC可通过作用于VEGFR2/3促进成骨细胞成骨。
Objective:To study the effect of the metanephric mesenchymal cell(MMC)and their supernatant and active components on osteoblast osteogenesis.Methods:MMC was mixed with osteoblast cell line MC3T3-E1,the activity and staining of osteogenesis marker alkaline phosphatase(ALP)was detected.Alizarin red staining was used to observe the formation of mineralized nodules of osteoblasts.The supernatant of MMC was prepared,osteoblasts were divided into control group with osteogenic induced medium and MMC supernatant conditioned medium group.The cell activity was measured by CCK-8 method;The activity and staining of ALP and the formation of mineralized nodules of osteoblasts was detected;The expression levels of osteogenic related proteins Runx2,OCN and OPN were detected by Western blot.Ki8751 and MAZ51,the specific inhibitors of VEGFR2 and VEGFR3,were added to observe whether MMC supernatant’s osteogenic effect was weakened by detecting related indicators.Results:The mixed culture of MMC and osteoblasts increased the activity and staining of ALP,the number and the volume of mineralized nodules increased.After adding the MMC supernatant,the activity of osteoblasts increased,the activity and staining of ALP increased,the expression of osteogenic related proteins Runx2,OCN,OPN increased,the number and the volume of mineralized nodules on the cell surface increased.After inhibiting VEGFR2/3,the osteogenic effect of MMC supermatant was weakened the activity and staining of ALP decreased,the expression of Runx2,OCN,OPN decreased,the number and the volume of mineralized nodules decreased.Conclusion:MMC can promote the osteogenesis of osteoblasts by acting on VEGFR2/3.
作者
纪鹏程
国文凯
傅博
谢院生
JI Pengcheng;GUO Wenkai;FU Bo(Chinese PLA Medical School,Beijing,100853)
出处
《中国中西医结合肾病杂志》
2023年第3期198-201,I0001,I0002,共6页
Chinese Journal of Integrated Traditional and Western Nephrology
基金
国家自然科学基金资助项目(No.82174115,81774027)。
