摘要
背景:前期研究发现,骨碎补总黄酮可促进纳米骨材料表面MC3T3-E1细胞的成骨分化,其作用机制有待进一步研究。目的:探究骨碎补总黄酮联合纳米骨材料对MC3T3-E1细胞发挥作用的机制。方法:将MC3T3-E1细胞与纳米骨材料共培养,选取100mg/L和250mg/L骨碎补总黄酮进行药物干预,以10μg/L转化生长因子β刺激为阳性对照组。分组如下:①正常组;②DKK1组:Wnt通路抑制剂DKK1(0.1mg/L)阻断Wnt/β-catenin信号通路;③DKK1+转化生长因子β组;④DKK1+100mg/L骨碎补总黄酮组;⑤DKK1+250mg/L骨碎补总黄酮组;⑥DKK1+纳米骨+转化生长因子β组;⑦DKK1+纳米骨+100mg/L骨碎补总黄酮组;⑧DKK1+纳米骨+250mg/L骨碎补总黄酮组。在干预24,48 h后收获细胞,免疫荧光双染法观察Wnt/β-catenin通路中Wnt与LRP结合情况,Real-time PCR和Western blot检测β-catenin、LRP5、Gsk-3β、Cyclin D1、RUNX2的表达。结果与结论:①激光共聚焦扫描显微镜下显示DKK1+转化生长因子β组、DKK1+250mg/L骨碎补总黄酮组、DKK1+纳米骨+转化生长因子β组、DKK1+纳米骨+250mg/L骨碎补总黄酮组棕黄色染色较明显,表明Wnt与LRP结合较其他组更好;②Real-time PCR和Western blot结果显示,骨碎补总黄酮可促进β-catenin、LRP5、RUNX2的表达,下调GSK-3β的表达,说明骨碎补总黄酮通过激活经典Wnt/β-catenin信号通路促进成骨细胞增殖分化,且骨碎补总黄酮诱导的基因活化呈剂量依赖性。
BACKGROUND:Preliminary study has found that osteopractic total flavone can promote osteogenic differentiation of MC3T3-E1 cells on the surface of nano-bone material,but the underlying mechanism needs to be studied in depth.OBJECTIVE:To investigate the actin mechanism of osteopractic total flavone combined with nano-bone material on MC3T3-E1 cells.METHODS:MC3T3-E1 cells were co-cultured with nano-bone material,and 100mg/L and 250mg/L osteopractic total flavone were treated as drug intervention,including 10μg/L transforming growth factor-βas positive control.The samples were divided into eight groups:(1)Normal group;(2)DKK1 group:Wnt pathway inhibitor DKK1(0.1mg/L)blocks Wnt/β-catenin signaling pathway;(3)DKK1+transforming growth factor-βgroup;(4)DKK1+100mg/L osteopractic total flavone group;(5)DKK1+250mg/L osteopractic total flavone group;(6)DKK1+nano-hydroxyapatite/collagen+transforming growth factor-βgroup;(7)DKK1+nano-hydroxyapatite/collagen+100mg/L osteopractic total flavone group;(8)DKK1+nano-hydroxyapatite/collagen+250mg/L osteopractic total flavone group.Cells in each group were harvested after 24 and 48 hours of intervention.Immunofluorescence labeling was used to observe the binding of Wnt and LRP in osteoblasts in the Wnt/β-catenin pathway.The expression ofβ-catenin,LRP 5,GSK-3β,Cyclin D1,and RUNX2 was detected by real-time polymerase chain reaction and western blot assay.RESULTS AND CONCLUSION:(1)Confocal laser scanning microscope showed that obvious brown and yellow staining was shown in the DKK1+transforming growth factor-βgroup,DKK1+250mg/L osteopractic total flavone group,DKK1+nano-hydroxyapatite/collagen+transforming growth factor-βgroup,and DKK1+nano-hydroxyapatite/collagen+250mg/L osteopractic total flavone group,indicating that Wnt and LRP combined better than other groups.(2)Real-time polymerase chain reaction and western blot assay results showed that osteopractic total flavone could promote the expression ofβ-catenin,LRP5 and RUNX2,and downregulated GSK3βexpression.These findings confirm that osteopractic total flavone can promote the differentiation and proliferation of osteoblasts by activating the Wnt/β-catenin signaling pathway.Gene activation induced by osteopractic total flavone was dose-dependent.
作者
李晋玉
俞兴
姜俊杰
徐林
赵学千
孙旗
郑晨颖
白春晓
刘楚吟
贾育松
Li Jinyu;Yu Xing;Jiang Junjie;Xu Lin;Zhao Xueqian;Sun Qi;Zheng Chenying;Bai Chunxiao;Liu Chuyin;Jia Yusong(Dongzhimen Hospital,Beijing University of Chinese Medicine,Beijing 100700,China;Institute of Basic Research in Clinical Medicine,China Academy of Chinese Medical Sciences,Beijing 100700,China)
出处
《中国组织工程研究》
CAS
北大核心
2020年第7期1030-1036,共7页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金项目(81503601)，项目负责人：李晋玉
国家自然科学基金项目(81603517)，项目负责人：姜俊杰
北京中医药大学东直门医院青苗人才项目(DZMYS-201802)，项目负责人：李晋玉~~
