β-蜕皮甾酮促进小鼠前成骨细胞体外增殖及诱导成骨分化  

Beta-ecdysterone promotes in vitro proliferation and osteogenic differentiation of MC3T3-E1 cells

作　　者：严才平 陈路[1] 邓长弓 陈骞[1] 蒋科[1] 易源缘 李毓灵 Yan Caiping;Chen Lu;Deng Changgong;Chen Qian;Jiang Ke;Yi Yuanyuan;Li Yuling(Department of Orthopedics,Affiliated Hospital of North Sichuan Medical College,Nanchong 637000,Sichuan Province,China;Department of Neurosurgery,Affiliated Hospital of North Sichuan Medical College,Nanchong 637000,Sichuan Province,China)

机构地区：[1]川北医学院附属医院骨科,四川省南充市637000 [2]川北医学院附属医院神经外科,四川省南充市637000

出　　处：《中国组织工程研究》2020年第29期4605-4612,共8页Chinese Journal of Tissue Engineering Research

基　　金：四川省科技厅面上项目科研基金(2018JY0250),项目负责人:李毓灵。

摘　　要：背景:β-蜕皮甾酮作为“植物类雌激素”不仅具有刺激蛋白质合成,促进碳水化合物和脂质代谢,缓解高血糖、高脂血症,以及保护内皮细胞免于凋亡并诱导其增殖的多种生物活性,而且有学者报道其在治疗骨质疏松症、骨折和其他骨骼炎症性疾病方面也有着重要的作用。目的:观察β-蜕皮甾酮对小鼠前成骨细胞(MC3T3-E1细胞)体外增殖的影响,以及在安全剂量下,β-蜕皮甾酮是否对该细胞具有诱导成骨分化的作用。方法:取第4代MC3T3-E1细胞在成骨诱导分化培养基中培养7,10,14,21,28 d后,检测细胞不同时间段成骨分化蛋白(碱性磷酸酶、Ⅰ型胶原蛋白、骨桥蛋白以及钙化结节)的表达量,以鉴定该细胞是否具有成骨分化的能力;然后将MC3T3-E1细胞接种于含不同终浓度β-蜕皮甾酮(0.01,0.1,1,10,100μmol/L)的诱导培养基中,分别于第1,2,3,4,5,6,7天利用CCK8法检测细胞的增殖活性;最后设置对照组(普通诱导培养基组)和实验组(普通诱导培养基+β-蜕皮甾酮),在相同条件下进行培养,并测定不同时间段各组细胞成骨标志蛋白的表达量。结果与结论:①MC3T3-E1细胞在成骨诱导培养基刺激下,第10天碱性磷酸酶染色以及Ⅰ型胶原蛋白荧光染色表达较高,同时碱性磷酸酶活性检测也验证了这一结果(P<0.05);诱导培养第14天骨桥蛋白免疫细胞化学染色也有明显表达;茜红素染色显示成骨诱导后的细胞较对照组结节数量明显增加,第28天比第21天的钙结节形成数目更多、直径更大、颜色更深;②CCK 8法测得β-蜕皮甾酮对MC3T3-E1细胞作用5 d后增殖活性达到最佳,促增殖活性最佳剂量为0.01μmol/L、0.1μmol/L,2种浓度之间差异无显著性意义(P>0.05);③实验组细胞经β-蜕皮甾酮诱导培养第10天较对照组细胞碱性磷酸酶、Ⅰ型胶原蛋白表达更高;第14天实验组细胞内骨桥蛋白、骨钙素表达更高;第28天2组钙结节染色无明显差BACKGROUND:β-ecdysterone as a“phytoestrogen”has the ability to stimulate protein synthesis,promote carbohydrate and lipid metabolism,relieve hyperglycemia and hyperlipidemia,protect endothelial cells from apoptosis and induce their proliferation.Some scholars have reported that it also plays an important role in the treatment of osteoporosis,fractures and other bone inflammatory diseases.OBJECTIVE:To observe the effect of β-ecdystrone on the proliferation of mouse pre-osteoblasts(MC3T3-E1 cells)in vitro,and to explore whether β-ecdysterone can induce osteogenic differentiation of MC3T3-E1 at a safe dose.METHODS:The fourth generation MC3T3-E1 cells were cultured in the osteogenic induction medium for 7,10,14,21,and 28 days.The osteogenic differentiation proteins(alkaline phosphatase,type I collagen,osteopontin,and calcified nodules)were detected at different time points,to identify whether the cells have the ability of osteogenic differentiation.MC3T3-E1 cells were then seeded into the induction medium containing different final concentrations of β-ecdysterone(0.01,0.1,1,10,100μmol/L).The proliferation activity of the cells was detected by cell counting kit-8 method at days 1,2,3,4,5,6,and 7 after induction.The control group(general induction medium group)and the experimental group(general induction medium+β-ecdysterone)were cultured under the same conditions,and the expression levels of osteogenic marker proteins in each group of cells at different time periods were determined.RESULTS AND CONCLUSION:In the MC3T3-E1 cells stimulated by the osteogenic induction medium,alkaline phosphatase staining and type I collagen florescence staining showed higher expression at day 10 of induction,and this was also confirmed by detection of alkaline phosphatase activity(P<0.05).At day 14 of induction,osteopontin immunocytochemical staining also indicated significant expression.Alizarin red staining results demonstrated that the number of calcified nodules increased significantly after osteogenic induction,and there we

关 键 词：β-蜕皮甾酮 MC3T3-E1细胞 成骨分化 碱性磷酸酶 Ⅰ型胶原蛋白 骨桥蛋白 骨钙蛋白 

分 类 号：R446[医药卫生—诊断学] R496[医药卫生—临床医学]