摘要
目的:探究龙血素B对小鼠胚胎成骨细胞前体细胞(MC3T3-E1细胞)成骨分化和骨形成的影响以及其通过P38MAPK信号通路调控成骨分化和骨形成的机制。方法:培养MC3T3-E1细胞,加入不同浓度(0、15、30、60、90、120μmol/L)龙血素B,采用CCK8法和流式细胞术(FCM)检测不同时间(24h、48h、72h)MC3T3-E1细胞增殖能力和凋亡情况,确定龙血素B促MC3T3-E1细胞成骨最佳的药物浓度及作用时间。培养MC3T3-E1细胞并分为空白组(不作干预),龙血素B组(加入龙血素B共培养),龙血素B+阻断剂组(加入龙血素B和P38抑制剂SB203580共培养),进行干预实验;采用碱性磷酸酶(ALP)活性检测试剂盒测定各组细胞ALP活性,qRT-PCR法检测各组细胞骨桥蛋白(OPN)基因、骨钙素(OCN)基因、骨唾液蛋白(BSP)基因表达水平,茜素红染色法观察各组细胞骨形成能力。结果:龙血素B可促进MC3T3-E1细胞增殖并抑制其凋亡,效果与浓度和干预时间相关,在90μmol/L浓度下干预48h促MC3T3-E1细胞生长作用最强;干预结束后第1、3、5天,龙血素B组细胞ALP活性较空白组升高,龙血素B+阻断剂组细胞ALP活性较龙血素B组降低(P<0.05);龙血素B组细胞OPN、OCN、BSP基因表达水平较空白组升高,龙血素B+阻断剂组细胞OPN、OCN、BSP基因表达水平较龙血素B组降低(P<0.05);干预结束后第21天,龙血素B组细胞钙化结节区域面积较空白组增大,龙血素B+阻断剂组细胞钙化结节区域面积较龙血素B组减小(P<0.05)。结论:龙血素B可提高MC3T3-E1细胞增殖活性,并通过激活P38MAPK信号通路促进MC3T3-E1细胞成骨分化和骨形成。
Objective:To investigate the effect of loureirin B on osteogenic differentiation and osteogenesis in MC3T3-E1 cells,and its mechanism of regulating osteogenic differentiation and osteogenesis via P38MAPK signaling pathway.Methods:Mouse embryonic osteoblast precursor cells(MC3T3-E1 cells)were cultured and different concentrations(0μmol/L,15μmol/L,30μmol/L,60μmol/L,90μmol/L and 120μmol/L)of loureirin B were added to the cells.The proliferation and apoptosis of MC3T3-E1 cells at different time(24 hours,48 hours and 72 hours)were detected by CCK8 method and flow cytometry(FCM).The optimal concentration and action time of loureirin B to promote osteogenesis of MC3T3-E1 cells were determined.MC3T3-E1 cells were divided into the blank group(no intervention),the loureirin B group(co-cultured with loureirin B)and the loureirin B+blocker group(co-cultured with loureirin B and P38 inhibitor SB203580).The activity of alkaline phosphatase(ALP)in each group was detected using an ALP activity detection kit.The expression levels of osteopontin(OPN)gene,osteocalcin(OCN)gene and bone sialoprotein(BSP)gene were measured by qRT-PCR method.Osteogenesis of each group was observed by alizarin staining method.Results:Loureirin B can promote the proliferation of MC3T3-E1 cells and inhibit their apoptosis.The effect was related to concentration and intervention time.Intervention at the concentration of 90μmol/L for 48 hours could achieve the strongest effect on promoting the growth of MC3T3-E1 cells.On the 1st,3rd and 5th day after intervention,the activity of ALP in the loureirin B group increased compared to the blank group,and the activity of ALP in the loureirin B+blocker group decreased compared to the loureirin B group(P<0.05).The expression levels of OPN gene,OCN gene and BSP gene in the loureirin B group were higher than those in the blank group.The expression levels of OPN gene,OCN gene and BSP gene in the loureirin B+blocker group were lower than those in the loureirin B group(P<0.05).After 21st day after intervention,the area of calcified nodules in the loureirin B group increased compared to the blank group.The area of calcified nodules in the loureirin B+blocker group decreased compared to the loureirin B group(P<0.05).Conclusion:Loureirin B can enhance the proliferation of MC3T3-E1 cells,and promote osteogenic differentiation and osteogenesis of MC3T3-E1 cells by activating P38MAPK signaling pathway.
作者
李经堂
涂乐佳
陈淼
魏鹏
周璟瑜
LI Jingtang;TU Lejia;CHEN Miao(Jiangxi Provincial People’s Hospital,the First Affiliated Hospital of Nanchang Medical College,Nanchang City,Jiangxi Province 330006;不详)
出处
《医学理论与实践》
2024年第7期1081-1084,共4页
The Journal of Medical Theory and Practice
基金
江西省中医药管理局科技计划(2022B935)。
