骨髓基质干细胞中TRPV4转导力学信号的研究
TRPV4-mediated mechanotransduction in marrow stromal cells
严旭 1付苏 1谢莹 2姜岩 1宁永明 1尚春风 1陈松峰 1毛克亚 3刘宏建1
作者信息
- 1. 郑州大学第一附属医院骨科医学部,郑州 450052
- 2. 郑州大学第一附属医院输血科,郑州 450052
- 3. 中国人民解放军总医院骨科医学部,北京 100853
- 折叠
摘要
目的 探讨TRPV4调控骨髓基质干细胞(MSC)成骨分化过程中与F-激动蛋白(F-actin)的定位关系及钙离子内流机制.方法 使用购买的大鼠MSC,分为对照组和拉伸力学组,体外进行成骨诱导培养7、14、21 d,进行茜素红染色和聚合酶链反应(PCR)检测成骨基因骨形成蛋白-2(BMP-2)和Runx2活性,同时通过蛋白质印迹法(Western blot)检测碱性磷酸酶(ALP)及TRPV4蛋白表达.使用钙离子荧光探针(Fura-4)检测对照组和拉伸力学组的细胞内钙离子内流,并通过免疫荧光染色TRPV4及F-actin蛋白,观察细胞内定位关系.初步确定TRPV4影响后,使用TRPV4激动剂GSK101处理MSC,分为对照组和GSK101组,检测成骨基因BMP-2及Runx2变化.两组间比较行配对t检验.结果 大鼠MSC行成骨诱导培养后,拉伸力学刺激显著促进MSC成骨分化,茜素红染色增强,且力学刺激于术后7 d显著促进BMP-2基因和Runx2基因转录活性增加(t=5.252、2.851,P＜0.05),变化倍数分别为(3.97±0.82)倍和(3.47±0.42)倍.ALP蛋白表达增多(t=4.069,P＜0.05),变化倍数为(2.42±0.86)倍.此时,TRPV4蛋白表达量维持不变(t=1.115,P＞0.05),变化倍数为(1.21±0.78)倍.免疫荧光染色结果证实力学刺激后TRPV4及力学反应性F-actin蛋白表达定位增强,Fura-4探针结果显示,拉伸力学刺激促进了 MSC体内钙离子内流(t=7.048,P＜0.01),变化倍数为(4.29±0.72)倍.使用TRPV4激动剂GSK101处理MSC后,成骨基因BMP-2及ALP的转录活性显著增强(t=3.762、3.977,P＜0.05),变化倍数分别为(3.68±1.05)倍和(4.33±0.63)倍.结论 拉伸力学刺激可激活MSC的TRPV4蛋白,于F-actin共定位,介导钙离子内流,从而提高成骨分化活性.
Abstract
Objective Marrow stromal cells(MSCs)are key cells in the process of fracture heal-ing,and mechanical stimulation can regulate the process of osteogenic differentiation.This study explores the localization of TRPV4 and F-actin as well as the calcium influx during osteogenic differentiation of MSCs.TRPV4 is therefore proposed as a potential clinical therapeutic target.Methods In vitro,the rat MSCs were divided into a control group and a stretching mechanical group.Osteogenesis induction culture was conducted in vitro for 7 days,14 days,and 21 days.Alizarin red staining and polymerase chain reac-tion(PCR)were performed to detect the activity of osteogenic genes[bone morphogenetic protein-2(BMP-2)and Runx2],and Western blotting was used to detect the expression of alkaline phosphatase(ALP)and TRPV4 proteins.A calcium ion fluorescence probe(Fura-4)was used to detect the intracellu-lar calcium ion influx in the control group and the stretching mechanical group,and the intracellular locali-zation was studied by immunofluorescence staining of TRPV4 and F-actin protein.After preliminarily deter-mining the impact of TRPV4,MSCs were treated with TRPV4 agonist(GSK101)and divided into a control group and GSK101 group.Changes in osteogenic genes(BMP-2 and Runx2)were detected.The paired t-test was done for comparison between two groups.Results After osteogenic induction culture of rat MSCs,tensile mechanical stimulation significantly promoted osteogenic differentiation of MSCs,with en-hanced Alizarin Red staining.Mechanical stimulation significantly increased the transcriptional activity of BMP-2 gene and Runx2 gene at 7th day after surgery(t=5.252,2.851,P＜0.05),with changes of(3.97±0.82)times and(3.47±0.42)times,respectively.The expression of ALP protein increased(t=4.069,P＜0.05),with a fold change of 2.42±0.86.At this time,the expression level of TRPV4 protein remained unchanged(t=1.115,P＞0.05),with a change multiple of(1.21±0.78)times.The results of immunofluorescence staining confirmed that the expression and localization of TRPV4 and mechanical re-sponsive F-actin protein were enhanced after mechanical stimulation.Fura-4 probe results showed that me-chanical stimulation promoted calcium ion influx in MSCs(t=7.048,P＜0.01),with a change multiple of 4.29±0.72.After treating MSCs with TRPV4 agonist GSK101,the transcriptional activity of osteogenic genes(BMP-2 and ALP)was significantly enhanced(t=3.762,3.977,P＜0.05),with changes of(3.68±1.05)times and(4.33±0.63)times,respectively.Conclusion Stretching stimulation can acti-vate the TRPV4 protein in MSCs,co localize with F-actin,mediate calcium ion influx,and thereby en-hance osteogenic differentiation activity.
关键词
骨髓基质干细胞/成骨分化/F-激动蛋白/钙离子Key words
Marrow stromal cells/Osteogenic differentiation/F-actin/Calcium引用本文复制引用
基金项目
河南省医学科技攻关计划项目(LHGJ20190168)
出版年
2024
NETL
NSTL
万方数据