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

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。

Objective Marrow stromal cells(MSCs)are key cells in the process of fracture healing,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 reaction(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 intracellular calcium ion influx in the control group and the stretching mechanical group,and the intracellular localization was studied by immunofluorescence staining of TRPV4 and F-actin protein.After preliminarily determining 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 enhanced 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 responsive F-actin protein were enhanced after mechanical stimulation.Fura-4 probe results showed that mechanical 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 activate the TRPV4 protein in MSCs,co localize with F-actin,mediate calcium ion influx,and thereby enhance osteogenic differentiation activity.