RhoA和核骨架-细胞骨架连接复合体在力学调控生长板软骨细胞肥大分化中的作用及其机制

The role and related mechanism of RhoA and linker of nucleoskeleton and cytoskeleton complex in mechanical strain-regulated hypertrophy differentiation of growth plate chondrocytes

目的:研究核骨架-细胞骨架连接复合体(LINC complex)在周期性张应力调控大鼠生长板软骨细胞肥大分化中的作用。方法:采用四点弯曲张力仪,对体外培养的SD大鼠(华中科技大学同济医学院动物实验中心提供)生长板软骨细胞行周期性张应力,采用小干扰RNA(siRNA)抑制LINC complex(SUN1和SUN2)的活性,实验分组为对照组、对照组+siSUN、应力组和应力组+siSUN,蛋白质印迹法(Western blot)检测力学刺激对FAK、YAP和RhoA蛋白活性的改变,QPCR检测肥大分化指标Ihh和COL X的mRNA表达变化。两组间比较采用t检验。结果:应力组FAK、YAP和RhoA的蛋白活化明显高于对照组[磷酸化FAK(p-FAK):0.993±0.082比0.423±0.111,t=36.539,P<0.01;磷酸化YAP(p-YAP):0.734±0.071比1.635±0.127,t=19.722,P<0.01;活化RhoA(Active RhoA):1.288±0.076比0.646±0.132,t=18.157,P<0.01],同时应力组Ihh和COL X的mRNA表达水平低于对照组(Ihh:0.973±0.227比3.634±0.381,t=104.589,P<0.01;COL X:1.957±0.274比5.991±0.421,t=47.177,P<0.01),给予细胞siSUN抑制LINC complex(SUN1和SUN2)的活性,应力组FAK、YAP和RhoA蛋白的活化与对照组比较,差异无统计学意义(p-FAK:0.559±0.062比0.527±0.073,t=1.595,P>0.05;p-YAP:1.258±0.064比1.342±0.075,t=1.334,P>0.05;active RhoA:0.494±0.065比0.508±0.094,t=2.245,P>0.05),同时应力组Ihh和COL X的mRNA的表达与对照组比较,差异也无统计学意义(Ihh:1.264±0.262比1.719±0.345,t=2.197,P>0.05;COL X:1.747±0.185比2.361±0.367,t=2.175,P>0.05)。结论:持续的周期性张应力通过LINC complex调控大鼠生长板软骨细胞的肥大分化。

Objective To investigate the role of LINC complex(Linker of nucleoskeleton and cytoskeleton complex)in mechanical strain-regulated hypertrophy differentiation of growth plate chondrocytes.Methods Rat growth plate chondrocytes were subjected to cyclic tensile strain.The cells were divided into four groups:The control group,the control group+siSUN,the strain group,the strain group+siSUN.Western blotting was used to examine the change of the activation of FAK,YAP and RhoA protein by cyclic tensile strain.And the mRNA expression of Ihh and COL X were examined by QPCR.T-test was used for comparison between the two groups.Results The activation of FAK,YAP and RhoA protein in strain group were higher than the control group[phosphoylated FAK(p-FAK):0.993±0.082 vs.0.423±0.111,t=36.539,P<0.01,phosphoylated YAP(p-YAP):0.734±0.071 vs.1.635±0.127,t=19.722,P<0.01,Active RhoA:1.288±0.076 vs.0.646±0.132,t=18.157,P<0.01].Meanwhile,the mRNA expression of Ihh and COL X in strain group were lower than the control group(Ihh:0.973±0.227 vs.3.634±0.381,t=104.589,P<0.01,COL X:1.957±0.274 vs.5.991±0.421,t=47.177,P<0.01).If the cells were treated by siSUN,there were no difference of the activation of FAK,YAP and RhoA protein between the strain group and contrl group(p-FAK:0.559±0.062 vs.0.527±0.073,t=1.595,P>0.05,p-YAP:1.258±0.064 vs.1.342±0.075,t=1.334,P>0.05,active RhoA:0.494±0.065 vs.0.508±0.094,t=2.245,P>0.05).Meanwhile,there were no difference of the mRNA expression of Ihh and COL X between the strain group and contrl group(Ihh:1.264±0.262 vs.1.719±0.345,t=2.197,P>0.05,COL X:1.747±0.185 vs.2.361±0.367,t=2.175,P>0.05).Conclusion Cyclic tensile strain could regulate the hypertrophy differentiation of rat growth plate chondrocytes through LINC complex.