基质相互作用分子2对软骨细胞钙离子释放激活钙通道的调控作用

The regulatory effects of stromal interactional molecule-2on Ca2+ release activated Ca2+ channel of chondrocytes

目的利用软骨细胞构建基质相互作用分子2(stromal interaction molecule-2,STIM2)过表达模型探讨STIM2蛋白在软骨细胞钙离子释放激活钙(Ca2+ release activated Ca2+,CRAC)通道中的调控作用。方法骨关节炎软骨细胞及正常软骨细胞分别取自全膝关节置换术中软骨退变及正常区域,采用胞内钙离子信号实时荧光检测技术对骨关节炎和正常软骨细胞CRAC通道功能进行检测;采用蛋白质印迹技术检测骨关节炎与正常软骨细胞中STIM2蛋白的表达情况。构建质粒并进行转染上调正常软骨细胞STIM2蛋白水平,使用细胞荧光、Real-time PCR与蛋白质印迹验证转染效率;采用胞内钙离子信号实时荧光检测技术对STIM2转染组与空载质粒组软骨细胞中CRAC通道功能的改变进行检测。结果在毒胡萝卜素处理后,骨关节炎软骨细胞的内质网释放钙离子信号的峰值强度[(2.13±0.12)倍]较正常软骨细胞[(2.87±0.32)倍]下降;在外源性钙离子处理后,骨关节炎软骨细胞钙离子信号荧光强度到达峰值所需的时间[(336.85±15.88)s]较正常软骨细胞[(231.96±17.82)s]增加。蛋白质印迹显示骨关节炎软骨细胞中STIM2蛋白表达增加。质粒转染后,STIM2转染组的软骨细胞荧光强度增加,STIM2基因相对表达量增加,STIM2蛋白表达增加。在毒胡萝卜素处理后STIM2转染组软骨细胞内质网释放钙离子信号的峰值强度[(2.60±0.19)倍]较空载质粒组[(4.58±0.28)倍]下降;外源性钙离子处理后STIM2转染组软骨细胞内质网释放钙离子信号的峰值强度[(2.24±0.19)倍]较空载质粒组[(4.34±0.33)倍]下降。结论软骨细胞中STIM2蛋白负向调控CRAC通道功能,减少内质网内储存性钙离子的浓度。

Objective To investigate the regulatory effects of stromal interaction molecule-2(STIM2)on Ca2+ release acti- vated Ca2+(CRAC)channel of chondrocytes using STIM2overexpression model.Methods Chondrocytes were harvested from degenerative (osteoarthritic chondrocytes)or normal regions (normal chondrocytes)of articular cartilage during total knee arthroplasty.CRAC channel function of chondrocytes was tested with Real-time calcium imaging.The protein expression level of STIM2was examined with Western Blot.The STIM2overexpression plasmid was constructed and transfected into chondrocytes.The transfect efficiency was verified by cell fluorescence,Real-time PCR,and Western Blot.Results Compared with normal chondrocytes (2.87±0.32folds,231.96±17.82s),osteoarthritic chondrocytes (2.13±0.12folds,336.85±15.88s)demonstrated lower peak intensity of calcium signal in the presence of thapsigargin,and required longer duration to reach saturated calcium concentration after exogenous calcium addition.STIM2expression level was increased in osteoarthritic chondrocytes.After plasmid transfection,the fluorescence intensity,gene expression and protein level of STIM2in chondrocytes were increased.Compared with chondrocytes in control group (4.58±0.28folds,4.34±0.33folds),chondrocytes of STIM2overexpression group (2.60±0.19folds,2.24±0.19folds) showed decreased calcium signal peak intensity upon either thapsigargin stimulation or exogenous calcium addition.Conclusion STIM2negatively regulates CRAC channel function and decreases stored-Ca2+ in chondrocytes.