目的:考察30 kPa流体静压力调控髁突软骨细胞增殖及凋亡的分子机制.方法:体外培养大鼠髁突软骨细胞,分为4组:正常压力组、30 kPa压力组、KN93对照组、30 kPa压力+KN93组.5-乙炔基-2'脱氧尿嘧啶核苷(5-ethynyl-2-deoxyuridine,EdU)标记法测定细胞增殖.流式细胞术法检测细胞凋亡比例.荧光探针法检测细胞内 Ca2+水平.Western blot 法检测细胞钙调蛋白依赖性蛋白激酶 Ⅱ(calcium/calmodulin-dependent protein kinaseⅡ,CaMK Ⅱ)、p-c-Jun、c-Jun、磷酸化的细胞外信号调节激酶 1/2(phosphorylated extracellular signal-regulated ki-nase 1/2,p-ERK1/2)、细胞外信号调节激酶 1/2(extracellular signal-regulated kinase 1/2,ERK1/2)、p-p38、p38 蛋白表达.结果:与正常压力组相比,30 kPa压力组细胞增殖率均降低;凋亡率均提高;细胞内Ca2+水平升高;CaMKⅡ蛋白表达上调,(p-c-Jun)/(c-Jun)、(p-ERK1/2)/(ERK1/2)、(p-p38)/(p38)的比值均增加,差异具有统计学意义(P＜0.01).与30 kPa压力组相比,30 kPa压力+KN93组细胞增殖率均提高;凋亡率均降低;细胞内Ca2+水平降低;CaMK Ⅱ蛋白表达下调,(p-c-Jun)/(c-Jun)、(p-ERK1/2)/(ERK1/2)、(p-p38)/(p38)的比值均降低,差异具有统计学意义(P＜0.01).结论:30 kPa静压力持续作用髁突软骨细胞,可抑制细胞增殖,促进细胞凋亡,提高细胞内Ca2+水平,其作用机制为Ca2+通过CaMK Ⅱ激活丝裂原活化蛋白激酶(mitogen activated protein kinases,MAPK)信号通路.
Hydrostatic Pressure Regulates Proliferation and Apoptosis of Condylar Chondrocytes through Ca2+/CaMK Ⅱ/MAPK Signaling Pathway
Objective:To investigate the molecular mechanism of 30 kPa hydrostatic pressure regulating the proliferation and apoptosis of condylar chondrocytes.Methods:Rat condylar chondrocytes were cultured in vitro.Cells divided into four groups:normal pressure group,30 kPa pressure group,KN93 control group,and 30 kPa pressure+KN9 3 group.EdU labeling method was used to measure cell proliferation.Flow cytometry was used to detect the proportion of cell ap-optosis.Fluorescence probe method was used to detect intracellular Ca2+levels.Western blot was used to detect the expression of CaMK Ⅱ,p-c-Jun,c-Jun,p-ERK1/2,ERK1/2,p-p38,and p38 proteins in cells.Results:Compared with the normal pressure group,the cell proliferation rate in the 30 kPa pressure group decreased,the cell apopto-sis rate increased significantly,the intracellular Ca2+ levels increased significantly,the expression of CaMK Ⅱ pro-tein was upregulated,and the ratios of(p-c-Jun)/(c-Jun),(p-ERK1/2)/(ERK1/2),and(p-p38)/(p38)were all increased(P＜0.01).Compared with the 30 kPa pressure group,the cell proliferation rate in the 30 kPa pressure+KN93 group was increased,the cell apoptosis rate decreased,the intracellular Ca2+level decreases,the expression of CaMK Ⅱ protein was downregulated,and the ratios of(p-c-Jun)/(c-Jun),(p-ERK1/2)/(ERK1/2),and(p-p38)/(p38)were all reduced(P＜0.01).Conclusion:The sus-tained effect of 30 kPa hydrostatic pressure can inhibit con-dylar chondrocytes proliferation,promote cell apoptosis,and increase intracellular Ca2+ levels.The mechanism of action may be that Ca2+ activates the MAPK signaling pathway through CaMK Ⅱ.
NETL
NSTL
万方数据
