泼尼松龙可抑制转化生长因子β激活激酶1诱导的成骨细胞凋亡  

Prednisolone inhibites osteoblast apoptosis induced by transforming growth factor beta activated kinase 1

作　　者：张雯 张蕾 任守忠[1] 刘日升 Zhang Wen;Zhang Lei;Ren Shouzhong;Liu Risheng(the First Affiliated Hospital of Hainan Medical University,Haikou 570102,Hainan Province,China;Hainan Cancer Hospital,Haikou 570102,Hainan Province,China)

机构地区：[1]海南医学院第一附属医院,海南省海口市570102 [2]海南省肿瘤医院,海南省海口市570102

出　　处：《中国组织工程研究》2019年第23期3630-3635,共6页Chinese Journal of Tissue Engineering Research

基　　金：海南省卫生计生行业科研项目(16A200077),项目负责人:张蕾~~

摘　　要：背景:研究显示转化生长因子β激活激酶1(transforming growth factor beta activated kinase 1,TAK1)在骨、关节的发育以及骨形态信号转导中发挥着重要作用,与骨关节炎的发生也存在一定的相关性。目的:探究泼尼松龙通过抑制TAK1表达对诱导成骨细胞凋亡的影响。方法:将M3T3-E1成骨细胞经原代培养后传代培养。取第3代细胞分为3组,正常细胞组(对照组)、阴性转染+泼尼松龙组、TAK1 siRNA转染+泼尼松龙组。采用碱性磷酸酶染色和钙结节染色评估成骨细胞分化能力的变化;采用免疫印迹法(Western blot)检测细胞内磷酸化(p)-TAK1、TAK1、磷酸化c-jun氨基末端激酶(p-JNK)、JNK蛋白表达,MTT法检测M3T3-E1细胞增殖情况;流式细胞仪检测细胞周期以及细胞凋亡变化。结果与结论:①TAK1 siRNA转染+泼尼松龙组细胞的碱性磷酸酶红染程度较少,阴性转染+泼尼松龙组次之,正常细胞组碱性磷酸酶染色最多;钙结节染色显示与正常细胞组相比,阴性转染+泼尼松龙组钙结节数量明显减少,TAK1 siRNA转染+泼尼松龙组结节数量最少;②荧光显微镜显示,阴性转染+泼尼松龙组细胞出现破碎,形态发生改变,TAK1 siRNA转染+泼尼松龙组破碎细胞数量明显增加;③Western Blot显示,3组间p-TAK1、p-JNK蛋白表达量逐渐降低(P<0.05);④MTT检测显示,3组间TAK1 siRNA转染+泼尼松龙组细胞增殖抑制率最高(P<0.05);在12-48h随着时间的延长,细胞增殖抑制率呈逐渐上升趋势,在72h时开始下降;⑤流式细胞仪检测结果显示,TAK1 siRNA转染+泼尼松龙组G2期细胞比例高于其他2组,S期细胞比例显著低于其他2组(P<0.05);⑥TAK1 siRNA转染+泼尼松龙组细胞凋亡率明显高于正常细胞组、阴性转染+泼尼松龙组(P<0.05);⑦结果说明,沉默TAK1表达后能够增强泼尼松龙诱导成骨细胞凋亡的作用,可能与JNK信号通路相关。BACKGROUND:Transforming growth factor beta activated kinase 1(TAK1)has been shown to play important roles in development of bone and joint and bone morphology signal transduction,and is related to osteoarthritis.OBJECTIVE:To explore the influence of prednisolone on osteoblast apoptosis induced by inhibiting TAK1 expression.METHODS:M3T3-E1 osteoblasts were cultured and passaged.The third generation of cells were divided into three groups:control group(normal group),negative transfection + prednisolone group,and TAK1 siRNA transfection + prednisolone group.The changes of osteoblast differentiation ability were evaluated by alkaline phosphatase staining and calcium nodule staining.Intracellular phosphorylation(p)-TAK1,and TAK1,phosphorylated c-jun amino,terminal kinase(p-JNK),and JNK protein expression levels were detected by western blot assay.MTT assay was used for M3T3-E1 cell proliferation.Flow cytometry was used to detect the cell cycle and apoptosis.RESULTS AND CONCLUSION:(1)Alkaline phosphatase staining showed that the number of stained cells in the TAK1 siRNA transfection + prednisolone group was least,followed by negative transfection + prednisolone group,and most in the control group.Calcium nodule staining showed that the number of intracellular calcium nodules in the negative transfection + prednisolone and TAK1 siRNA transfection + prednisolone groups decreased compared with the control group,especially the TAK1 siRNA transfection + prednisolone group.(2)Fluorescence microscope showed that the cells in the negative transfection + prednisolone group were broken and the morphology changed.The number of broken cells in the TAK1 siRNA transfection + prednisolone group increased significantly.(3)Western blot assay showed that the expression levels of p-TAK1 and p-JNK protein decreased gradually(P<0.05).(4)MTT assay showed that TAK1 siRNA transfection + prednisolone group had highest cell inhibition rate(P<0.05).Within 12 to 48 hours,the cell inhibition rate was on a rise,and then decreased at 72 hours.(5)The

关 键 词：转化生长因子β激活激酶1 泼尼松龙 成骨细胞 细胞凋亡 JNK信号通路 TAK1 基因沉默 

分 类 号：R446[医药卫生—诊断学]