外源性Klotho对糖尿病肾病小鼠成骨细胞甲状旁腺激素刺激的分化活性调控作用  

Regulatory effect of Klotho on changes of osteoblast differentiation activity induced by PTH(1-34)in diabetic nephropathy mice

作　　者：刘建梅[1] 贾俊亚[1] 闫铁昆[1] 于亚民 韦丽[1] 商文雅[1] 林珊[1] LIU Jianmei;JIA Junya;YAN Tiekun;YU Yamin;WEI Li;SHANG Wenya;LIN Shan(Nephrology Department,General Hospital of Tianjin Medical University,Tianjin 300052,China)

机构地区：[1]天津医科大学总医院肾内科,天津300052

出　　处：《山东医药》2021年第33期20-23,共4页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(30800529,81270791);天津市科委基金项目(14JCYBJC27900)。

摘　　要：目的观察外源性Klotho对糖尿病肾病小鼠成骨细胞甲状旁腺激素(PTH)刺激的分化活性的调控作用。方法18周龄KK-Ay糖尿病肾病模型小鼠及C57BL/6J对照小鼠各12只,处死后取股骨,采用骨片贴壁分离筛选法获取成骨细胞,分为A、B组,KK-Ay小鼠成骨细胞为A组,C57BL/6J小鼠成骨细胞为B组。A组成骨细胞分别加入不同浓度的PTH(1-34)、Klotho,分别记为A1、A2、A3、A4、A5组,其中A1组加入0μg/mL的PTH(1-34),A2组加入0.05μg/mL的PTH(1-34),A3组加入0.1μg/mL的PTH(1-34),A4组加入0.2μg/mL的PTH(1-34),A5组加入0.1μg/mL的PTH(1-34)和100 ng/mL的Klotho。B组成骨细胞参照A组各亚组给予相同处理,分别记为B1组、B2组、B3组、B4组、B5组。采用ELISA法检测各组细胞中环磷酸腺苷(cAMP),采用蛋白印迹法检测各组细胞中磷酸化蛋白激酶A(p-PKA)、磷酸化细胞外调节蛋白激酶(p-ERK)1/2,作为成骨细胞对PTH刺激反应性的指标。结果A1组、A2组、A3组、A4组细胞中cAMP浓度、p-PKA相对表达量、p-ERK1/2相对表达量组间相比,P均>0.05。B1组、B2组、B3组、B4组细胞中cAMP浓度、p-PKA相对表达量、p-ERK1/2相对表达量组间相比,P均<0.05。A5组细胞中cAMP浓度、p-PKA相对表达量、p-ERK1/2相对表达量与B5组相比,P均>0.05。结论正常小鼠成骨细胞对PTH刺激反应性呈剂量依赖性上升,而糖尿病肾病小鼠成骨细胞对PTH刺激无反应;加入外源性Klotho可促进糖尿病肾病小鼠成骨细胞对PTH的刺激反应。Objective To investigate the regulatory effect of exogenous Klotho on the activity of osteoblasts in diabetic nephropathy mice induced by recombinant parathyroid hormone(PTH)(1-34).Methods Twelve 18-week-old KK-Ay diabetic nephropathy model mice and 12 C57BL/6J mice were sacrificed to collect their femora,and osteoblasts were cultured and identified by the method of adherent separation and screening of bone fragments.Osteoblasts were then divided into two groups:the osteoblasts from KK-Ay mice were taken as the group A and osteoblasts from C57BL/6J mice as group B.The osteoblasts in the group A were treated with different concentrations of PTH(1-34)and Klotho,which were marked as groups A1,A2,A3,A4,andA5.Osteoblasts in the group A1 were added with 0μg/mL PTH(1-34),group A2 with 0.05μg/mL PTH(1-34),group A3 with 0.1μg/mL PTH(1-34),group A4 with 0.2μg/mL PTH(1-34),and group A5 with 0.1μg/mL PTH(1-34)and 100 ng/mL Klotho.The osteoblasts in the group B were divided into groups B1,B2,B3,B4,and B5,which were treated with the same methods of subgroups of group A.Intracellular cyclic adenosine monophosphate(cAMP)levels were detected by ELISA,and the levels of phosphorylated PKA(p-PKA)and phosphorylated extracellular signal-regulated kinase(p-ERK)1/2 in osteoblasts were detected by Western blotting,which were taken as the indicators of osteoblasts’response to PTH stimulation.Results There were no significant differences in the levels of intracellular cAMP,p-PKA or p-ERK1/2 between groups A1,A2,A3,and A4(all P>0.05).Significant differences were found in the levels of intracellular cAMP,p-PKA and p-ERK1/2 between groups B1,B2,B3,and B4(all P<0.05).There were no significant differences in the levels of intracellular cAMP,p-PKA,or p-ERK1/2 between group A5 and group B5(all P>0.05).Conclusions The osteoblasts of normal mice show a dose-dependent increase in the response to PTH stimulation,while the osteoblasts of diabetic nephropathy mice do not respond to PTH stimulation.Adding Klotho can improve the osteoblasts’resp

关 键 词：KLOTHO 成骨细胞 糖尿病并发症 糖尿病肾病 甲状旁腺激素 

分 类 号：R587.1[医药卫生—内分泌]