慢性肾功能不全幼鼠胫骨生长板软骨细胞糖原合成酶激酶3β表达水平变化对生长板发育的影响  

Expression level of glycogen synthase kinase 3βin growth plate chondrocyte on the development of tibial growth plate in young rats with chronic renal insufficiency

作　　者：余建平[1] 王小健[1] 安雪军[1] 贾中伟[1] 郭松佳 路晓 郭秀生[1] 苏云星[1] Yu Jianping;Wang Xiaojian;An Xuejun;Jia Zhongwei;Guo Songjia;Lu Xiao;Guo Xiusheng;Su Yunxing(Department of Orthopedic Surgery;Shanxi Provincial Key Laboratory of Nephrology,Shanxi Provincial People's Hospital,Taiyuan 030012,China)

机构地区：[1]山西省人民医院骨科,太原030012 [2]山西省人民医院肾脏病山西省重点实验室,太原030012

出　　处：《中华小儿外科杂志》2021年第12期1124-1130,共7页Chinese Journal of Pediatric Surgery

基　　金：山西省自然科学基金(201701D121161);山西省重点研发计划项目(201803D31160)。

摘　　要：目的观察慢性肾功能不全(chroic renal insufficiency,CRI)幼鼠胫骨生长板软骨细胞糖原合成酶激酶3β(glycogen synthase kinase 3β,GSK3β)表达水平变化对生长板发育的影响。方法将40只4周龄雄性SD幼鼠,按随机数字表法平均分为CRI组(腺嘌呤150 mg·kg^(-1)·d^(-1)灌胃,n=20)和对照组(同剂量蒸馏水灌胃,n=20),连续灌胃6周后处死全部幼鼠,取双侧胫骨制作组织学切片、番红固绿染色后比较两组幼鼠胫骨近端生长板宽度,应用免疫组织化学技术检测GSK3β表达部位及水平;提取两组胫骨生长板软骨细胞进行体外培养至第3代,观测软骨细胞内GSK3β、磷酸化GSK3β(p-GSK3β)、Wnt/β通路关键蛋白β联蛋白(β-catenin)、基质金属蛋白酶-13(matrix metalloproteinase-13,MMP-13)表达水平,裂解细胞后应用蛋白质印迹法技术检测GSK3β、p-GSK3β、β-catenin、MMP-13表达水平。两组间比较采用成组t检验。结果幼鼠胫骨组织学切片显示CRI组生长板相对宽度为(0.52±0.14),对照组为(1.00±0.07),组间比较差异有统计学意义(P<0.001)。幼鼠胫骨组织学切片免疫组织化学结果显示CRI组软骨细胞GSK3β评分为(1.8±0.4)分,对照组为(7.1±2.2)分,组间差异有统计学意义(P<0.001)。体外细胞实验免疫组织化学结果显示CRI组软骨细胞内GSK3β评分为(0.94±0.32)分,对照组为(5.63±1.71)分,组间比较差异有统计学意义(P<0.001);CRI组p-GSK3β评分为(4.97±1.10)分,对照组为(1.58±0.51)分,组间比较差异有统计学意义(P<0.001);CRI组β-catenin评分为(5.11±2.03)分,对照组为(1.43±0.31)分,组间比较差异有统计学意义(P<0.001);CRI组MMP-13评分为(6.25±2.33)分,对照组为(2.57±1.13)分,组间比较差异有统计学意义(P<0.001)。蛋白质印迹法结果显示,CRI软骨细胞内GSK3β表达水平为(0.13±0.04),对照组为(0.38±0.11),组间比较差异有统计学意义(P<0.001);CRI软骨细胞内p-GSK3β表达水平为(0.52±0.18),对照组为(0.19±0.08),组Objective To observe the effect of glycogen synthase kinase 3β(GSK3β)on the development of tibial growth plate in young rats with chronic renal insufficiency(CRI).Methods Forty 4-week-old male Sprague-Dawley(SD)rats were randomly divided into two groups(n=20 each).Control group received an intragastric infusion of distilled water while CRI group had adenine suspension 150 mg/(kg·d).All young rats were sacrificed after a 6-week continuous gavage.Bilateral tibias were collected and the width of growth plate was measured in histological sections.Immunohistochemistry(IHC)was employed for detecting the expression site/level of GSK3βin tibial growth plate.Chondrocytes isolated from growth plate were cultured in vitro to P3 generation and the levels of GSK3β,p-GSK3β,β-catenin(key protein of Wnt/βsignaling pathway)and MMP-13 in chondrocytes were observed by IHC.Western blot was utilized for detecting the relative levels of GSK3β,p-GSK3β,β-catenin and matrix metalloproteinase-13(MMP-13).Paired t test was used for inter-group comparison.Results Tibial histological sections indicated that the relative width of growth plate was narrower in CRI group than that of control group[(0.52±0.14)vs(1.00±0.07)]and the inter-group difference was statistically significant(P<0.001).IHC results showed that GSK3βscore expressed by chondrocytes was lower in CRI group than that in control group[(1.8±0.4)vs(7.1±2.2)]and the inter-group difference was statistically significant(P<0.001).IHC results of chondrocyte experiments in vitro revealed that GSK3βscore was lower in CRI group than that in control group[(0.94±0.32)vs(5.63±1.71)]and the inter-group difference was statistically significant(P<0.001).IHC results of chondrocyte experiments in vitro indicated that p-GSK3βscore was higher in CRI group than that in control group[(4.97±1.10)vs(1.58±0.51)]and the inter-group difference was statistically significant(P<0.001).IHC results of chondrocyte experiments in vitro showed thatβ-catenin score was higher in CRI group than t

关 键 词：肾功能不全 生长板 软骨细胞 

分 类 号：R692[医药卫生—泌尿科学]