慢性肾衰竭幼鼠胫骨生长板软骨细胞初级纤毛高表达加速软骨细胞分化的机制研究  

Mechanism of highly expressed primary cilia in tibial growth plate chondrocyte accelerating chondrocyte differentiation in young rats with chronic renal failure

作　　者：王小健[1] 李荣山[2] 田伟 郑刚 毕宏[3] 王艳红[2] 董丽娜[2] 郭松佳 路晓 周晓霜[2] Wang Xiaojian;Li Rongshan;Tian Wei;Zheng Gang;Bi Hong;Wang Yanhong;Dong Lina;Guo Songjia;Lu Xiao;Zhou Xiaoshuang(Department of Orthopaedic Surgery,Shanxi Provincial People's Hospital,Taiyuan 030012,China;Shanxi Provincial Key Laboratory of Nephrology,Shanxi Provincial People's Hospital,Taiyuan 030012,China;Department of Pathology,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 [3]山西省人民医院病理科,太原030012

出　　处：《中华肾脏病杂志》2021年第9期758-764,共7页Chinese Journal of Nephrology

基　　金：中国博士后科学基金(2020M680910);山西省自然科学基金(201701D121161);山西省重点研发计划项目(201803D31160,201703D321027-4)。

摘　　要：目的探讨慢性肾衰竭(chronic renal failure,CRF)幼鼠胫骨生长板软骨细胞初级纤毛高表达加速软骨细胞分化的机制。方法选取雄性4周龄SD大鼠40只,体重(98±3)g,随机分为对照组(蒸馏水,n=20)和CRF组(腺嘌呤150 mg·kg^(-1)·d^(-1),n=20),连续灌胃6周后处死幼鼠,采用胫骨制作组织学切片比较生长板长度,免疫荧光检测软骨细胞初级纤毛表达率、Wnt/β-catenin信号通路关键蛋白β联蛋白(β-catenin)表达水平;体外培养两组幼鼠胫骨生长板软骨细胞至第3代,免疫荧光检测软骨细胞初级纤毛表达率及印度刺猬蛋白(Indian hedgehog,IHH)和Wnt/β-catenin通路拮抗蛋白糖原合成酶激酶3β(glycogen synthase kinase 3β,GSK3β)表达水平;应用免疫共沉淀检测IHH与GSK3β之间的作用关系。结果与对照组相比,CRF组组织学切片生长板相对长度变短[(0.51±0.11)比(1.00±0.08),t=16.11,P<0.001],软骨细胞初级纤毛表达率增高[(26.3±5.5)%比(7.6±1.9)%,t=14.37,P<0.001],软骨细胞β-catenin蛋白表达增多[(7.1±2.0)分比(3.6±1.0)分,t=7.10,P<0.001]。体外培养结果显示,与对照组相比,CRF组软骨细胞初级纤毛表达率增高[(31.4±8.2)%比(12.5±3.1)%,t=9.64,P<0.001],IHH蛋白表达增多[(1360±270)比(310±84),t=16.61,P<0.001],而两组GSK3β蛋白表达差异无统计学意义[(850±195)比(780±140),t=1.30,P=0.200]。免疫共沉淀检测结果显示,CRF组GSK3β蛋白与IHH蛋白在软骨细胞内存在直接相互作用。结论CRF幼鼠胫骨生长板软骨细胞初级纤毛表达增多,相关蛋白IHH表达增多,IHH蛋白与Wnt/β-catenin信号通路拮抗蛋白GSK3β存在直接作用,导致Wnt/β-catenin信号通路激活、软骨细胞加速分化,最终导致生长板出现闭合趋势。Objective To explore the mechanism of highly expressed primary cilia in tibial growth plate chondrocytes accelerating chondrocytes differentiation in young rats with chronic renal failure(CRF).Methods Forty male 4-week-old SD rats weighing(98±3)g were randomly divided into control group(intragastric administration with distilled water,n=20)and CRF group(intragastric administration with adenine suspension 150 mg·kg^(-1)·d^(-1),n=20).All the young rats were sacrificed after continuous gavage for 6 weeks.The length of the growth plate was measured with histological sections.Immunofluorescence(IF)was used to detect the expression rate of primary cilia and the level ofβ-catenin,the key protein of Wnt/β-catenin signaling pathway in tibial growth plate chondrocytes.Chondrocytes isolated from growth plate in two groups were cultured in vitro to P3 generation,and the expression rate of primary cilia in chondrocytes,the levels of Indian hedgehog(IHH)and glycogen synthase kinase 3β(GSK3β)were detected by IF.Co-immunoprecipitation was used to detect the relationship between IHH and GSK3β.Results Compared with the control group,the relative length of the growth plate was shorter in histological sections[(0.51±0.11)vs(1.00±0.08),t=16.11,P<0.001],the expression rate of primary cilia was higher[(26.3±5.5)%vs(7.6±1.9)%,t=14.37,P<0.001],and the level ofβ-catenin increased[(7.1±2.0)scores vs(3.6±1.0)scores,t=7.10,P<0.001]in CRF group.In vitro,the expression rate of primary cilia was higher in CRF group chondrocytes[(31.4±8.2)%vs(12.5±3.1)%,t=9.64,P<0.001]than that in control group.The level of IHH in CRF group increased than that in control group[(1360±270)vs(310±84),t=16.61,P<0.001].There was no significant difference in GSK3βlevel of chondrocytes between the two groups[(850±195)vs(780±140),t=1.30,P=0.200].There was a direct interaction between IHH and GSK3βin CRF group chondrocytes.Conclusions The expression levels of primary cilia and related protein IHH increase in tibial growth plate chondrocytes of CRF

关 键 词：肾功能衰竭 慢性 软骨细胞 WNT信号通路 生长板 初级纤毛 印度刺猬蛋白 

分 类 号：R692.5[医药卫生—泌尿科学]