慢性肾衰竭幼鼠胫骨生长板软骨细胞线粒体自噬增强对软骨细胞凋亡的影响  

Effect of increased mitophagy of chondrocytes in tibial growth plate of young rats with chronic renal failure on apoptosis

作　　者：王小健[1,2] 徐威威 李荣山[2] 李爱中 路晓 马月红 田伟 张宇明[1] 常峰[1] 苏云星[1] Wang Xiaojian;Xu Weiwei;Li Rongshan;Li Aizhong;Lu Xiao;Ma Yuehong;Tian Wei;Zhang Yuming;Chang Feng;Su Yunxing(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)

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：《中华肾脏病杂志》2022年第3期211-217,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线粒体自噬水平及对细胞凋亡的影响。方法雄性4周龄Sprague-Dawley(SD)幼鼠40只, 按照随机数字表法分为正常对照组(蒸馏水灌胃)和CRF组(腺嘌呤150 mg·kg-1·d-1灌胃), 连续灌胃6周后处死幼鼠, X线测量胫骨长度, 组织学切片测量比较胫骨生长板宽度, 应用原位末端转移酶标记技术(TUNEL)检测生长板软骨细胞凋亡率;体外培养正常对照组和CRF组幼鼠胫骨生长板软骨细胞至第3代, 应用TUNEL技术检测软骨细胞凋亡率, 应用荧光双染技术观测软骨细胞线粒体与自噬溶酶体共定位情况, 应用Western印迹法检测线粒体标志蛋白线粒体外膜转位酶(translocase of the outer mitochondrial membrane-20, Tom-20)和自噬标志轻链蛋白-3(light chain-3, LC-3)表达情况, 应用透射电镜观察软骨细胞线粒体自噬情况。结果与正常对照组相比, CRF组幼鼠胫骨长度较短[(27.32±5.81)mm比(35.43±3.61)mm, t=5.226, P<0.001], 生长板相对宽度较窄(0.56±0.19比1.00±0.21, t=6.744, P<0.001), 软骨细胞凋亡率较高(17.2%±4.8%比5.1%±3.4%, t=6.505, P<0.001)。CRF组体外培养生长板软骨细胞凋亡率高于正常对照组(11.8%±6.2%比3.1%±1.2%, t=4.357, P<0.001), 荧光双染技术结果显示CRF组软骨细胞线粒体与自噬溶酶体出现明显的共定位现象, CRF组软骨细胞LC-3蛋白(t=8.944, P<0.001)及Tom-20蛋白(t=6.708, P<0.001)表达均少于正常对照组, 电镜结果显示CRF组软骨细胞内出现较多的自噬囊泡吞噬线粒体并降解的现象。结论 CRF幼鼠胫骨生长板软骨细胞线粒体自噬水平出现增高现象, 导致线粒体减少, 引起软骨细胞凋亡、数量减少, 最终导致胫骨发育不良。Objective To explore the level of tibial growth plate chondrocyte mitophagy in young rats with chronic renal failure(CRF)and its effect on chondrocyte apoptosis.Methods Male 4-week-old Sprague-Dawley rats were randomly divided into two groups according to random number table method:normal control group(n=20,intragastric administration with distilled water)and CRF group(n=20,given adenine suspension 150 mg·kg-1·d-1).All the young rats were sacrificed after continuous gavage for 6 weeks.The length of tibia was measured on X ray film,the width of tibia growth plate was measured and compared on histological section,and the apoptosis rate of chondrocytes in growth plate was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling(TUNEL)assay.The growth plate chondrocytes of two groups were isolated and cultured to the third generation in vitro,and the apoptosis rate of chondrocytes was detected by TUNEL assay.The co-localization of mitochondria and autophagy lysosomes in chondrocytes was observed by double fluorescence staining.Western blotting was used to detect the level of mitochondrial marker protein translocate of the outer mitochondrial membrane-20(Tom-20)and autophagy marker light chain-3 protein(LC-3).The mitophagy of growth plate chondrocytes was observed by transmission electron microscope.Results Compared with the normal control group,the tibia length of CRF group was shorter[(27.32±5.81)mm vs(35.43±3.61)mm,t=5.226,P<0.001],and the relative width of growth plate in histological section was narrower(0.56±0.19 vs 1.00±0.21,t=6.744,P<0.001).The apoptosis rate of chondrocytes in growth plate in CRF group was higher than that in the normal control group(17.2%±4.8%vs 5.1%±3.4%,t=6.505,P<0.001).The apoptosis rate of chondrocytes cultured in vitro in CRF group was higher than that in the normal control group(11.8%±6.2%vs 3.1%±1.2%,t=4.357,P<0.001).The result of double influorescence staining showed that there was co-localization between mitochondria and autophagy lysosomes

关 键 词：肾功能不全 慢性 线粒体 自噬 生长板 

分 类 号：R692.5[医药卫生—泌尿科学]