软骨终板细胞印度刺猬蛋白表达对软骨终板退变的意义  

Effect of Indian hedgehog protein in cartilage endplate cells on cartilage endplate degeneration

作　　者：张健 宋洁富 王小健 秦德安 安奇君 赵中涛 梁庆元 安雪军 Zhang Jian;Song Jiefu;Wang Xiaojian;Qin Dean;An Qijun;Zhao Zhongtao;Liang Qingyuan;An Xuejun(Department of Orthopedics Surgery,People’s Hospital Affiliated to Shanxi Medical University,Taiyuan 030012,China)

机构地区：[1]山西医科大学附属人民医院骨科,太原030001

出　　处：《中华实验外科杂志》2020年第11期2004-2007,共4页Chinese Journal of Experimental Surgery

基　　金：山西省基础研究项目(2015011121);山西省重点研发计划项目(201803D31160)。

摘　　要：目的探讨细胞内印度刺猬蛋白(Ihh)表达水平变化对软骨终板细胞的影响与意义。方法2018年5月至12月手术收集山西医科大学附属山西省人民医院骨科31例软骨终板(CEP)组织标本,体外分离人CEP细胞培养至P3代,随机分为过表达组、空载体组和敲减组,分别添加Ihh信使RNA质粒、空载体和Ihh沉默RNA质粒,质粒转染培养48 h后,使用蛋白质印迹法(Western blot)和实时定量聚合酶链反应法(RT-qPCR)检测标本Ihh及相关指标的蛋白和mRNA表达,免疫荧光和原位杂交染色观测细胞表型。3组实验数据因方差不齐,呈正态性分布,选择Welch检验,Games-Howell法组间多重比较。结果质粒干预CEP软骨细胞Ihh水平表达,Westernblot结果显示过表达组、空载体组和敲减组3组细胞中Ihh(0.79±0.09,0.31±0.20,0.04±0.03)、Ⅱ型胶原(ColⅡ)(0.18±0.08,0.49±0.14,1.13±0.27)和基质金属蛋白酶-13(MMP-13)(0.78±0.17,0.51±0.12,0.37±0.06)灰度值差异有统计学意义(F=176.670、37.650、26.070,P<0.01)。RT-qPCR结果显示过表达组、空载体组和敲减组3组细胞中Ihh mRNA(3.13±1.21,0.92±0.18,0.39±0.22)、ColⅡmRNA(0.66±0.13,1.03±0.24,1.64±1.32)和MMP-13 mRNA(9.17±2.35,1.04±0.27,3.56±0.26)相对表达水平差异有统计学意义(F=17.170、15.890、34.732,P<0.01)。免疫荧光和原位杂交结果显示与空载体组比较,Ihh和MMP-13在过表达组最强,敲减组最弱,而ColⅡ在敲减组中最强,过表达组最弱。结论下调软骨细胞内Ihh表达可明显增加ColⅡ,减少MMP-13,维持细胞表型,进一步延缓CEP软骨细胞退变。Objective To observe the effect of Indian hedgehog(Ihh)expression changes on the cartilage endplate cells.Methods Thirty-one cartilage endplate(CEP)specimens were collected from May to December 2018,then they were isolated and cultured to P3 generation in vitro.They were divided into 3 groups:over-expression group(Ihh mRNA plasmid transfection),empty vector group and knockdown group(Ihh silencing RNA,siRNA).After 48 h of plasmid transfection,Ihh and related indexes were detected by Western blotting and real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR).Chondrocytes were stained by immunofluorescence and in situ hybridization.The relative data were analyzed.The experimental data of 3 groups had uneven variance and normal distribution,and Welch test and Games-Howell method was used.Results After interfering the expression of Ihh in CEP chondrocytes by plasmids,the results of Western blotting showed that Ihh(0.79±0.09,0.31±0.20,0.04±0.03),Collagen typeⅡ(ColⅡ)(0.18±0.08,0.49±0.14,1.13±0.27)and matrix metalloproteinase-13(MMP-13)(0.78±0.17,0.51±0.12,0.37±0.06)showed significant difference among the three groups(Asymptotical F=63.130,80.650,58.640,P<0.01).RT-qPCR showed that there were significant differences in the relative expression levels of Ihh mRNA(3.13±1.21,0.92±0.18,0.39±0.22),ColⅡmRNA(0.66±0.13,1.03±0.24,1.64±1.32)and MMP-13 mRNA(9.17±2.35,1.04±0.27,3.56±0.26)among the three groups(AsymptoticalF=176.670,37.650,26.070,P<0.01).The results of immunofluorescence and in situ hybridization showed that Ihh and MMP-13 had the strongest fluorescence in the over-expression group and the weakest in the knockdown group,and ColⅡhad the strongest fluorescence in the knockdown group and the weakest in the over-expression group.Conclusion Down-regulation of Ihh expression in chondrocytes can significantly increase ColⅡand reduce MMP-13 to maintain their phenotype,and then further delay the degeneration of CEP chondrocytes.

关 键 词：印度刺猬蛋白 软骨终板 Modic退变 质粒转染 

分 类 号：R681.53[医药卫生—骨科学]