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(Col Ⅱ) (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)、Col Ⅱ mRNA(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Col Ⅱ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Col Ⅱ，减少MMP-13，维持细胞表型，进一步延缓CEP软骨细胞退变。

abstractsObjective: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 type Ⅱ (Col Ⅱ) (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), Col ⅡmRNA (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 (Asymptotical F=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 Col Ⅱ had 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 Col Ⅱ and reduce MMP-13 to maintain their phenotype, and then further delay the degeneration of CEP chondrocytes.