软骨调节素在成人退变椎间盘中的表达

摘要

目的 利用分子生物学及免疫组化方法研究软骨调节素(ChM-Ⅰ)在成人退变椎间盘细胞及椎间盘组织中的表达情况.方法 2009年3月至4月取3例因腰椎间盘退变性疾病而需行后路椎间融合患者的椎间盘组织分别进行髓核及纤维环细胞培养.取部分原代细胞利用RT-PCR和Western blot研究ChM-Ⅰ mRNA和蛋白在成人退变椎间盘细胞中的表达情况.利用real-time PCR和Western blot研究不同浓度碱性成纤维细胞生长因子(bFGF)对髓核细胞和纤维环细胞ChM-Ⅰ mRNA和蛋白表达的影响.收集2008年10月至2009年10月因腰椎间盘退变性疾病而行手术切除的椎间盘组织标本26例,根据MRI表现退变程度为Ⅲ～V级,作为退变组.收集同期因脊柱肿瘤行手术治疗时切除的椎间盘标本6例,退变程度Ⅰ级,作为对照组.利用免疫组化方法研究ChM-Ⅰ在不同退变程度椎间盘组织中的表达情况.结果 RT-PCR、Western blot显示ChM-Ⅰ在椎间盘髓核细胞及纤维环细胞中均有表达.bFGF可抑制ChM-Ⅰ在髓核及纤维环细胞中的表达,且呈剂量依赖性(P＜0.05).ChM-Ⅰ在对照组椎间盘组织中表达量很低,阳性细胞率为0.12±0.03,而在椎间盘发生退变后其表达量明显升高,退变组与对照组相比差异有统计学意义(P＜0.05).结论 髓核细胞及纤维环细胞可表达ChM-Ⅰ,bFGF可明显抑制ChM-Ⅰ mRNA及蛋白的表达.椎间盘发生退变后ChM-Ⅰ表达明显升高,提示其可能在椎间盘退变的病理过程中发挥一定的作用.%Objectives To investigate the expression of chondromodulin-1(ChM-Ⅰ)in human adult degenerative intervertebral disc(IVD)cells and the relationship between ChM-Ⅰ expression and disc degeneration.Methods Three degenerated disc specimens obtained from patients in the treatment of disc degenerative disease from March to April 2009 were used for cell culture.ChM-Ⅰ expression in ⅣD cells was examined by RT-PCR and Western blot.The effect of basic fibroblast growth factor(bFGF)on the expression of ChM-Ⅰ was assessed by real-time PCR and Western blot.From October 2008 to October 2009,26 human ⅣD tissues were obtained from patients in the surgical treatment of disc degenerative disease at different stage of degeneration according to MRI.Six IVD tissues removed from patients with metastatic spinal tumor were used as normal control.The expression of ChM-Ⅰ determined by immunohistochemical analysis was correlated with MRI degeneration grade.Results RT-PCR and Western blot examination showed that ChM-Ⅰ was expressed in both adult degenerative anulus fibrosus and nucleus pulposus cells.The mRNA and protein expression of ChM-Ⅰ were both down-regulated by administration of bFGF with dose-dependent way(P＜0.05).Immunohistochemical analysis showed the percent of ChM-Ⅰ immunopositive cells in the control group was 0.12 ± 0.03,and the number increased significantly in the advanced degeneration group(P＜ 0.05).Conclusions The current results demonstrate that ⅣD cells express ChM-Ⅰ.Administration of bFGF down-regulates the expression of ChM- Ⅰ.The expression of ChM-Ⅰ is correlated with the degree of ⅣD degeneration which means it may involve in the process of ⅣD degeneration.