腰椎黄韧带增生肥厚中氧自由基H2O2介导转化生长因子β1表达的作用及意义

摘要

背景:目前腰椎黄韧带肥厚的病理机制尚不明确,转化生长因子β1在肥厚黄韧带中表达明显增加.研究表明体内氧自由基堆积与组织器官退变密切相关,氧自由基可能通过介导转化生长因子β1表达参与腰椎黄韧带肥厚的进展.目的:观察氧自由基H2O2介导的人黄韧带细胞中转化生长因子β1、Ⅰ型胶原表达增加在腰椎黄韧带肥厚过程中的影响及意义.方法:腰椎后路减压术中取影像学上无黄韧带肥厚的单纯腰椎间盘突出症患者的腰椎黄韧带标本1例,进行体外细胞分离、培养,采用经培养的第4-6代细胞,应用H2O2对其进行干预,建立低氧诱导模型.设立正常对照组、H2O2浓度50μmol/L组、100μmol/L组、150μmol/L组、200μmol/L组,干预72 h,通过实时定量PCR(RT-qPCR)和Western blot方法分别检测转化生长因子β1、Ⅰ型胶原的mRNA及蛋白表达.结果与结论:①RT-qPCR检测结果显示:H2O2浓度150μmol/L组、200μmol/L组转化生长因子β1 mRNA表达显著增加(P<0.05);实验组Ⅰ型胶原mRNA表达均高于对照组(P<0.05),且200μmol/L组表达显著高于其余实验组,差异有显著性意义(P<0.05);②Western blot检测结果显示:随H2O2浓度增加,转化生长因子β1、Ⅰ型胶原蛋白水平表达显著增加(P<0.05);③结果表明,氧自由基H2O2通过刺激人腰椎黄韧带细胞产生转化生长因子β1,上调Ⅰ型胶原表达,进而促进腰椎黄韧带增生肥厚.%BACKGROUND:The pathogenesis of ligamentum flavum hypertrophy remains poorly understood, and the expression of transforming growth factor beta1 (TGF-β1) is increased notably. Reactive oxygen species (ROS) accumulation is associated with tissue degeneration, which may accelerate the progression of ligamentum flavum hypertrophy by upregulating TGF-β1 expression. OBJECTIVE:To clarify the effect and significance of ROS H2O2-mediated up-regulation of TGF-β1 and collagen type Ⅰ in the progress of ligamentum flavum hypertrophy. METHODS:Ligamentum flavum was removed from a case of acquired lumbar disc herniation with normal ligamentum flavum during lumbar posterior decompression surgery, and then separated and cultured in vitro to the 4-6 generations, followed by exposure to H2O2 at various concentrations (0, 50, 100, 150, 200μmol/L) for 72 hours. The mRNA and protein expression levels of TGF-β1 and collagen type Ⅰ were detected by real-time PCR and western blot assay, respectively. RESULTS AND CONCLUSION:Real-time quantitative PCR showed that the mRNA expression level of TGF-β1 was significantly increased in the 150 and 200μmol/L groups (P<0.05). The mRNA expression level of collagen type Ⅰ was significantly higher in the experimental groups than that in the control group, especially in the 200μmol/L group (P<0.05). Western blot assay revealed that the protein expression levels of TGF-β1 and collagen type Ⅰ were significantly increased in a dose-dependent manner (P<0.05). These findings indicate that H2O2 may accelerate the progression of ligamentum flavum hypertrophy by up-regulating the expression levels of TGF-β1 and collagen type Ⅰ.