摘要目的 探讨椎间盘组织中转化生长因子-β (transforming growth factor-β,TGF-β)通路调节胸腺基质淋巴细胞生成素(thymic stromal lymphopoietin,TSLP)表达的相关分子机制.方法 在体外培养小鼠椎间盘组织,用肿瘤坏死因子-α(tumor necrosis factora,TNF-α)模拟椎间盘突出时的炎症微环境,利用TGF-β通路的抑制剂或核因子-κB (nuclear factor-kappaB,NF-κB)抑制剂培养后,用ELISA法、免疫组化法及Western法测定椎间盘组织中TSLP的变化.结果 (1)TNF-α组的磷酸化NF-κB的阳性细胞率为92.4％±1.4％,对照组为9.4％±3.3％.加入TGF-β Ⅰ型受体抑制剂的HTS组磷酸化NF-κB的阳性细胞率为74.1％±6.7％;Western blot结果显示:HTS组和TNF-α组分别与对照组比较,表达磷酸化NF-κB明显增高,差异有统计学意义.(2)ELISA结果显示:HTS组表达TSLP为(482.8±44.5) pg/ml,对照组为(66.8±4.2) pg/ml,加入IMD和MG后,其表达TSLP下调.(3)TGF-β通路主要因子Smad 2/3的免疫组化结果显示:对照组中磷酸化Smad 2/3的阳性细胞率为46.6％±3.3％,HTS组为23.7％±10.9％,两者比较差异有统计学意义;Western blot结果显示:HTS组与对照组相比,表达磷酸化Smad 2/3明显下调.(4)ELISA结果显示:HTS组上调了TSLP的表达,MCP-1、IL-6均表达下调,MMP-3的表达无变化.结论 在生理条件下,椎间盘组织中内源性TGF-β通过抑制NF-κB的活性限制了TSLP的表达;当椎间盘突出时,内源性的稳态系统被打破,TGF-β活性下降或缺失,抑制NF-κB的能力下降,而后者促使TSLP生成增加,促进椎间盘突出重吸收.

abstractsObjective To illuminate the underlying mechanism of thymic stromal lymphopoietin (TSLP)expression in intervertebral disc tissue.Methods Simulate mice intervertebral disc tissue with tumor necrosis factor α (TNF-α) in vitro tomimic the inflammatory micro-environment in intervertebral disc herniation.Afterwards,inhibit transforming growth factor-β (TGF-β) or nuclear factor-kappa B (NF-κB) pathways with specific inhibitors and exam the alteration of TSLP expression using ELISA,immunohistochemistry and Western blot analysis.Results (1) After stimulation of TNF-α,the positive cells rate was 92.4％±1.4％,and there were 9.4％±3.3％ positive cells in control cultured tissue.After blockade of TGF-β activity by HTS,the positive cells rate was 74.1％±6.7％.Consistently,the Western blot analysis of phosphorylated NF-κB expression showed a dramatic elevation in HTS and TNF-α treated tissue comparing to control.(2) Using ELISA analysis,the expression of TSLP in HTS treated tissue was evaluated as 482.8±44.5 pg/ml.The control was evaluated as 66.8±4.2 pg,/ml,which was decremented by IMD and MG.(3) As the main molecule in TGF-β pathway,phosphorylated Smad 2/3 positive cell rate was evaluated by immunohistochemistry.In control tissue,the positive cells was up to 46.6％±3.3％ but in HTS treated tissue the positive cells decreased to 23.7％±10.9％.Consistently,the Western blot analysis of phosphorylated Smad 2/3 expression showed a dramatic elimination in HTS treated tissue comparing to control.(4) In ELISA analysis,it showed an increment of TSLP expression in HTS treatment,but decrements in MCP-1 and IL-6 expression.MMP-3 expression level was not altered.Conclusion In physical conditions,endogenous TGF-β limits TSLP expression by the inhibition of NF-κB activity in intervertebral disc tissue.However,the homeostasis is disrupted during intervertebral disc herniation.The activity of TGF-β decreases or is impaired,and the inhibition of NF-κB is eliminated.As a consequent,TSLP expression elevates to improve the re-absorption of herniated intervertebral disc.