Th17、Treg在肺纤维化小鼠外周血、肺组织中的表达及意义

Expression and significance of Th17 and Th17/Treg cells in a murine model with bleomycin subcutaneous injection induced pulmonary fibrosis

目的观察CD4+IL-17+辅助性T细胞(Th17)及Th17/CD4+CD2+5Foxp3+调节性T细胞(Treg)在博来霉素皮下注射致小鼠肺纤维化模型外周血及肺组织的表达及意义。方法 20只雌性BALB/c小鼠随机分为模型组和对照组各10只:模型组皮下注射博莱霉素4周,对照组相同条件下注射0.1 mL PBS。采用HE染色和Masson's染色观察两组肺组织炎症和纤维化的病理改变;流式细胞术检测两组肺组织及外周血Th17、Treg及Th17/Treg的表达;应用酶联免疫吸附试验检测两组血清IL-17水平;免疫荧光定量PCR法检测两组肺实质组织中IL-17和维甲酸相关孤独受体(RORγt)的mRNA的表达,并分析上述各指标的相互关系。结果模型组肺部纤维化评分、肺组织炎症程度和羟脯氨酸的含量均显著高于对照组(P均<0.01);外周血和肺组织Th17、Th17/Treg及外周血IL-17含量较对照组均显著增高(P均<0.01);肺实质RORγt和IL-17 mRNA水平较对照组明显增高(P均<0.05)。相关性分析结果显示,外周血和肺组织Th17及Th17/Treg、外周血IL-17的水平与肺组织羟脯氨酸水平、肺部纤维化评分、肺组织炎症反应均呈显著正相关(P均<0.01)。结论肺纤维化模型Th17及Th17/Treg表达均增高;Th17可能通过分泌IL-17参与肺纤维化的发病,Th17/Treg失衡可能通过加重肺纤维化免疫炎症,促进肺部胶原纤维的沉积,在肺纤维化的发病发挥重要作用。

Objective To investigate the expression and significance of CD4^＋ IL-17 ^＋ T helper cells （Th17） and Th17/CD4^＋ CD25^＋Foxp3^＋ regulatory T cells（Treg） in the peripheral blood and lung tissue of a murine model with bleomycin subcutaneous injection induced pulmonary fibrosis. Methods A total of 20 female BALB / c mice were randomly divided into two groups： the model group and the control group, 10 in each group. Bleomycin was injected subcutaneously for 4 weeks in the model group, and meanwhile, the mice in the control group were injected with 0.1mL PBS. The lung tissue inflammation and fibrosis of the pathological changes in both the two groups were detected by HE staining and Masson staining; the expression of Th17 and Th17/Treg in the lung tissue and peripheral blood of the two groups was detected by flow cytometry; the serum level of IL-17 in both the two groups was detected by ELISA; the expression of IL-17 in the lung parenchymal tissue and mRNA expression in retinoic acid-related orphan receptors （RORYt） in two groups were detected by quantitative PCR assay. Then, the relationship between these indicators was analyzed. Results The pulmonary fibrosis score, inflammation of the lung tissue and hydroxyproline content in the model group were significantly higher than those in the control group （ all P 〈0.01 ）. The levels of Th17, Th17/Treg in peripheral blood and lung tissue,and IL-17 level in the peripheral blood of the model group were significantly increased compared with the control group （ P 〈0.01 ）. Compared with the control group, lung parenchyma RORγt and the IL-17 mRNA level of the model group were significantly increased （all P 〈 0.05 ）. The correlation analysis showed that the levels of Th17 and Thl7/Treg in the peripheral blood and lung tissue and the IL-17 level in peripheral blood were all positively correlated to hydroxyproline level in the lung tissue, pulmonary fibrosis score and lung tissue inflammatory response （ all P 〈 0.01 ）. Conclusion The expression of Thl7 and Thl7/Treg in the murine model with pulmonary fibrosis was all increased. Th17 may be involved in the pathogenesis of pulmonary fibrosis through the secretion of IL-17, and the imbalance of Thl7/Treg may play an important role in the pathogenesis of pulmonary fibrosis possibly by means of aggravating pulmonary fibrosis immune inflammation and promoting the lung deposition of collagen fibers.