间充质干细胞对延缓大鼠放射性肺纤维化进展的作用研究

Infusion of mesenchymal stem cells limits fibrogenesis in irradiated rat lung

目的 评价人脂肪来源间充质干细胞对大鼠放射性肺纤维化进展的抑制作用.方法 选用雄性SD大鼠,共48只.采用随机数字表法从中选出36只大鼠,给予右侧全肺15 Gy X射线照射.造模结束后2h,随机数字表法将受照大鼠分为3组：PBS对照组、成纤维细胞治疗组和干细胞治疗组,每组12只,剩余12只未受照大鼠作为健康对照组.于照射后第24周分别对受照右肺进行影像学及病理学检测.后者包括HE染色,Masson染色,免疫组织化学染色（α-SMA及TGF-β1）.分别获取受照大鼠的外周血及支气管-肺泡灌洗液样本,采用ELISA法检测样本中的肝细胞生长因子（HGF）和转化生长因子-β1（TGF-β1）的含量.采用实时荧光定量PCR法对受照肺组织内Ⅰ型胶原-α1（Collagen Ⅰ-α1）及Ⅲ型胶原-α1（CollagenⅢ-α1）基因的表达量进行检测.结果 影像学检查结果显示,受照肺在造模后第24周出现高密度影,病变以右下叶最为明显.PBS对照组及成纤维细胞治疗组的大鼠右肺下叶受损程度较干细胞治疗组重.病理结果提示,PBS对照组及成纤维细胞治疗组的大鼠右肺下叶结构被破坏,表现为肺泡塌陷及肺泡间隔增宽.两组大鼠右肺下叶细胞外基质沉积明显;组织内Collagen Ⅰ-α1及CollagenⅢ-α1的表达量较干细胞治疗组提高（F =4.39、7.73,P＜0.05）.PBS对照组及成纤维细胞治疗组的大鼠右肺下叶α-SMA及TGF-β1的表达明显.ELISA结果提示,干细胞治疗组的大鼠血清中及支气管-肺泡灌洗液中HGF的浓度显著高于其余两组（F=14.97、41.13,P＜0.05）;TGF-β1的浓度低于其余两组（F=172.49、62.82,P＜0.05）.结论 人脂肪来源间充质干细胞能够抑制放射性肺纤维化的进展,对受照肺组织具有一定的保护作用.

Objective To evaluate the inhibitory effect of human adipose tissue-derived mesenchymal stem cells on radiation-induced pulmonary fibrosis.Methods A total of 48 male SpragueDawley rats were enrolled in this study.For establishing the model of radiation-induced lung injury,36 rats were randomly selected to administrate a dose of 15 Gy to their whole right lungs.Thereafter,the irradiated rats were randomly classified into three groups.Each group contained 12 rats.Phosphate buffered sodium （PBS group）,human skin-derived fibroblasts （fibroblast group） and human adipose tissue-derived mesenchymal stem cells （Ad-MSC group） were injected into rat through the tail vein respectively.The rest 12 rats without semi-thoracic irradiation were set as healthy controls.At 24 weeks post-irradiation,radiological imaging and pathological analyses were used for evaluating the histological changes in irradiated lungs.The pathological analyses included H＆amp;E staining for reflecting pulmonary architecture,Masson＆#39;s Trichrome （M.T） staining for evaluating extracellular matrix （ECM） formation and IHC-staining for identifying the expressions of alpha-smooth muscle actin （α-SMA） and TGF-β1 in irradiated lungs.Additionally,the concentrations of HGF and TGF-β1 in serum and BALF were tested by ELISA assay.Quantitative real-time PCR assay was used to test the gene-expressions in irradiated lungs,including Collagen Ⅰ-α1 and Collagen Ⅲ-α1.Results The chest radiological imaging revealed that high-density shadows were presented in the irradiated lungs at 24 weeks post-irradiation.The lower lobes were predominant disease.However,the lesion of high-density shadow was not obvious in Ad-MSC group.Histological analysis by H＆amp;E staining indicated that the irradiated lungs in both PBS and fibroblast groups lost their architectures,representing by alveolar collapse and thickening alveolar septa.Moreover,compared to Ad-MSC group,excessive ECM formation together with significantly up-regulated expressions of Collagen Ⅰ-α1 and Collagen Ⅲ-α1 （F =4.39,7.73,P 〈 0.05） were also found within the injured tissue treated with PBS or fibroblasts.Apart from this,obvious expressions of α-SMA and TGF-β1 in the injured tissue were the common features after treating with PBS or fibroblasts.Thus,with the treatment of Ad-MSCs,the concentrations of HGF in serum and BALF were significantly higher than those in PBS and fibroblast groups （F =14.97,41.13,P 〈 0.05）,and the concentration of TGF-β1 significantly lower than those in PBS and fibroblast groups （F =172.49,62.82,P 〈 0.05）.Conclusions Infusion of human Ad-MSCs could protect lungs against radiation-induced fibrosis.