人脐血间充质干细胞治疗大鼠肺纤维化的实验研究

Experimental treatment of pulmonary interstitial fibrosis with human umbilical cord blood mesenchymal stem cells

目的 探讨人脐血间充质干细胞对博来霉素所致大鼠肺纤维化的影响及可能机制.方法 取第2代人脐血间充质干细胞培养至第4代;取清洁级健康雄性SD大鼠60只随机分为博来霉素组、干细胞治疗组、地塞米松治疗组和阴性对照组,每组15只.博来霉素组、干细胞治疗组和地塞米松治疗组分别经气管注入博来霉素制造肺纤维化模型,干细胞治疗组造模后立即经鼠尾静脉注入5-溴-2-脱氧尿嘧啶核苷（Brdu）标记的干细胞,地塞米松治疗组造模后第2天开始连续7d腹腔注射地塞米松,阴性对照组经气管注入等量生理盐水,在第7、14、28天处死各组大鼠5只,HE、Masson染色、免疫组织化学法观察转化生长因子（TGF）-β1表达及标记细胞情况、酸解法测定肺羟脯氨酸的含量.结果 干细胞治疗组第7、14、28天肺组织均可见标记的干细胞.与阴性对照组比较,博来霉素组、干细胞治疗组和地塞米松治疗组肺泡炎评分及肺纤维化评分均明显升高,差异均有统计学意义（P＜0.05）.不同处理后7、14、28 d,干细胞治疗组与地塞米松治疗组肺泡炎评分均低于博来霉素组,第28天干细胞治疗组和地塞米松治疗组肺纤维化评分低于博来霉素组,差异均有统计学意义（P＜0.05）.与地塞米松治疗组比较,干细胞治疗组肺泡炎评分及肺纤维化的差异无统计学意义（P＞0.05）.与博来霉素组比较,各个时间点干细胞治疗组和地塞米松治疗组TGF-β1阳性细胞数和羟脯氨酸含量均减少,差异均有统计学意义（P＜0.05）;与地塞米松治疗组比较,干细胞治疗组肺组织TGF-β1阳性细胞数和羟脯氨酸含量的表达差异无统计学意义（P＞0.05）.结论 人脐血间充质干细胞可以定植于受损的肺组织中,并可以在肺纤维化早期有效减轻肺泡炎及肺纤维化,其可能的作用机制与抑制TGF-β1的表达及减少胶原的形成有关有关.

Objective To investigate the therapeutic effect and possible action mechanism of human umbilical cord blood mesenchymal stem cells （hUCB-MSCs） in the treatment of bleomycin-induced pulmonary fibrosis in rats.Methods The second generation of hUCB-MSCs was cultured to the fourth generation.Sixty healthy male Sprague-Dawley rats （clean grade） were randomly and equally divided into 4 groups：bleomycin group,stem cell treatment group,dexamethasone treatment group,and negative control group.A pulmonary fibrosis model was established by intratracheal instillation of bleomycin in the bleomycin group,stem cell treatment group,and dexamethasone treatment group.The stem cell treatment group was injected with stem cells labeled with 5-bromo-2-deoxyuridine （Brdu） via the caudal vein immediately after the model was established.The dexamethasone treatment group was intraperitoneally injected with dexamethasone for 7 d from the next day after the model was established.The negative control group was given an equal volume of normal saline by intratracheal instillation.In each group,5 rats were sacrificed in the 7th,14th,and 28th days.The expression of transforming growth factor β1 （TGF-β1） and Brdu-labeled stem cells were observed by HE and Masson staining and immunohistochemistry.Lung hydroxyproline content was determined by acid hydrolysis.Results The stem cell treatment groups had Brdu-labeled stem cells seen in lung tissue in the 7th,14th,and 28th days.Compared with the negative control group,the bleomycin group,stem cell treatment group,and dexamethasone treatment group had significantly increased scores of alveolitis and pulmonary fibrosis （P＜0.05）.In the 7th,14th,and 28th days,the scores of alveolitis in stem cell treatment group and dexamethasone treatment group were significantly lower than those in bleomycin group （P＜0.05）; in the 28th day,the scores of pulmonary fibrosis in stem cell treatment group and dexamethasone treatment group were significantly lower than that in bleomycin group （P＜0.05）.There were no significant differences in scores of alveolitis and pulmonary fibrosis between the dexamethasone treatment group and stem cell treatment group （P＞0.05）.Compared with the bleomycin group,the stem cell treatment group and dexamethasone treatment group had significantly decreased number of TGF-β1-positive cells and hydroxyproline content in lung tissue at all time points （P＜0.05）.There were no significant differences in number of TGF-β1-positive cells and hydroxyproline content in lung tissue between the stem cell treatment group and dexamethasone treatment group （P＞0.05）.Conclusion hUCB-MSCs can be transplanted into damaged lung tissue and effectively reduce alveolitis and pulmonary fibrosis in the early stage of pulmonary fibrosis.The action mechanism of hUCB-MSCs may involve inhibiting the expression of TGF-β1 and reducing the formation of collagen.