气管内移植人脐血间充质干细胞对博来霉素致小鼠肺纤维化的作用

摘要

目的：：探讨气管内移植人脐血间充质干细胞( HUCBMSCs ）对于博来霉素诱导的小鼠肺纤维化的作用，比较气管内及尾静脉移植HUCBMSCs对肺纤维化的治疗效果。方法：取第2代HUCBMSCs培养至4代6；0只无特定病原体的雄性昆明小鼠随机分成4组：阴性对照组( Cont组）、博来霉素组( BLM组）、HUCBMSCs移植Ⅰ组( MⅠ组）和HUCBMSCs移植Ⅱ组( MⅡ组），每组15只；后3组小鼠采用气管内插管灌注博来霉素制造肺纤维化模型2，4 h后，MⅠ组各小鼠气管内灌注5-溴-2-脱氧尿嘧啶核苷(Brdu）标记的HUCBMSCs，MⅡ组各小鼠尾静脉内注射Brdu标记的HUCBMSCs，分别于模型建造后的第7、14、28天每组各处死5只小鼠取材。采用HE染色和Masson染色观察肺组织形态学变化；免疫组织化学法检测间充质干细胞在肺组织的定位和分布；碱水解法测定羟脯氨酸的水平；免疫印迹法检测TGF-β1、α-SMA的蛋白表达量。结果：两组间充质干细胞移植组第7、14、28天均可见Brdu标记的细胞；3个时间点的MⅠ组、Ⅱ组的肺泡炎症和肺纤维化程度均分别较博来霉素组轻，差异有统计学意义(P＜0.05）。肺组织中TGF-β1、α-SMA的表达情况：BLM组＞MⅠ组、MⅡ组＞Cont组(P＜0.05）；MⅠ组和Ⅱ组表达量差异无统计学意义( P＞0.05）。结论：气管内移植HUCBMSCs可以定植于受损的肺组织中，能够减轻博来霉素诱导的小鼠肺纤维化。%Objective:To explore the effect of intratracheal transplantation human umbilical cord blood mesenchymal stem cells on pulmonary interstitial fibrosis by bleomycin in mice,compare the treatment in pulmonary fibrosis of intratracheal transplantation with tail vain injection human umbilical cord blood mesenchymal stem cells.Methods:The second generation of HUCBMSCs were cultured to the fourth generation.Sixty specific pathogen free male Kunming mice were randomly divided into 4 groups:negative control group ( Cont group) ,bleomycin group( BLM group) ,HUCBMSCs transplantation groupⅠ( MⅠgroup) and HUCBMSCs transplantation groupⅡ(MⅡ group),each group 15 mice.Pulmonary fibrosis models were induced by bleomycin via intratracheal perfusion in the latter three groups.Twenty four hours after model establishment,5-bromo-2-deoxyuridine( Brdu) marked HUCBMSCs were poured in trachea in MⅠgroup ,the same were injected into tail vein in MⅡ group.At the 7th,14th,28th day,5 mice in each group were executed re-spectively.The morphological changes of the lung tissues were observed by HE staining and Masson staining.The localization and distribution of human umbilical cord blood mesenchymal stem cells were determined by the method of immunohistochemistry.The hydroxyproline contents were measured by alkali hydrolysis assay.The protein levels of transforming growth factor β-1 ( TGF-β1 ) and smooth muscle alpha-actin(α-SMA) were detected by Western blot.Results:In the two mesenchymal stem cell transplantation groups, there were Brdu marked cells at the 7th,14th,28th days in lung tissue.The alveolitis and fibrosis in lung of the two mesenchymal stem cell transplantation groups were milder than which of the the bleomycin group(P<0.05).Hydroxyproline levels in the bleomycin groups at 3 time points showed a gradually rising trend,the highest level was at 28th day( P<0.01).The protein levels of TGF-β1 andα-SMA in the lung tissues ranked as follows:BLM group>MⅠ,MⅡgroup>Cont group(P<0.05),the difference between MⅠgroup and MⅡgroup was not statistically significant(P>0.05).Conclusion:The colonization of human umbilical cord blood mesenchymal stem cells can be seen in the damaged tissue via intratracheal transplantation which can alleviate pulmonary fibrosis in mice caused by bleomycin.