间充质干细胞移植减轻大鼠盐酸吸入性肺损伤

摘要

目的 研究骨髓间充质干细胞(BMSCs)移植对大鼠盐酸吸入性肺损伤的作用与机制.方法 本实验于复旦大学附属中山医院实验研究中心进行.改良贴壁法分离培养Sprague-Dawley (SD)大鼠BMSCs.24只SD大鼠随机(随机数字法)分为3组(n=8):对照组,损伤组和移植组.损伤组和移植组气道内滴注盐酸(1.2 ml/kg,pH=1.5),对照组则滴入等量磷酸缓冲盐溶液(PBS),用动脉血气、肺组织湿干比和病理改变评价模型是否成功建立.移植组于颈外静脉注射5×106个BMSCs,其他2组注入0.5 ml的PBS.移植6h后行动脉血气分析,酶联免疫吸附法(ELISA)测定血清和支气管肺泡灌洗液(BALF)的肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)和白介素-10 (IL-10)质量浓度,观察肺组织病理改变和测定肺组织湿干比.体外实验中,BMSCs与损伤肺细胞或正常肺细胞于Transwell体系共培养36 h,观察损伤肺影响BMSCs迁移情况；另取BMSCs与损伤肺细胞昆合培养或于Transwell中培养,正常肺细胞或损伤肺细胞单独培养作对照,6h后检测上清液中TNF-α、IL-6和IL-10质量浓度.用方差分析比较多组间的差异,独立样本t检验比较两组间差异.结果 BMSCs移植改善了肺损伤大鼠的低氧血症(P＜0.01)和肺组织损伤,降低了肺组织湿干比(P＜0.01),降低了血清和BALF的TNF-α水平(P＜0.05；P＜0.01),升高了血清和BALF的IL-10质量浓度(P＜0.01),对IL-6水平无明显影响(P＞0.05).体外实验中,BMSCs向损伤肺迁移明显多于向正常肺迁移(P＜0.01),BMSCs与肺细胞接触或不接触的共培养均降低了上清液TNF-α质量浓度(P＜0.01),升高了IL-10质量浓度(P＜0.01),对IL-6质量浓度无影响(P＞0.05).结论 BMSCs移植可以减轻大鼠盐酸吸入性肺损伤,可能机制为干细胞通过旁分泌途径下调炎症反应.%Objective To investigate the effects and the possible mechanism of bone mesenchymal stem cells (BMSCs) transplantation in rat model of acid aspiration induced lung injury.Methods BMSCs cultures were obtained from bone marrow of Sprague-Dawley (SD) rats.Twenty-four SD rats were randomly divided into 3 groups:Control,Injury and Injury + BMSCs groups,and hydrochloric acid (HCl) (1.2ml/kg,pH =1.5) or the same volume of phosphate buffered saline (PBS) instead were instilled into trachea of rats to make injury models or for control group respectively.Then,5 × 106 BMSCs or 0.5 ml PBS were injected into jugular vein of rats.Rats were exsanguinated at 6 h after injury.Arterial blood gas,wet/dry ratio and histological changes of lung tissue were determined.Bronchoalveolar lavage fluid (BALF) and serum were collected for the measurement of Tumor necrosis factor-α (TNF-α),Interleukin-6 (IL-6) and Interleukin-10 (IL-10) by Enzyme-linked immuno-sorbent assay (ELISA).In vitro,lung cells from normal rats and from HCl injury rats were co-cultured with BMSCs in a Transwell system (8 μm pore size).Thirty-six hours later,the numbers of migrated BMSCs were counted.In addition,lung cells from HCl injured rats were co-cultured with BMSCs in either a standard single well or in a Transwell (0.4 μm pore size).Control wells were prepared with only lung cells from normal rats or HC; injured rats.After incubation for 6 hours,the cell culture supernalants were then collected to assay the levels of TNF-α,IL-6 and IL-10using ELISA.Comparisons among multiple groups were performed using One-way analysis of variance (ANOVA).Comparisons between groups were carried out using independent-sample t-test.Results BMSCs transplantation attenuated histological lung injury and hypoxia caused by HCl instillation.Injury +BMSCs group decreased wet/dry ratio compared with Injury group.BMSCs administration mediated a down-regulation of inflammation by deceasing TNF-α concentration and increasing IL-10 in BALF and serum.Invitro,co-culture studies of BMSCs with lung cells provided evidence that lung cells stimulated BMSCs migration towards injured lung.Besides,the anti-inflammatory effect of BMSCs was not cell contact dependent.Conclusions BMSCs transplantation prevented lung injury caused by HCl aspiration,and the preventive effects of BMSCs were partly driven by the paracrine to down-regulate the inflammatory response to lung injury induced by HCl.