骨髓间充质干细胞对矽尘所致急性肺损伤的疗效及其机制研究

发布时间：2018-01-22 16:07

本文关键词： 骨髓间充质干细胞二氧化硅急性肺损伤巨噬细胞白细胞介素-1β　出处：《山西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:研究BMSC移植对矽尘所致急性肺损伤的疗效及作用机制。方法:采用随机分配法将60只小鼠分为对照组、SiO_2组和BMSC移植组3个组。气管暴露法建立实验动物模型,对照组经气管注射0.90%氯化钠20μL;SiO_2组和BMSC移植组经气管注射20μL浓度为250 g/L的SiO_2混悬液,即5 mg/只,6 h后,上述两组小鼠分别经尾静脉给予100μL的0.90%氯化钠或细胞密度为5×109个/L的BMSC。常规喂养3天后处死小鼠,观察3组小鼠肺组织损伤情况,Wright-Giemsa染色BALF中细胞并计数。ELISA检测小鼠血清、BALF中细胞因子表达水平,qPCR检测小鼠肺组织中细胞因子mRNA的表达情况;FCM检测小鼠BALF中巨噬细胞百分比,免疫组化检测肺组织中巨噬细胞数目,WB检测肺组织中IL-1β、Caspase-1蛋白表达水平。Transwell共培养BMSC和巨噬细胞,ELISA检测巨噬细胞培养上清中IL-1β表达水平,WB检测巨噬细胞中IL-1β、Caspase-1蛋白表达水平。结果:肉眼观察可见对照组和BMSC移植组小鼠肺脏表面光滑,SiO_2组小鼠肺脏出现明显的肿胀、充血。病理切片显示对照组和BMSC移植组小鼠肺组织结构无破坏,肺泡腔大小均匀、完整,肺泡结构完整;SiO_2组小鼠肺组织有大量炎性细胞浸润、聚集,肺泡腔有炎性渗出物,肺泡塌陷。SiO_2组小鼠肺系数高于对照组(P0.05),BMSC移植组较SiO_2组降低(P0.05)。SiO_2组小鼠BALF中总细胞数目和巨噬细胞数目分别高于对照组(P0.05),BMSC移植组小鼠上述两个指标分别低于SiO_2组(P0.05)。SiO_2组小鼠血清、BALF中IL-1β、IL-6水平分别高于对照组,BMSC移植组上述两个指标较SiO_2组降低(P0.05)。Si O2组小鼠肺组织中IL-1β、IL-6 m RNA相对表达水平分别高于对照组(P0.05),BMSC移植组上述两个指标较SiO_2组降低(P0.05)。SiO_2组小鼠BALF中巨噬细胞百分比较对照组升高,BMSC移植组巨噬细胞百分比较SiO_2组降低。免疫组化结果显示SiO_2组小鼠肺组织中有较多巨噬细胞聚集,BMSC移植组巨噬细胞较少。SiO_2组小鼠BALF中CCL-3表达水平高于对照组(P0.05),BMSC移植组较SiO_2组降低(P0.05)。SiO_2组小鼠肺组织中IL-1β、Caspase-1表达较对照组升高,BMSC移植组上诉两个指标较SiO_2组降低;而Pro-IL-1β、Pro-Caspase-1表达水平在3组间比较无差异。体外实验发现不同浓度SiO_2作用巨噬细胞后IL-1β表达水平升高,并呈浓度依赖;分别与BMSC共培养后IL-1β表达水平降低。巨噬细胞中IL-1β、Caspase-1表达情况与体内实验结果一致。结论:1、BMSC可以减轻矽尘所致的急性肺损伤。2、其作用机制可能是BMSC通过作用于巨噬细胞NLRP3炎性小体通路,减少IL-1β的产生,从而减轻炎症反应。
[Abstract]:Objective: to study the effect and mechanism of BMSC transplantation on acute lung injury induced by silica dust. Methods: 60 mice were randomly divided into control group. The experimental animal model was established by trachea exposure in SiO_2 group and BMSC transplantation group, and the control group was injected with 20 渭 L sodium chloride 0.90% 渭 L through trachea. In SiO_2 group and BMSC transplantation group, 20 渭 L SiO_2 suspension (250g / L) was injected through trachea for 6 h. The mice in the two groups were given 100 渭 L sodium chloride (0.90% 渭 L) via tail vein or BMSCs with cell density of 5 脳 10 9 / L. The mice were sacrificed after 3 days of routine feeding. The lung tissue injury in the three groups was observed and the expression of cytokines in the serum of BALF was detected by Wright-Giemsa staining. The expression of cytokine mRNA in lung tissue of mice was detected by qPCR. The percentage of macrophages in BALF and the number of macrophages in lung tissue were detected by FCM and WB respectively. Expression level of Caspase-1 protein. Transwell co-cultured BMSC and macrophages were used to detect the expression of IL-1 尾 in the supernatant of macrophage culture by enzyme-linked immunosorbent assay (Elisa). Results: the lung surface of mice in control group and BMSC transplantation group was smooth. The lung of SiO_2 group showed obvious swelling and hyperemia. The pathological sections showed that the lung tissue structure of the control group and BMSC transplantation group was not damaged, the alveolar cavity was uniform and intact, and the alveolar structure was intact. In SiO_2 group, there were a lot of inflammatory cells in lung tissue, aggregation, inflammatory exudate in alveolar cavity, lung coefficient in group 2 was higher than that in control group (P0.05). Compared with SiO_2 group, the total number of BALF cells and the number of macrophages in BALF of BMSC transplantation group were significantly lower than those of control group (P 0.05). The above two indexes in BMSC transplantation group were lower than those in SiO_2 group (P 0.05). Sio group 2 mice were higher than control group in IL-1 尾 -IL-6 level in serum. Compared with SiO_2 group, the above two indexes in BMSC group decreased the IL-1 尾 in lung tissue of P0.05U 路Sio _ 2 group. The relative expression level of IL-6 m RNA was higher than that of control group (P 0.05). Compared with SiO_2 group, the percentage of macrophages in BALF of BMSC transplantation group was lower than that of SiO_2 group, and the percentage of macrophage in BALF of BALF group was higher than that of control group. The percentage of macrophages in the BMSC group was lower than that in the SiO_2 group. The immunohistochemical results showed that there were more macrophages in the lung tissue of the SiO_2 group. The expression of CCL-3 in BALF of mice in BMSC transplantation group was higher than that in control group (P 0.05). Compared with SiO_2 group, the expression of IL-1 尾 -caspase-1 in lung tissue of BMSC transplantation group was lower than that of SiO_2 group. The two indexes of appeal in BMSC transplantation group were lower than those in SiO_2 group. And Pro-IL-1 尾. There was no difference in the expression of Pro-Caspase-1 between the three groups. In vitro, it was found that the expression of IL-1 尾 in macrophages with different concentrations of SiO_2 increased in a dose-dependent manner. The expression level of IL-1 尾 was decreased after co-culture with BMSC, and the expression of IL-1 尾 -caspase-1 in macrophages was consistent with the experimental results in vivo. Conclusion: 1. BMSC can attenuate acute lung injury induced by silica. The mechanism may be that BMSC reduces the production of IL-1 尾 by acting on the inflammatory corpuscle pathway of NLRP3 in macrophages. This reduces the inflammatory response.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R135.2

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