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目的:观察骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)对矽肺大鼠肺泡巨噬细胞自噬活化的影响,探讨骨髓间充质干细胞对矽肺的疗效及其机制。方法:体外分离、培养雄性3-5周龄SD大鼠,获取BMSCs原代,培养至3代。将60只雌性SD大鼠随机分为3组(n=20):对照组,矽肺模型组,BMSCs治疗组。采用非暴露气管灌注法建立大鼠矽肺病模型,于造模成功后12 h给予BMSCs干预治疗(3×106/ml)。分别于1 d、7 d、14 d及28 d处死各组大鼠,应用原位支气管肺泡灌洗技术,获取大鼠肺泡巨噬细胞(Alveolar macrophages,AM)。使用细胞流式仪鉴定BMSCs;HE染色观察AM形态学改变;免疫细胞化学法与Westernblotting法检测LC-3、Beclin-1分布、定位和定量。结果:模型组与对照组相比AM体积较大,胞质丰富,部分细胞内可见矽尘吞噬颗粒;模型组大鼠肺组织LC-3、Beclin-1在各时间点的表达较对照组均增多(P<0.05),1 d即开始增多,至14 d时达高峰,28d时回落,但仍高于对照组的表达水平;BMSCs治疗组大鼠肺组织较模型组病理改变有明显的缓解,各时间点LC-3、Beclin-1蛋白的表达显著下调(P<0.05)。结论:在矽肺大鼠AM中存在自噬的激活,BMSCs可有效抑制自噬的激活,进而缓解矽肺的病理进程。
OBJECTIVE: To observe the effect of bone marrow mesenchymal stem cells (BMSCs) on the activation of alveolar macrophages in silicotic rats, and to explore the therapeutic effect and mechanism of bone marrow mesenchymal stem cells on silicosis. Methods: Male Sprague-Dawley rats, 3-5 weeks old, were isolated and cultured. Primary BMSCs were obtained and cultured to the third generation. Sixty female SD rats were randomly divided into three groups (n = 20): control group, silicosis model group and BMSCs treatment group. The rats model of silicosis was established by non-exposed tracheal perfusion. BMSCs were given 12 × 10 6 / ml after 12 h of successful modeling. The rats in each group were sacrificed on days 1, 7, 14, and 28, respectively. Alveolar macrophages (AM) were obtained by in situ bronchoalveolar lavage. BMSCs were identified by flow cytometer. The morphological changes of AM were observed by HE staining. The distribution and localization of LC-3 and Beclin-1 were detected by immunocytochemistry and Western blotting. Results: Compared with the control group, the model group had larger AM volume and abundant cytoplasm, and some cells could see the phagocytosis of silica particles. The expression of LC-3 and Beclin-1 in the model group at each time point were significantly higher than those in the control group (P <0.05), which began to increase on the first day and peaked on the 14th day, and then decreased on the 28th day, but still higher than that of the control group. The lung tissue in the BMSCs-treated group was significantly relieved compared with the model group At each time point, the expression of Beclin-1 protein was significantly down-regulated (P <0.05). CONCLUSIONS: Autophagy is present in AM of silicotic rats. BMSCs can effectively inhibit the activation of autophagy, and then alleviate the pathological process of silicosis.