间充质干细胞微泡对ARDS肺纤维化影响的实验研究

doi:10.3877/cma.j.issn.1674-0785.2024.01.011

目的

明确间充质干细胞微泡（MSC-MVs）对急性呼吸窘迫综合征（ARDS）肺纤维化小鼠肺纤维化的影响。

方法

采用慢病毒转染技术获取肝细胞生长因子（HGF）低表达的MSC-MVs（siHGF-MSC-MVs）；三次注射脂多糖（LPS）法建立ARDS肺纤维化小鼠模型，然后从ARDS肺纤维化小鼠尾静脉注入MSC-MVs或siHGF-MSC-MVs。使用免疫组织化学、western blot、ELISA等方法检测肺纤维化相关蛋白表达，比较MSC-MVs对ARDS肺纤维化小鼠肺纤维化及纤维化相关指标的影响。

结果

ARDS肺纤维化组肺纤维化评分明显高于对照组，MSC-MVs注入肺纤维化小鼠尾静脉后，肺纤维化评分明显降低，然而siHGF-MSC-MVs组肺纤维化评分明显高于MSC-MVs组。与肺纤维化组比较，MSC-MVs组Ⅰ型、Ⅲ型胶原蛋白抗原、TGF-β、α-SMA蛋白表达量明显降低，而siHGF-MSC-MVs组较MSC-MVs组显著增加。ARDS肺纤维化组肺湿/干和Evans蓝漏出量高于对照组，MSC-MVs注射后湿/干和Evans蓝漏出量明显降低，然而，HGF低表达后MSC-MVs的作用被明显抑制。

结论

MSC-MVs部分通过分泌HGF抑制ARDS肺纤维化小鼠肺纤维化。

关键词: 间充质干细胞微泡, 急性呼吸窘迫综合征, 肺纤维化, 肝细胞生长因子

Objective

To investigate the effect of mesenchymal stem cell microvesicles (MSC-MVs) on pulmonary fibrosis in mice with acute respiratory distress syndrome (ARDS).

Methods

MSC-MVs with low expression of hepatocyte growth factor (siHGF-MSC-MVs) were obtained by lentivirus transfection. A mouse model of ARDS pulmonary fibrosis was established by injecting lipopolysaccharide (LPS) three times, and then MSC-MVs or siHGF-MSC-MVs were injected from the tail vein of ARDS mice with pulmonary fibrosis. Immunohistochemistry, Western blot, ELISA, and other methods were used to detect the expression of pulmonary fibrosis related proteins, and to compare the effects of MSC-MVs on pulmonary fibrosis and fibrosis related indicators in ARDS mice with pulmonary fibrosis.

Results

The pulmonary fibrosis score in the pulmonary fibrosis group was significantly higher than that of the control group. After MSC-MVs were injected into the tail vein of pulmonary fibrosis mice, the pulmonary fibrosis score was significantly reduced. However, the siHGF-MSC-MVs group had a significantly higher pulmonary fibrosis score than the MSC-MVs group. Compared with the pulmonary fibrosis group, the expression levels of type Ⅰ and Ⅲ collagen antigens, TGF-β, α-SMA protein were significantly reduced in the MSC-MVs group, while these indicators significantly increased in the siHGF-MSC-MVs group compared to the MSC-MVs group. The amount of lung wet/dry and Evans blue leakage in the ARDS pulmonary fibrosis group was higher than that in the control group. After MSC-MVs injection, the wet/dry weight ratio and the amount of Evans blue leakage was significantly reduced. However, the effect of MSC-MVs was significantly inhibited after HGF knockdown.

Conclusion

MSC-MVs inhibit pulmonary fibrosis in ARDS mice partly by secreting HGF.

Key words: Mesenchymal stem cell microvesicles, Acute respiratory distress syndrome, Pulmonary fibrosis, Hepatocyte growth factor

图1 MSC-MVs对ARDS肺纤维化肺损伤的影响。图a为各组肺组织病理切片Masson染色；图b为各组肺损伤评分。与对照组比较，*P<0.05；与肺纤维化比较，^#P<0.05；与MSC-MVs组比较，^&P<0.05

图2 免疫组化检测MSC-MVs对ARDS肺纤维化小鼠肺纤维化相关指标的影响。图a为各组肺组织病理切片免疫组织化学图片；图b为α-SMA蛋白表达量；图c为Ⅰ型胶原蛋白表达量；图d为Ⅲ型胶原蛋白表达量；图e为TGF-β蛋白表达量。与对照组比较，*P<0.05；与肺纤维化比较，^#P<0.05；与MSC-MVs组比较，^&P<0.05

图3 Western Blot检测MSC-MVs对ARDS肺纤维化小鼠肺纤维化相关蛋白的影响。图a为各组肺组织纤维化相关蛋白的western blot条带图；图b为各纤维化相关蛋白相对表达量。与对照组比较，*P<0.05；与肺纤维化比较，^#P<0.05；与MSC-MVs组比较，^&P<0.05

图4 MSC-MVs对ARDS肺纤维化小鼠肺血管内皮通透性的影响。图a为各组肺湿干重比；图b为Evans蓝检测各组血管内皮通透性。与对照组比较，*P<0.05；与肺纤维化比较，^#P<0.05；与MSC-MVs组比较，^&P<0.05

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Effect of mesenchymal stem cell microbubbles on pulmonary fibrosis in mice with acute respiratory distress syndrome: an experimental study

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Effect of mesenchymal stem cell microbubbles on pulmonary fibrosis in mice with acute respiratory distress syndrome: an experimental study