骨髓间充质干细胞来源的外泌体对哮喘模型小鼠气道阻力和肺组织病变的改善作用及其机制

doi:10.13481/j.1671-587X.20260303

摘要/Abstract

摘要：

目的探讨骨髓间充质干细胞（BMSCs）来源的外泌体（BMSC-exos）对小鼠哮喘的影响，并阐明其机制。方法提取BMSCs，检测其标志物表达情况。将BMSCs分别转染微小RNA（miR）-26a mimic（mimic-miR-26a）、miR-NC和miR-26a inhibitor（inh-miR-26a），并提取相应外泌体（mimic-miR-26a exos、miR-NC exos和inh-miR-26a exos），小鼠给予卵清蛋白（OVA）和氢氧化铝构建小鼠哮喘模型，造模成功的50只小鼠随机分为模型组、BMSC-exos组、miR-NC exos组、mimic-miR-26a exos组和inh-miR-26a exos组，每组10只，未造模10只小鼠作为对照组，并每天分别给予10 μg BMSC-exos、miR-NC exos、mimic-miR-26a exos、inh-miR-26a exos和等体积磷酸盐缓冲液（PBS），采用HE染色观察各组小鼠肺组织病理形态表现，使用小动物肺功能检测仪检测各组小鼠气道阻力，实时荧光定量PCR（RT-qPCR）法检测各组小鼠肺组织中miR-26a表达水平，双萤光素酶报告基因实验验证miR-26a与Toll样受体4（TRL4）的靶向关系，Western blotting法检测各组小鼠肺组织中TRL4蛋白表达水平。结果提取的BMSCs高表达其标志物分化簇（CD）44和CD105，不表达CD45和CD34，提示BMSCs提取成功。BMSC-exos粒径约为100 nm，双层膜，呈“杯托”样，符合外泌体的结构特征，BMSC-exos表达肿瘤易感基因101（TSG101）、CD9和CD81，符合外泌体的生物学特征。HE染色，对照组小鼠肺组织结构正常，肺泡间隔无明显增宽，未见明显炎症细胞浸润；模型组小鼠肺组织结构紊乱，肺泡结构损伤明显，肺泡间隔明显增宽，存在大量的炎症细胞浸润，杯状细胞大量增生，肺损伤明显，造模成功；BMSC-exos组小鼠肺组织结构紊乱明显缓解，炎症细胞浸润减少，肺泡结构损伤减少；miR-26a BMSC-exos处理后，对照组小鼠肺组织结构正常；模型组小鼠肺组织炎症细胞大量浸润，肺泡结构损伤明显，杯状细胞明显增生，肺泡间隔明显增宽，肺损伤明显；与模型组比较，miR-NC exos组小鼠肺组织病变程度减轻，mimic-miR-26a exos组小鼠肺组织病变程度进一步减轻。在6.25、12.50、25.00和50.00 g·L^-1乙酰甲胆碱（Mch）作用下，与对照组比较，模型组小鼠气道阻力明显升高（P<0.05）；与模型组比较，BMSC-exos组小鼠气道阻力明显降低（P<0.05）。在6.25、12.50、25.00和50.00 g·L^-1 Mch作用下，与模型组比较，miR-NC exos组小鼠气道阻力明显降低（P<0.05）；与miR-NC exos组比较，mimic-miR-26a exos组小鼠气道阻力明显降低（P<0.05），inh-miR-26a exos组小鼠气道阻力明显升高（P<0.05）。RT-qPCR法，与miR-NC exos比较，mimic-miR-26a exos中miR-26a表达水平明显升高（P<0.05），inh-miR-26a exos中miR-26a表达水平明显降低（P<0.05）；与对照组比较，模型组小鼠肺组织中miR-26a表达水平明显降低（P<0.05）；与模型组比较，BMSC-exos组小鼠肺组织中miR-26a表达水平明显升高（P<0.05）；与miR-NC exos组比较，mimic-miR-26a exos组小鼠肺组织中miR-26a表达水平明显升高（P<0.05），inh-miR-26a exos组小鼠肺组织中miR-26a表达水平明显降低（P<0.05）。双萤光素酶报告基因实验，与转染TLR4野生型（WT）和miR-NC组比较，转染TLR4 WT和miR-26a组细胞相对萤光素酶活性明显降低（P<0.05），提示TLR4为miR-26a靶基因。Western blotting法，与对照组比较，模型组小鼠肺组织中TLR4蛋白表达水平明显升高（P<0.05）；与模型组比较，BMSC-exos组小鼠肺组织中TLR4蛋白表达水平明显降低（P<0.05）；与miR-NC exos组比较，mimic-miR-26a exos组小鼠肺组织中TLR4蛋白表达水平明显降低（P<0.05），inh-miR-26a exos组小鼠肺组织中TLR4蛋白表达水平明显升高（P<0.05）。结论 BMSC-exos可降低哮喘模型小鼠的气道阻力，减轻肺组织病变程度，其机制可能与BMSC-exos促使miR-26a传递至肺组织并抑制TLR4表达有关。

关键词: 骨髓间充质干细胞, 外泌体, 哮喘, 气道阻力, 微小RNA-26a, Toll样受体4

Abstract:

Objective To discuss the effect of exosomes derived from bone marrow mesenchymal stem cells（BMSCs）（BMSC-exos） on asthma in the mice， and to clarify the mechanism. Methods The BMSCs were extracted， and the expressions of its markers were detected. The BMSCs were transfected with microRNA （miR）-26a mimic （mimic-miR-26a）， miR-NC and miR-26a inhibitor （inh-miR-26a）， respectively， and the corresponding exosomes （mimic-miR-26a exos， miR-NC exos and inh-miR-26a exos） were extracted. Mouse asthma model was established by administering ovalbumin （OVA） and aluminum hydroxide to the mice. The 50 successfully modeled mice were randomly divided into model group， BMSC-exos group， miR-NC exos group， mimic-miR-26a exos group and inh-miR-26a exos group， with 10 mice in each group， and 10 non-modeled mice were regarded as control group. Each group was administered daily with 10 μg of BMSC-exos， miR-NC exos， mimic-miR-26a exos， inh-miR-26a exos and an equal volume of phosphate buffered saline （PBS）， respectively. HE staining was used to observe the pathomorphological manifestations of lung tissue of the mice in various groups； small animal lung function test instrument was used to detect the airway resistance of mice in various groups； real-time fluorescence quantitative PCR（RT-qPCR） method was used to detect the expression levels of miR-26a in the lung tissue of mice in various groups； dual luciferase reporter gene assay was used to verify the targeting relationship between miR-26a and Toll-like receptor 4 （TLR4）； Western blotting method was used to detect the expression level of TLR4 protein in the lung tissue of mice in various groups. Results The extracted BMSCs highly expressed their markers cluster of differentiation（CD）44 and CD105， but did not express CD45 and CD34， indicating that BMSCs were successfully extracted. The particle size of BMSC-exos was about 100 nm， with a bilayer membrane and a “cup-shaped” morphology， which conformed to the structural characteristics of exosomes. BMSC-exos expressed tumor susceptibility gene 101 （TSG101）， CD9 and CD81， which conformed to the biological characteristics of exosomes. HE staining results showed that the lung tissue structure of mice in control group was normal， the alveolar septum was not significantly widened， and no obvious inflammatory cell infiltration was observed； the lung tissue structure of mice in model group was disordered， the alveolar structure was obviously damaged， the alveolar septum was significantly widened， there was a large amount of inflammatory cell infiltration， goblet cells were hyperplastic， and lung injury was obvious， indicating successful modeling； in BMSC-exos group， the lung tissue structure disorder of mice was significantly alleviated， the inflammatory cell infiltration was reduced， and the alveolar structure damage was reduced； after treatment with miR-26a BMSC-exos， the lung tissue structure of mice in control group was normal； in model group， a large amount of inflammatory cell infiltration was observed in the lung tissue， the alveolar structure was obviously damaged， goblet cells were obviously hyperplastic， the alveolar septum was significantly widened， and lung injury was obvious； compared with model group， the degree of lung tissue lesions in miR-NC exos group was alleviated， and the degree of lung tissue lesions in mimic-miR-26a exos group was further alleviated. The small animal lung function test results showed that under the action of 6.25， 12.50， 25.00 and 50.00 g·L^-1 methacholine （Mch）， compared with control group， the airway resistance of mice in model group was significantly increased （P<0.05）； compared with model group， the airway resistance of mice in BMSC-exos group was significantly decreased （P<0.05）. Under the action of 6.25， 12.50， 25.00 and 50.00 g·L^-1 Mch， compared with model group， the airway resistance of mice in miR-NC exos group was significantly decreased （P<0.05）； compared with miR-NC exos group， the airway resistance of mice in mimic-miR-26a exos group was significantly decreased （P<0.05）， and the airway resistance of mice in inh-miR-26a exos group was significantly increased （P<0.05）. The real-time fluorescence quantitative PCR （RT-qPCR） method results showed that compared with miR-NC exos， the expression level of miR-26a in mimic-miR-26a exos was significantly increased （P<0.05）， and the expression level of miR-26a in inh-miR-26a exos was significantly decreased （P<0.05）； compared with control group， the expression level of miR-26a in the lung tissue of mice in model group was significantly decreased （P<0.05）； compared with model group， the expression level of miR-26a in the lung tissue of mice in BMSC-exos group was significantly increased （P<0.05）； compared with miR-NC exos group， the expression level of miR-26a in the lung tissue of mice in mimic-miR-26a exos group was significantly increased （P<0.05）， and the expression level of miR-26a in the lung tissue of mice in inh-miR-26a exos group was significantly decreased （P<0.05）. The dual luciferase reporter gene assay results showed that compared with the cells transfected with TLR4 wild type （WT） and miR-NC group， the relative luciferase activity of cells in group transfected with TLR4 WT and miR-26a was significantly decreased （P<0.05）， indicating that TLR4 was the target gene of miR-26a. The Western blotting method results showed that compared with control group， the expression level of TLR4 protein in the lung tissue of mice in model group was significantly increased （P<0.05）； compared with model group， the expression level of TLR4 protein in the lung tissue of mice in BMSC-exos group was significantly decreased （P<0.05）； compared with miR-NC exos group， the expression level of TLR4 protein in the lung tissue of mice in mimic-miR-26a exos group was significantly decreased （P<0.05）， and the expression level of TLR4 protein in the lung tissue of mice in inh-miR-26a exos group was significantly increased （P<0.05）. Conclusion BMSC-exos can reduce airway resistance and alleviate the degree of lung tissue lesions in the asthmatic model mice， its mechanism may be related to BMSC-exos promoting the delivery of miR-26a to the lung tissue and inhibiting the expression of TLR4.

Key words: Bone marrow mesenchymal stem cells, Exosomes, Asthma, Airway resistance, MicroRNA-26a, Toll-like receptor 4

中图分类号:

徐丽娜,何小双,辛雯艳,邬超. 骨髓间充质干细胞来源的外泌体对哮喘模型小鼠气道阻力和肺组织病变的改善作用及其机制[J]. 吉林大学学报(医学版), 2026, 52(3): 602-611.

Lina XU,Xiaoshuang HE,Wenyan XIN,Chao WU. Improvement effect of bone marrow mesenchymal stem cell-derived exosomes on airway resistance and lung tissue lesion in mice of asthmatic models and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2026, 52(3): 602-611.

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Group	Airway resistance
Group	Mch[ρ_B/(g·L^－1)] 0	6.25	12.50	25.00	50.00
Control	2.50±0.50	3.20±0.20	8.27±0.31	9.00±0.20	12.13±0.15
Model	3.16±0.15^*	6.58±0.20^*	12.33±0.30^*	13.02±0.17^*	17.25±0.44^*
BMSC-exos	2.53±0.15^△	5.65±0.14^△	9.42±0.21^△	11.60±0.23^△	15.09±0.17^△

Group	Airway resistance
Group	Mch[ρ_B/(g·L^－1)] 0	6.25	12.50	25.00	50.00
Control	2.60±0.47	3.50±0.22	4.35±0.30	6.05±0.16	10.07±0.16
Model	3.22±0.17^*	7.03±0.21^*	11.34±0.20^*	14.16±0.33^*	17.62±0.26^*
MiR-NC exos	2.55±0.12^△	4.88±0.10^△	8.55±0.23^△	10.61±0.16^△	14.62±0.22^△
Mimic-miR-26a exos	2.05±0.11^#	3.33±0.12^#	5.25±0.15^#	8.47±0.19^#	12.01±0.20^#
Inh-miR-26a exos	3.03±0.12^#	8.01±0.18^#	10.83±0.25^#	14.7±0.33^#	17.5±0.26^#

Exosome	Expression level of miR-26
MiR-NC exos	1.00±0.08
Mimic-miR-26a exos	3.45±0.16^*
Inh-miR-26a exos	0.58±0.18^*