尾静脉注射LR-MSCs对哮喘小鼠气道炎症、气道高反应性的影响

发布时间：2018-11-23 08:12

【摘要】：目的:气道炎症、气道高反应性是支气管哮喘的两个关键的病理生理特征。前期实验发现间充质干细胞对支气管哮喘具有有效的治疗作用。因而本研究通过分离、培养并鉴定小鼠肺间充质干细胞(LR-MSCs),并探讨尾静脉注射LR-MSCs对哮喘模型小鼠气道炎症及气道高反应性的影响及哮喘治疗的新途径。方法:采用流式细胞分选仪从经过酶混合体消化的小鼠肺组织单细胞悬液中分选Sca-1+CD31-CD45-细胞群(即LR-MSCs)后进行体外培养,再将稳定传代的LR-MSCs通过流式细胞表面分析检测技术进行间充质干细胞表面标记的鉴定;采用腹腔注射和反复雾化吸入鸡卵清白蛋白OVA的方法建立急性哮喘模型、同时利用NS建立正常对照组、并通过尾静脉注射LR-MSCs入哮喘模型小鼠体内建立LR-MSCs干预组、NS处理小鼠尾静脉注射LR-MSCs建立LR-MSCs对照组;利用BUXCO无创肺功能检测仪测定各组小鼠气道高反应性的Penh值;通过支气管肺泡灌洗术获得支气管肺泡灌洗液BALF,计数各组小鼠BALF的细胞总数、中性粒细胞数、嗜酸性粒细胞数以及单核细胞数;测定各组小鼠BALF中Th2型炎症因子IL4、IL-5、IL-13的表达;通过ELISA测定各组小鼠血清中炎症因子OVAsIgE以及OVAsIgG1的水平;HE染色观察大体组织改变及炎性浸润程度。结果:(1)成功分离Sca-1+CD31-CD45-细胞群然后对其进行体外培养,并通过流式细胞仪检测Sca-1+CD31-CD45-细胞群表面间充质干细胞表面标记的表达,验证了Sca-1+CD31-CD45-细胞群即LR-MSCs,该细胞群阳性表达CD29、Sca-1,阴性表达CD31和CD45,符合间充质干细胞表面标记的表达情况;(2)成功通过OVA致敏及激发建立急性哮喘小鼠模型,表现为哮喘模型组的气道高反应性Penh值明显高于正常对照组,BALF中Th2型炎症因子IL4、IL-5、IL-13的水平,BALF中细胞总数、中性粒细胞计数、嗜酸性粒细胞计数以及单核细胞计数,血清中炎症因子OVAsIgE以及OVAsIgG1的水平明显高于正常对照组,且哮喘模型组较正常对照组支气管壁及血管周围有大量炎症细胞浸润;(3)通过尾静脉注射LR-MSCs进行干预可有效降低气道高反应性并减轻气道炎症水平,表现为LR-MSCs干预组的气道高反应性Penh值较哮喘模型组明显下降,LR-MSCs干预组BALF中Th2型炎症因子IL4、IL-5、IL-13的水平,BALF中细胞总数、中性粒细胞计数、嗜酸性粒细胞计数以及单核细胞计数,血清炎症因子中OVAs IgE以及OVAs IgG1水平明显低于哮喘模型组,且LR-MSCs干预组较哮喘模型组支气管壁及血管周围炎症细胞浸润不明显,而正常对照组与LR-MSCs对照组之间气道高反应性与气道炎症水平无明显差异。结论:(1)成功分离获得肺间充质干细胞LR-MSCs,提示小鼠肺内存在LR-MSCs。(2)LR-MSCs干预能在一定程度上减轻哮喘小鼠气道炎症及气道高反应性,可望成为治疗哮喘的新途径,具有广阔应用前景。
[Abstract]:Objective: airway inflammation and airway hyperresponsiveness are two key pathophysiological features of bronchial asthma. Previous studies have found that mesenchymal stem cells have an effective therapeutic effect on bronchial asthma. In this study, we isolated, cultured and identified mouse pulmonary mesenchymal stem cells (LR-MSCs), and explored the effects of tail vein injection of LR-MSCs on airway inflammation and airway hyperresponsiveness in asthmatic mice and a new approach to the treatment of asthma. Methods: Sca-1 CD31-CD45- cell group (LR-MSCs) was isolated from mouse lung tissue single cell suspension digested by enzyme mixture by flow cytometry and cultured in vitro. The stable passage of LR-MSCs was detected by flow cytometry and the surface markers of mesenchymal stem cells were identified. The acute asthma model was established by intraperitoneal injection and repeated atomization inhalation of chicken ovalbumin OVA, and the normal control group was established by NS, and the LR-MSCs intervention group was established by tail vein injection of LR-MSCs into the asthmatic model mice. LR-MSCs was injected into tail vein of mice treated with NS to establish LR-MSCs control group. The airway hyperresponsiveness (Penh) of mice in each group was measured by BUXCO noninvasive pulmonary function detector. The BALF, count of bronchoalveolar lavage fluid (BALF) was obtained by bronchoalveolar lavage. The total number of BALF cells, the number of neutrophils, the number of eosinophilic granulocytes and the number of monocytes in each group; The expression of Th2 type inflammatory factor (IL4,IL-5,IL-13) in BALF of each group was determined; the levels of OVAsIgE and OVAsIgG1 in serum of each group were measured by ELISA; the changes of gross tissue and the degree of inflammatory infiltration were observed by HE staining. Results: (1) Sca-1 CD31-CD45- cells were isolated and cultured in vitro, and the expression of surface markers of mesenchymal stem cells (MSCs) on the surface of Sca-1 CD31-CD45- cells was detected by flow cytometry. The positive expression of CD29,Sca-1, and CD45, in Sca-1 CD31-CD45- cell group, that is, LR-MSCs, were consistent with the expression of surface markers of mesenchymal stem cells. (2) the acute asthma model was successfully established by OVA sensitization and stimulation. The results showed that the airway hyperreactive Penh value in the asthma model group was significantly higher than that in the normal control group, and the level of Th2 type inflammatory factor IL4,IL-5,IL-13 in BALF was significantly higher than that in the normal control group. The levels of total cell count, neutrophil count, eosinophilic granulocyte count, monocyte count, serum inflammatory factor OVAsIgE and OVAsIgG1 in BALF were significantly higher than those in the control group. Compared with the normal control group, a large number of inflammatory cells infiltrated the bronchial wall and blood vessels in the asthmatic model group. (3) injection of LR-MSCs via caudal vein could effectively reduce airway hyperresponsiveness and reduce airway inflammation, which showed that the Penh value of airway hyperresponsiveness in LR-MSCs intervention group was significantly lower than that in asthmatic model group. The levels of Th2 type inflammatory factor IL4,IL-5,IL-13 in BALF, the total number of cells in BALF, neutrophil count, eosinophil count and monocyte count in LR-MSCs intervention group. The levels of OVAs IgE and OVAs IgG1 in serum inflammatory factors were significantly lower than those in asthmatic model group, and the infiltration of inflammatory cells in bronchial wall and blood vessel in LR-MSCs intervention group was less than that in asthma model group. There was no significant difference in airway hyperresponsiveness and airway inflammation between normal control group and LR-MSCs control group. Conclusion: (1) the successful isolation of lung mesenchymal stem cells (LR-MSCs,) suggests that the presence of LR-MSCs. (2) LR-MSCs can alleviate airway inflammation and airway hyperresponsiveness in asthmatic mice to some extent. It is expected to be a new approach to the treatment of asthma and has broad application prospects.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R562.25

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