肺脏2型固有淋巴细胞或可通过M2型巨噬细胞促进小鼠肺泡化进程

Pulmonary type 2 innate lymphoid may promote the process of alveolarization in mice through type 2 macrophages

目的探讨小鼠肺发育过程中2型固有淋巴细胞(ILC2)、M1和M2型巨噬细胞(AMs)在肺组织中的动态改变及与肺发育的关系。方法新生C57BL/6小鼠于出生后1、3、7、14和21 d随机取15只处死后取肺组织,流式细胞术检测肺组织ILC2、M1及M2型巨噬细胞数量,酶联免疫吸附试验(ELISA)和实时荧光定量PCR(RT-qPCR)检测肺组织IL-33、IL-13及IL-4表达,流式细胞术和Western blot分别检测体外IL-33干预巨噬细胞RAW264.7后表型变化与诱导型一氧化氮合酶(iNOS)和精氨酸酶-1(Arg-1)蛋白水平表达。结果小鼠出生后肺组织中ILC2数量先升后降,在14 d达到峰值;M1型巨噬细胞数量于3 d内升高,以后降低,M2型巨噬细胞先升后降,14 d达高峰;IL-33于出生后14 d内高水平表达,21 d出现降低;IL-13和IL-4表达水平均先升后降,14 d达到峰值;尽管IL-13+ILC2/IL-13+和IL-4+ILC2/IL-4+百分比均在21 d内逐渐增加,但IL-13+ILC2和IL-4+ILC2数量均呈先升后降趋势,峰值出现在14 d;体外IL-33干预RAW264.7表型无明显变化,iNOS和Arg-1蛋白水平无明显变化。结论在新生小鼠肺发育过程中,ILC2峰值落在快速肺泡化期末;M2型巨噬细胞在肺泡化期占主导地位;体外IL-33直接刺激巨噬细胞无明显作用。

This study was designed to investigate the dynamic changes of type 2 innate lymphoid cells(ILC2),type 1 and 2 macrophages(AMs) in lung tissues and their relationship with lung development. Flow cytometry was used to count ILC2, type 1 and 2 macrophages in lung tissues;ELASA and real-time quantitative PCR(RT-qPCR)were used to detect the expression of IL-33, IL-13 and IL-4 in lung tissues;flow cytometry and Western blot were used to detect the phenotype change and the protein levels of inducible nitric oxide synthetase(iNOS) and arginase-1(Arg-1) after IL-33 interference in vitro. Data showed that the amount of ILC2 in lung tissues first increased and then decreased after the mice were born, reaching the peak on day 14 of the postnatal period. The number of type 1 macrophage increased within 3 days after birth and then kept decreasing, while type 2 macrophage first increased and then decreased after peaking on day 14 after birth. IL-33 was highly expressed within 14 days after birth and decreased on day 21 of the postnatal period. The expression levels of IL-13 and IL-4 first increased and then decreased, peaking on day 14 after birth. Although the percentage of IL-13^+ILC2/ILC2/IL-13^+and IL-4^+ILC2/IL-4^+both increased gradually within 21 days after birth, the number of IL-13^+ILC2 and IL-4^+ILC2 both increased first and then decreased, and the peak appeared on day 14 of the postnatal period. There was no significant change in phenotype, iNOS and Arg-1 protein level of RAW264.7 macrophages after IL-33 intervention in vitro. In conclusion, during the process of lung development in neonatal mice, the peak value of ILC2 appears at the end of rapid alveolation, type 2 macrophage is dominant in the period of alveolation, and IL-33 had no significant effect on stimulating macrophages directly in vitro.