环境大气细颗粒物宫内暴露对大鼠子代免疫功能相关指标的影响

Effects of intrauterine exposure to ambient fine particles on immune function-related indicators in neonatal rats

目的观察大鼠宫内环境空气动力学当量直径〈2.5 μm的大气细颗粒物（fine particles，即PM2.5）暴露对致敏原卵清蛋白反应性的影响及其可能机制。 方法Sprague-Dawley孕鼠随机分为4组：过滤空气-生理盐水（简称空气-盐水）组、PM2.5-盐水组、空气-卵清蛋白组和PM2.5-卵清蛋白组，每组10只。PM2.5暴露系将孕鼠置于PM2.5与环境等浓度的染毒柜中，暴露时间为妊娠第1天至分娩；吸入空气的孕鼠置于去除颗粒物的洁净柜中。卵清蛋白致敏为妊娠第4和9天孕鼠腹腔注射50 μg卵清蛋白，第18~20天每日雾化吸入1%卵清蛋白30 min，未用卵清蛋白组以等量生理盐水处理。胎鼠自然娩出后即刻以酶联免疫吸附试验检测各组新生仔鼠血浆白细胞介素（interleukin，IL）-4、IL-5和干扰素-γ的含量，蛋白质印迹技术检测肺组织GATA-3、T-bet蛋白水平，实时荧光定量-聚合酶链反应技术检测脾组织微小RNA（microRNA，miR）-146a和miR-146b表达的变化，光学显微镜下观察肺组织学变化。采用单因素方差分析及LSD法进行统计学分析。 结果（1）血浆IL-4和IL-5水平：IL-4水平在PM2.5-盐水组[（18.56±7.04） ng/L]、空气-卵清蛋白组[（34.04±7.06） ng/L]和PM2.5-卵清蛋白组[（45.67±8.18） ng/L]较空气-盐水组[（10.51±2.88） ng/L]升高，在PM2.5-卵清蛋白组较PM2.5-盐水组和空气-卵清蛋白组升高（F=54.667，P〈0.001）。IL-5水平在空气-卵清蛋白组和PM2.5-卵清蛋白组较空气-盐水组升高（F=6.253，P=0.023）。血浆干扰素-γ水平在空气-卵清蛋白组较空气-盐水组、PM2.5-盐水组和PM2.5-卵清蛋白组升高（F=28.604，P〈0.001）。（2）肺组织GATA-3和T-bet蛋白水平：GATA-3水平在PM2.5-盐水组（31.09±3.54）、空气-卵清蛋白组（35.24±5.00）和PM2.5-卵清蛋白组（47.81±3.63）较空气-盐水组（24.19±3.12）升高，在PM2.5-卵清蛋白组较PM2.5-盐水组和空气-卵清蛋白组升高（F=96.581，P〈0.001）。T-bet水平在空气-卵清蛋白组和PM2.5-卵清蛋白组较空气-盐水组降低，在PM2.5-卵清蛋白组较PM2.5-盐水组和空气-卵清蛋白组降低（F=30.852，P〈0.001）。（3）脾组织miR-146a和miR-146b表达水平：miR-146a在PM2.5-盐水组（1.72±0.27）、空气-卵清蛋白组（1.56±0.37）和PM2.5-卵清蛋白组（3.06±0.52）较空气-盐水组（1.05±0.25）升高，在PM2.5-卵清蛋白组较PM2.5-盐水组和空气-卵清蛋白组升高（F=42.276，P〈0.001）。miR-146b表达变化趋势与miR-146a一致（F=28.776，P〈0.001）。（4）空气-卵清蛋白组、PM2.5-卵清蛋白组见肺泡腔狭窄或消失，肺间质炎症细胞浸润，肺泡间隔淤血。PM2.5-卵清蛋白组这些变化更为严重。 结论宫内环境水平PM2.5暴露可影响胎肺的正常发育，使部分免疫指标发生改变，在母体卵清蛋白致敏时其不良效应更为显著。

ObjectiveTo investigate the effects of maternal exposure to ambient fine particles （PM2.5） in Fuzhou during pregnancy on immune responses to ovalbumin （OVA） in neonatal rats and the possible mechanisms.MethodsPregnant Sprague-Dawley rats were randomly assigned into four groups （ten in each）： filtered air （FA） plus normal saline （NS）, airborne PM2.5 plus NS （PM2.5-NS）, FA plus OVA （FA-OVA） and PM2.5 plus OVA （PM2.5-OVA） groups. Pregnant dams in the PM2.5 exposure groups were placed in a PM2.5 exposure chamber in which the PM2.5 concentration was equal to the ambient air from the beginning of gestation till delivery, whereas the other dams inhaling air without particulate matters were put into a clean chamber. OVA sensitization was conducted through intraperitoneal injection of OVA at 50 μg per dam at 4 and 9 days of gestation, followed by inhalation of atomized 1% OVA for 30 min at 18, 19 and 20 days of gestation. Dams without OVA sensitization were given NS in the same way. Levels of interleukin （IL）-4, IL-5 and interferon-γ （IFN-γ） in neonatal rats＇ plasma were measured by enzyme-linked immunosorbent assay just after birth. Protein levels of transcription factors GATA-3 and T-bet in lung were analyzed by Western-blotting. Changes in microRNA（miR）-146a and miR-146b in spleen were detected by real-time polymerase chain reaction. Histological changes in lung were observed under light microscope. One-way analysis of variance and LSD test were used as statistical methods.Results（1） IL-4 level in plasma was significantly increased in PM2.5-NS [（18.56±7.04） ng/L], FA-OVA [（34.04±7.06） ng/L] and PM2.5-OVA groups [（45.67±8.18） ng/L] as compared with that in FA-NS group [（10.51±2.88） ng/L], and the level of IL-4 in PM2.5-OVA group was higher than that in PM2.5-NS and FA-OVA groups （F=54.667, P〈0.001）. Significantly increased IL-5 level in plasma was found in FA-OVA and PM2.5-OVA groups as compared with that in FA-NS group （F=6.253, P=0.023）. Among the four groups, FA-OVA group showed significantly increased IFN-γ level in plasma （F=28.604, P〈0.001）. （2） GATA-3 level in lung tissues was significantly increased in PM2.5-NS （31.09±3.54）, FA-OVA （35.24±5.00） and PM2.5-OVA groups （47.81±3.63） as compared with that in FA-NS group （24.19±3.12）, and higher in PM2.5-OVA group than in PM2.5-NS and FA-OVA groups （F=96.581, P〈0.001）. T-bet level was significantly lower in FA-OVA and PM2.5-OVA groups than in FA-NS group. Moreover, PM2.5-OVA group showed decreased T-bet level as compared with that in PM2.5-NS and FA-OVA groups （F=30.852, P〈0.001）. （3） Expression of miR-146a in spleen was significantly enhanced in PM2.5-NS （1.72±0.27）, FA-OVA （1.56±0.37） and PM2.5-OVA groups （3.06±0.52） than in FA-NS group （1.05±0.25）. Moreover, PM2.5-OVA group showed enhanced expression of miR-146a as compared with that in PM2.5-NS and FA-OVA groups （F=42.276, P〈0.001）. Changes in the expressions of miR-146b were similar to those in miR-146a （F=28.776, P〈0.001）. （4） Stenosis or disappearance of alveolar spaces, accompanied with infiltration of inflammatory cells in interstitial substance and congestion in alveolar septum, was seen in FA-OVA and PM2.5-OVA groups and conditions in the latter group were more severe.ConclusionsIntrauterine exposure to ambient PM2.5 negatively affects fetal lung development and immunological function in rats, especially when the dams are sensitized with OVA during pregnancy.