摘要目的 观察气管给予12-烷基化壳聚糖纳米粒(12-ACSs)包裹的反义内皮素转换酶(ECE)核酸表达质粒对小鼠变应性气道炎症的影响.方法 将40只健康雌性Balb/c小鼠按完全随机设计原则分为4组,每组10只,分为健康对照(N)组、OVA致敏激发并给予包裹反义ECE核酸的12-ACSs(NM)组、OVA致敏激发气道炎症模型(As)组及OVA致敏激发并给予裸反义ECE核酸(DNA)组.NM组、As组、DNA组在致敏后于首次激发前24 h分别通过气管灌注包裹反义ECE核酸的12-ACSs 100μl生理盐水100μl和反义ECE核酸5μg加生理盐水(总量100μl),N组用等体积生理盐水处理.末次激发后24 h收集BALF,计算BALF中细胞总数、分类计数.HE染色观察各组小鼠肺组织的病理变化.ELISA法检测肺组织匀浆、脾细胞培养卜清液白细胞介素(IL)-4、5、10、13、干扰素-γ(IFN-γ)和内皮素-1(ET-1)水平.流式细胞仪分别检测分泌IL-4、IFN-γ和IL-10脾细胞比例.对各组数据进行正态检验和单因素方差齐性检验,采用LSD法进行两两比较.结果 NM组小鼠BALF中细胞总数为(5.4±0.6)×105/ml、嗜酸性粒细胞为(1.8±0.4)×105/ml,均明显低于As组[分别为(8.5±0.9)×105/ml、(3.8±0.6)×105/ml]和DNA组[分别为(8.3±1.1)×105/ml、(3.8±0.5)×105/m1],均P<0.01;NM组小鼠肺组织的病理损害明显轻于As组和DNA组.肺匀浆N组ET-1水平为(11.7±1.7)ng/L,明显低于NM组的(18.5±1.4)ng/L、As组的(28.9±2.3)ng/L和DNA组的(27.1±1.6)ng/L,均P<0.01,而NM组ET-1水平则明显低于As组和DNA组(均P<0.01),As组和DNA组差异无统计学意义.N组IL-5水平均明显低于NM组、As组和DNA组(均P<0.01),而NM组IL-5水平则明显低于As组和DNA组(均P<0.05),Ag组和DNA组差异无统计学意义.IL-4、IL-13结果与IL-5、ET-1相似.各组IFN-γ的水平差异无统计学意义.NM组IL-10水平高于As组和DNA组(均P<0.01),与N组比较差异无统计学意义,脾细胞培养上清液细胞因子检测结果与肺匀浆结果相似.4组中CD4、IL-4表达均为阳性的细胞表达率分别为N组3.1%、NM组3.3%、As组5.9%、DNA组6.6%;表达IFN-γ、CD4阳性的细胞表达率分别为N组1.2%、NM组1.6%、As组1.7%、DNA组1.6%;表达IL-10、CD4、CD25阳性的细胞分别为N组1.9%、NM组4.3%、As组3.4%、DNA组3.1%.结论 气管内给予包裹反义ECE核酸表达质粒的12-ACSs纳米粒,可减少ET-1、IL-4、IL-5、IL-13的合成,增加IL-10的分泌,减轻变应性气道炎症.

abstractsObjective To explore a novel nonspecific immunomodulation for the treatment of allergic airway inflammation by RNA interference for endothelin converting enzyme(ECE) using 12-alkylated chitosans/plasmid-encoding antisense ECE complex nanoparticles. Methods Forty BALB/c mice were randomly divided into Group N( normal control), Group NM ( OVA + 12-ACSs/antisense-ECE plasmid), Group As(OVA) and Group DNA(OVA + antisense-ECE plasmid), and sensitized by intraperitoneal injection of OVA at day 1 and day 14, followed by challenge with aerosol of 1% OVA at day 24, 25 and 26,but with saline as a control (N). Supernatants from cultured splenocytes and lung homogenates were subjected to detection of the levels of interleukin-4, 5, 10, 13 (IL-4, 5, 10, 13), interferon-γ (IFN-γ) and endothelin-1 ( ET-1) by using ELISA. Lung tissues were embedded, sliced and HE stained for histopathologic examination. The cultured splenocytes were subjected to flow cytometry detection( IL-4 ,IL-10 and IFN-T). Results Mice in group NM showed a lower level of cell count than that in either group AS or group DNA. Compared with N group, the lung tissues taken from the mice in As and DNA groups displayed allergic inflammation with eosinophil infiltration, while the pulmonary inflammation was decreased significantly in group NM. The levels of ET-1, IL-4, IL-13 and IL-5 were down regulated in group NM compared to As and DNA groups(P<0.05 or P<0.01). After stimulation by OVA, the splenocytes from the mice in NM group produced a higher level of IL-10 than that from As and DNA groups(P<0.05 or P<0.01). The number of Th2 lymphocytes(CD4+T cells with IL-4 expression ) was significantly elevated in the mice of As, DNA and NM groups respectively, while the number of Th2 lymphocytes was lower in themice of group NM than in the mice of group As or group DNA. The number of CD4+CD25+cells with IL-10 expression was up-regulated in the mice of As, DNA and NM groups respectively compared to the control. The percentage of T regulating cells was higher in the mice of group NM compared with that in the mice of group As or group DNA. No detectable difference in the level of Thl cells( CD+4T cells with IFN-gamma expression) was found among the 4 groups. Conclusion 12-ACSs can encapsulate and deliver antisenceECE expression plasmid into bronchial epithelial cells in vitro and 12-ACSs/antisense ECE plasmid complex nanoparticles have the capability to down regulate the synthesis of ET-I and thus decrease the allergic airway inflammation in OVA-sensitized mice.