摘要目的 探究巨噬细胞活化在先天性巨结肠相关性小肠结肠炎(Hirschsprung's disease associated enterocolitis,HAEC)发病机制中的作用.方法 以SPF级Ednrb基因敲除小鼠为先天性巨结肠(Hirschsprung's disease,HSCR)模型鼠.采用抽签法,随机选择2周龄和3周龄EdnrB-/-小鼠各3只作为EdnrB-/-组,相应周龄野生型小鼠各3只作为对照组,氯膦酸二钠脂质体(clodronate li-posomes,CLOD)腹腔注射2周龄EdnrB-/-小鼠3只作为CLOD处理组.收集三组小鼠结肠肠管标本,并分为结肠远段 、近段.采用CD68及iNOS免疫荧光双染法观察小鼠结肠巨噬细胞及其活化.RT-qPCR分析小鼠结肠各段iNOS、TNF-α 的mRNA表达水平.结果 CD68及iNOS免疫荧光双染显示,2周龄EdnrB-/-组小鼠结肠近段的CD68阳性巨噬细胞百分比为(5.71±1.96)％,与同龄野生型小鼠结肠近段(1.31±0.81)％ 比较,差异有统计学意义(P<0.05).3周龄EdnrB-/-组小鼠结肠近段CD68阳性的巨噬细胞和iNOS阳性细胞百分比较高,分别为(12.81±2.28)％ 和(7.81±2.35)％,与同龄野生型小鼠(2.25±1.17)％ 和(1.22±0.66)％ 比较,差异均有统计学意义(P均<0.05).CLOD处理组结肠近段CD68阳性细胞百分比为(2.52±0.49)％,iNOS阳性细胞百分比为(1.29±0.38)％,与3周龄EdnrB-/-组小鼠近段比较,差异均有统计学意义(P均<0.05).RT-qPCR分析结果显示:2周龄EdnrB-/-小鼠结肠各段炎症因子表达无明显变化,而3周龄EdnrB-/-小鼠结肠近段肠管炎症因子的表达水平明显升高.CLOD处理组小鼠肠管炎症明显减轻 、炎性因子释放明显下降.结论 巨噬细胞活化及其分泌的促炎因子在HAEC的发生发展中起到一定作用.

abstractsObjective To explore the mechanism of macrophage activation in murine model of Hirschsprung-associated enterocolitis .Methods According to drawing lot method ,specific-pathogen-free level laboratory 2-week-old and 3-week-old EdnrB-/- mice were selected as experimental group (n= 3) ,wild-type mice of comparable age as control group (n = 3) and 2-week-old EdnrB-/- mice treated with clodronate liposomes (CLOD) as treatment group (n = 3) .All intestinal samples were prepared into expansion and narrow sections according to their morphological changes . The expressions of CD68 and inducible nitric oxide synthase (iNOS ) were detected by immunofluorescent staining for assessing the macrophage and the activation level of intestinal macrophage .And reverse transcription-polymerase chain reaction (RT-qPCR) was employed for detecting the expressions of iNOS and TNF-α at the mRNA level .Results As compared with 2-week-old wild-type murine intestinal expansion colonic sections ,the positive cells of CD68 in 2-week-old EdnrB-/- mice slightly increased [(1 .31 ± 0 .81)％ vs .(5 .71 ± 1 .96)％ ,P< 0 .05] .However ,for expansion colonic sections&nbsp;of 3-week-old EdnrB-/- mice ,the positive cells of CD68 [(12 .81 ± 2 .28)％ vs .(2 .25 ± 1 .17)％ ,P<0 .05] and Inos [(7 .81 ± 2 .35 )％ vs . (1 .22 ± 0 .66 )％ , P < 0 .05 ] significantly increased . The expansion colonic sections of CLOD-treated 3-week-old EdnrB-/- mice group , the positive cells of CD68 and iNOS were (2 .52 ± 0 .49)％ and (1 .29 ± 0 .38)％ .As compared with expansion colonic sections of 3-week-old EdnrB-/- , there were significant differences . In 2-week-old EdnrB-/- mice group ,RT-qPCR showed that iNOS ,TNF-α and IL-1β had no differences as compared with control group at the level of mRNA . Yet in 3-week-old EdnrB-/- mice , these cytokines significantly increased .In CLOD-treated 3-week-old EdnrB-/- mice group ,inflammatory injury markedly declined and the expressions of cytokines decreased obviously as compared with 3-week-old EdnrB-/- mice group .Conclusions Intestinal macrophage activation and secretion of proinflammatory cytokines may be associated with the occurrence and development of Hirschsprung -associated enterocolitis .