慢性旋毛虫感染对伯氏疟原虫共感染小鼠肝脏免疫病理的调节作用

Modulation of chronic Trichinella spiralis infection on liver immunopathology in mice co-infected with Plasmodium berghei ANKA

目的探讨慢性旋毛虫(Trichinella spiralis,Ts)感染对伯氏疟原虫(Plasmodium berghei ANKA,PbA)共感染小鼠肝脏免疫病理的调节作用及其机制。方法将64只SPF级6~8周龄雌性昆明小鼠按体重(22~25 g)采用随机数字表法分为4组:对照组,不做处理;Ts单感染组(Ts组),每只小鼠口饲感染30条Ts幼虫;PbA单感染组(PbA组),每只小鼠于实验第121天经腹腔注射0.1 ml含1×10^(6)个感染PbA红细胞的磷酸盐缓冲液(PBS);Ts与PbA共感染组(Ts+PbA组):每只小鼠于口饲感染30条Ts幼虫后第121天经腹腔注射0.1 ml含1×10^(6)个感染PbA红细胞的PBS溶液。每组16只小鼠,其中每组10只小鼠用于观察生存率。PbA组和Ts+PbA组于感染PbA后第3天起每隔1天取尾静脉血,制作薄血膜涂片,进行吉姆萨染色,光镜下计数外周血红细胞PbA感染率。在感染Ts后第135天和/或感染PbA后第15天处死小鼠,称取小鼠体重和肝脏重量,计算肝脏指数;取感染Ts小鼠的新鲜膈肌制作肌肉压片,光镜下观察Ts幼虫囊包;光镜下,苏木素-伊红(HE)染色观察各组小鼠肝脏组织病理改变,天狼星红染色观察比较各组肝脏组织的纤维化程度,免疫组织化学染色观察肝脏中F4/80+枯否氏细胞表达水平。结果感染Ts、PbA后,光镜下分别观察到膈肌组织中的Ts幼虫囊包和红细胞内的PbA。感染PbA后,PbA组和Ts+PbA组小鼠生存率比较,差异无统计学意义(P>0.05);与PbA组比较,Ts+PbA组小鼠外周血红细胞PbA感染率在感染PbA后的第11、15天均较低(%:27.104±7.623比45.032±9.849,60.218±2.776比76.778±6.351,P均<0.05),小鼠肝脏指数和肝脏组织病理学评分也均较低(P均<0.05)。天狼星红染色结果显示,Ts+PbA组小鼠肝脏组织的纤维化阳性面积显著高于PbA组(P<0.05)。免疫组织化学染色显示,Ts+PbA组小鼠肝脏组织中F4/80+枯否氏细胞的平均光密度值显著高于PbA组(P<0.01)。结论慢性Ts感染可降低PbA共感染小鼠外周血红细胞的PbA感染率,增强肝脏组织F4/80+枯否氏细胞的表达,减轻肝脏免疫病理改变。

Objective To investigate the regulation and mechanism of chronic Trichinella spiralis(Ts)infection on liver immunopathology in mice co-infected with Plasmodium berghei ANKA(PbA).Methods According to body weight,64 specific pathogen free female Kunming mice(6-8 weeks old,weighting 22-25 g)were divided into 4 groups by using random number table method.Control group:uninfected;Ts group:mice were mono-infected with 30 Ts larvae by oral gavage on day 0;PbA group:mice were mono-infected with 1×10^(6) PbA-infected red blood cells in 0.1 ml of phosphate buffer(PBS)administered by intraperitoneal injection on day 121;co-infected(Ts+PbA)group:mice were infected with 30 Ts larvae by oral gavage and intraperitoneal injected with 1×10^(6) PbA-infected red blood cells in 0.1 ml PBS on day 121 after Ts infection.There were 16 mice in each group,in which 10 mice in each group were monitored for the survival rate.The peripheral red blood cell parasitemia of PbA group and Ts+PbA group were monitored every other day by light microscope examination of Giemsa-stained thin tail-blood smears from day 3 after PbA infection.Mice were sacrificed at day 135 after Ts infection and/or at day 15 after PbA infection,the mouse body weight and liver weight were measured,and the liver index were calculated.Ts-infected mice were monitored by a light microscope examination of diaphragm compression slide.Under a light microscope,the liver pathology and liver fibrosis of mice were observed and compared with hematoxylin-eosin(HE)staining and Sirius red staining,respectively.The F4/80+Kupffer cells in liver of mice were examined by immunohistochemical staining.Results After infection with Ts or PbA,Ts larvae cysts were observed in diaphragm tissues and PbA were observed in red blood cells under the light microscope.After PbA infection,there was no significant difference in survival rate between PbA group and Ts+PbA group(P>0.05).Compared with PbA group,the peripheral red blood cell parasitemia was significantly decreased in Ts+PbA group on days 11 and 15 after PbA infection(%:27.104±7.623 vs 45.032±9.849,60.218±2.776 vs 76.778±6.351,P<0.05),and the liver index and the liver pathology score were significantly decreased in Ts+PbA group(P<0.05).Sirius red staining showed that the positive area of liver fibrosis in Ts+PbA group was significantly higher than that in PbA group(P<0.05).Immunohistochemical staining showed that the average optical density value of F4/80+Kupffer cells in Ts+PbA group was significantly higher than that in PbA group(P<0.01).Conclusion Chronic Ts infection may reduce the peripheral red blood cell parasitemia,increase F4/80+Kupffer cells expression in liver,and attenuate liver pathology in mice co-infected with PbA.