旋毛虫诱导的非特异性免疫对伯氏疟原虫感染小鼠小肠组织免疫反应的调节

Regulation of non-specific immunity induced by Trichinella spiralis to immune response in small intestinal tissue of Plasmodium berghei ANKA infected mice

目的探讨旋毛虫(Trichinella spiralis,Ts)诱导的非特异性免疫对伯氏疟原虫(Plasmodium berghei,Pb)ANKA感染小鼠小肠组织免疫反应的调节作用.方法36只SPF级雌性昆明小鼠(6~8周龄,体重为18~22 g),按体重采用随机数字表法分成4组,分别为对照组、Ts单感染组(Ts组)、PbANKA单感染组(Pb组)、Ts与PbANKA共感染组(Ts+Pb组),每组9只.小鼠正常进食、饮水,普通饲料喂养.对照组不做任何实验处理;Ts组在实验第1天经口感染20条Ts幼虫;Pb组在实验第9天经腹腔注射感染1×106个寄生PbANKA的红细胞;Ts+Pb组在实验第1天经口感染20条Ts幼虫,第9天经腹腔注射感染1×106个寄生PbANKA的红细胞.于感染Ts后第22天和/或感染PbANKA后第13天剖杀小鼠,采用透射电镜观察各组小鼠腹腔巨噬细胞的形态变化;采用实时荧光定量PCR(qRT-PCR)检测各组小鼠小肠组织M1型巨噬细胞标记物[诱导型一氧化氮合酶(iNOS)、白细胞介素-6(IL-6)]以及M2型巨噬细胞标记物[C型甘露糖受体2(Mrc-2)、类几丁质酶3(Ym1)]mRNA表达水平,并比较M2/M1型巨噬细胞标记物mRNA表达水平的比值.结果透射电镜观察发现,对照组小鼠腹腔巨噬细胞形态结构正常;Ts组小鼠腹腔巨噬细胞呈现较多伪足;Pb、Ts+Pb组小鼠腹腔巨噬细胞除呈现较多伪足外,并吞噬有疟原虫.4组小鼠小肠组织iNOS(1.000±0.290、1.277±0.251、3.088±1.110、2.604±0.773)、IL-6 mRNA表达水平(1.000±0.393、2.180±0.629、1.650±0.612、3.242±1.780)比较,差异有统计学意义(F=12.420、5.270,P均<0.05).与对照组比较,Pb、Ts+Pb组iNOS mRNA表达水平明显升高(P均<0.05);与Ts组比较,Ts+Pb组iNOS mRNA表达水平明显升高(P<0.05).与对照组比较,Ts+Pb组IL-6 mRNA表达水平明显升高(P<0.05).4组小鼠小肠组织Mrc-2、Ym1 mRNA表达水平比较,差异有统计学意义(F=9.890、20.500,P均<0.05).Ts+Pb组Mrc-2 mRNA表达水平明显高于对照、Ts、Pb组(P均<0.05).Pb组Ym1 mRNA表达水平明显高于对照组(P<0.05);Ts+Pb组Ym1 mRNA表达水平明显高于对照、Ts、Pb组(P均<0.05).4组小鼠小肠组织Mrc-2/iNOS、Ym1/iNOS比较,差异有统计学意义(F=3.642、22.360,P均<0.05).Ts+Pb组Mrc-2/iNOS明显高于对照、Pb组(P均<0.05).Ts+Pb组Ym1/iNOS高于对照、Ts、Pb组(P均<0.05).结论Ts诱导的宿主非特异性免疫参与PbANKA感染小鼠肠道免疫应答的调节,并促使其小肠组织巨噬细胞向M2型极化.

Objective To investigate the regulatory effect of non-specific immunity induced by Trichinella spiralis(Ts)on the immune response of small intestinal tissue of mice infected with Plasmodium berghei(Pb)ANKA.Methods Thirty-six specific pathogen free female Kunming mice(6-8 weeks old,weighting 18-22 g)were randomly divided into 4 groups according to body weight by the random number table method,including control group,Ts-mono-infected group(Ts group),PbANKA-mono-infected group(Pb group),and Ts+Pb-co-infected group(Ts+Pb group),9 mice in each group.The mice were fed normal food,water and normal feed.The control group was not given any experimental treatment;the Ts group was infected with 20 Ts larvae orally on the first day of the experiment;the Pb group was infected with 1×106 PbANKA erythrocytes by intraperitoneal injection on the 9th day of the experiment;the Ts+Pb group was infected with 20 Ts larvae orally on the first day of the experiment,and 1×106 PbANKA erythrocytes were given by intraperitoneal injection on the 9th day.Mice were sacrificed on 22th day after Ts infection and/or 13th day after PbANKA infection,the morphological changes of peritoneal macrophages in each group were observed by transmission electron microscope;the mRNA expression levels of M1-type macrophage markers[inducible nitric oxide synthase(iNOS)and interleukin-6(IL-6)],M2-type macrophage markers[mannose receptor C type 2(Mrc-2)and chitinase-like 3(Ym1)]in the small intestinal tissue of mice in each group were detected by quantitative real-time PCR(qRT-PCR),and the ratios of mRNA expression levels of M2/M1 macrophage markers were compared.Results Transmission electron microscope showed that the morphology and structure of peritoneal macrophages in the control were normal;more pseudopodia were observed in the peritoneal macrophages in Ts group;and more pseudopodia and engulfed Plasmodium parasites were observed in the peritoneal macrophages in Pb group and Ts+Pb group.The iNOS(1.000±0.290,1.277±0.251,3.088±1.110,2.604±0.773)and IL-6 mRNA expression levels(1.000±0.393,2.180±0.629,1.650±0.612,3.242±1.780)of small intestinal tissue were compared among the 4 groups,the differences were statistically significant(F=12.420,5.270,P<0.05).Compared with the control group,the iNOS mRNA expression levels in the Pb and Ts+Pb groups were significantly increased(P<0.05);compared with the Ts group,the iNOS mRNA expression level in the Ts+Pb group was significantly increased(P<0.05).Compared with the control group,the IL-6 mRNA expression level in the Ts+Pb group was significantly increased(P<0.05).The mRNA expression levels of Mrc-2 and Ym1 of small intestinal tissue in the 4 groups were significantly different(F=9.890,20.500,P<0.05).The Mrc-2 mRNA expression level in the Ts+Pb group was significantly higher than those in the control,Ts,and Pb groups(P<0.05).The Ym1 mRNA expression level in the Pb group was significantly higher than that in the control group(P<0.05);the Ym1 mRNA expression level in the Ts+Pb group was significantly higher than those in the control,Ts,and Pb groups(P<0.05).The Mrc-2/iNOS,Ym1/iNOS of small intestinal tissue in the 4 groups were significantly different(F=3.642,22.360,P<0.05).The Mrc-2/iNOS in the Ts+Pb group was significantly higher than those in the control and Pb groups(P<0.05).The Ym1/iNOS in the Ts+Pb group was higher than those in the control,Ts,and Pb groups(P<0.05).Conclusion The non-specific immunity induced by Ts infection is involved in the regulation of intestinal immune response of mice infected with PbANKA,which may promote M2 polarization of macrophages in the small intestinal tissue.