腹腔感染棘球蚴微囊对小鼠肝脏多房棘球蚴感染及致病的影响

Effect of intraperitoneal inoculation with Echinococcus microcysts on the infection and pathogenicity of E. multilocularis in mouse liver

目的探讨腹腔感染细粒棘球蚴(Eg)或多房棘球蚴(Em)微囊对小鼠肝脏Em感染及致病的影响。方法收集Eg和Em原头节,分别体外成囊培养6周和8周。将60只C57雌性小鼠随机分为Em/Em感染组、 Eg/Em感染组、单纯Em肝脏感染组和假手术组等4组,每组15只。Em/Em感染组、 Eg/Em感染组、单纯Em肝脏感染组小鼠分别腹腔注射感染Em微囊50个(1×PBS缓冲液稀释至1 ml)、 Eg微囊50个(1×PBS缓冲液稀释至1 ml)、生理盐水1 ml,1个月后小鼠开腹,经肝门静脉注射感染2 000个(200μl)Em原头节;假手术组小鼠腹腔和肝门静脉注射等量生理盐水。小鼠腹腔感染前及感染后第1、 3和6个月尾静脉采血,ELISA检测血清中抗包囊囊液蛋白抗体情况。肝门静脉感染后1、3和6个月,每组各剖检5只小鼠,肉眼观察肝脏病灶感染情况,并进行量化计分。取各组小鼠大叶肝脏,进行石蜡切片和HE染色,观察肝脏病灶组织情况。采用SPSS 21.0进行统计学分析,数据采用独立样本t检验。结果 体外培养6周的Eg微囊直径为300~500μm,培养8周的Em微囊直径为150~300μm。ELISA结果显示,腹腔感染棘球蚴微囊后1、 3、 6个月,Em/Em感染组和Eg/Em感染组小鼠血清中抗包囊囊液蛋白抗体水平(A405值)均上升且维持较高水平,其中感染后1个月的A405值分别为2.77±0.62、 2.35±0.23,较假手术组的0.15±0.02升高10倍以上。肝门静脉感染1、 3和6个月后剖检,Eg/Em感染组腹腔可见Eg包囊形成,Em/Em感染组、单纯Em肝脏感染组和假手术组则均未见有包囊形成。肝门静脉感染后1、 3、 6个月,单纯Em肝脏感染组小鼠的肝脏病灶最为严重,肝脏表面大多有数十个点状或大小不一泡状病灶;Eg/Em感染组小鼠肝脏病灶较弱或正常,肝脏表面有1~3个微小病灶,且随感染周期延长趋于正常。Em/Em组和假手术组小鼠肝脏在不同时期均未见病灶。病灶严重程度评分结果显示,单纯Em肝脏感染组各时期评分(4.8±1.3、5.0±1.5、4.5±1.4)均高于Eg/Em感染组(1.6±0.9、 1.2±1.1、 1.6±0.9)(t=4.92、 4.81、 5.24,均P <0.01)。HE染色结果显示,肝门静脉感染Em原头节后1个月,单纯Em肝脏感染组病灶有大量炎性细胞浸润,以嗜酸粒细胞为主;Eg/Em感染组病灶的炎性细胞浸润较少;感染后3个月,单纯Em肝脏感染组病灶的炎性细胞相对前期减少,以中性粒细胞居多,病灶周边形成胶原纤维层;Eg/Em感染组病灶以胶原纤维层为主;感染后6个月,Eg/Em感染组病灶恢复正常趋势较明显,以结缔组织增生为主。结论 腹腔Em感染可完全阻断小鼠肝脏Em再感染,腹腔Eg感染可较大程度抑制小鼠肝脏Em感染。

Objective To investigate the effect of intraperitoneal inoculation of Echinococcus granulosus(Eg)or E.multilocularis(Em)micorcysts on hepatic infection and pathogenesis of Em in mice liver.Methods The protoscoleces(PSCs)of Eg and Em were collected and cultured in vitro for 6 and 8 weeks to encyst,respectively.Sixty C57 female mice were randomly assigned to 4 groups,which were Em/Em infection,Em/Eg infection,pure Em infection and sham surgery groups,with 15 mice in each group.The mice of designated infection groups were intraperitoneally injected with 50 Em microcysts(diluted in 1 ml 1×PBS),the mice in the Eg/Em infection group were intraperitoneally inoculated with 50 Eg microcysts,and 1 ml of normal saline,respectively.After 1 month,the mice in all three groups underwent laparotomy and were injected with 2000 Em PSCs(200µl)through hepatic portal vein(HPV).The mice in the sham operation group were injected with the same amount of normal saline into HPV in abdominal cavity.Mice blood was collected from the tail vein before intraperitoneal inoculation and at 1,3 and 6 months after inoculation.In the collected blood samples,serum antibodies against hydatid cyst fluid antigen were detected by ELISA.At 1,3 and 6 months after HPV infection,5 mice in each group were dissected to inspect the liver lesions of infection with naked eyes and conduct quantitative scoring.The big lobe of liver was collected from mice to prepare paraffin section,and stain with HE to observe the changes in liver lesion tissue.SPSS 21.0 was used for statistical analysis,and an independent sample t-test was used to compare the mean between the two groups.Results The diameter of Eg microcysts cultured for 6 weeks in vitro is about 300 to 500µm,and the diameter of Em microcysts cultured for 8 weeks is about 150 to 300µm.ELISA results showed that after 1,3,and 6 months of intraperitoneal infection with echinococcus microcapsules,the levels of anti-cyst fluid protein antibodies(A405 values)in the serum of mice infected with Em/Em and Eg/Em elevated and maintained at high level.One month after intraperitoneal inoculation of microcysts,the serum anti cyst fluid protein antibody levels(A405 values)in mice infected with Em/Em and Eg/Em groups were 2.77±0.62 and 2.35±0.23,respectively,which were more than 10 times higher than 0.15±0.02 in the sham operation group.After 1,3,and 6 months of HPV infection,the autopsy was performed.Eg cysts were seen in the abdominal cavity of the Eg/Em infected group,while no cysts were seen in the Em/Em group,the Em liver infected group and the sham group.Liver infection at different stages was the most severe in the Em liver infection group,where the liver surface was mostly covered with punctate or vesicular lesions of varying sizes.In the Eg/Em infection group,the infected lesions were weak or normal,and there were 1-3 small lesions on the liver surface that tend to become normal as the infection cycle extends.No infectious lesions were found in the Em/Em group and the sham group at different stages.The results of the lesion severity score showed that the scores of each stage in the group with Em liver infection(4.8±1.3,5.0±1.5,4.5±1.4)were higher than those in the group with Eg/Em infection(1.6±0.9,1.2±1.1,1.6±0.9)(t=4.92,4.81,5.24,all P<0.01).The results of HE staining showed that 1 month after HPV infection with Em PSCs,there was a large number of inflammatory cells infiltrating the lesions in the Em liver infection group,mainly eosinophils;In the Eg/Em infected group,there was less infiltration of inflammatory cells in the lesion.At 3 months after infection,the inflammatory cells in the focus of the simple Em liver infection group decreased relative to the prophase,with neutrophils predominating,and a collagen fibre layer formed around the focus;In the Eg/Em infection group,the focus was mainly collagen fibre layer;At 6 months after infection,the lesions in both the Em liver infection group and the Eg/Em infection group showed a trend of recovery,mainly with connective tissue hyperplasia.Conclusion Em microcysts infection via abdominal cavity can completely block Em reinfection in mice liver,and Eg infection via abdominal cavity can suppress Em infection in mice liver to a greater extent.