【作者】 张慧；

【导师】 张文宝；

【作者基本信息】 新疆医科大学 ， 病原生物学， 2022， 博士

【摘要】 目的:棘球蚴病（Echinococcosis）是棘球绦虫幼虫（metacestode）寄生于中间宿主肝脏、肺脏等器官引起的一种人兽共患寄生虫病,呈世界性流行,严重危害人类生命健康。人类棘球蚴病主要包括两种类型,分别是由细粒棘球绦虫（Echinococcos granulosus sensu stricto,E.granulosus s.s.）引起的囊型棘球蚴病（Cystic echinococcosis,CE）和由多房棘球绦虫（Echinococcus multilocularis,E.multilocularis）引起的泡型棘球蚴病（Alveolar echinococcosis,AE）。两型棘球绦虫与宿主肝脏界面的病理学表型有明显的差异,炎症细胞的募集在病灶肉芽肿形成中起着重要的作用。本研究旨在鉴定两型棘球绦虫感染病灶周围“炎症微环境”中的关键免疫细胞亚群,研究虫体抗原和宿主细胞因子对炎症细胞的趋化作用,探索两型棘球绦虫-宿主肝脏界面微环境不同炎症病理表型的可能机制。方法:第一部分建立经肝门静脉感染两型棘球绦虫小鼠模型,病理切片观察感染中后期肝脏病灶周围炎性细胞浸润情况和肝脏病理表现,采用免疫组织化学确定肝脏病灶周围“炎症微环境”的关键免疫细胞亚群。第二部分通过体外培养两型棘球绦虫微囊,采用x CELLigence RTCA DP系统实时监测细胞因子、两型棘球绦虫微囊、虫体分泌抗原丝氨酸蛋白酶（Serine protease,SP）、SP与补体活性片段C5a相互作用后对巨噬细胞和嗜酸性粒细胞等炎症细胞的趋化作用。第三部分通过Worm Base Para Site网站下载细粒棘球绦虫和多房棘球绦虫Kunitz型结构域丝氨酸蛋白酶抑制剂（Kunitz-type domain protease inhibitors,KDPIs）基因序列并进行生物信息学的比较,采用最大似然法构建系统发育进化树,以评估这些KDPIs基因的进化关系。对前期转录组分析显示表达上调的KDPIs基因采用定量PCR技术在E.granulosus s.s.和E.multilocularis虫体发育不同阶段（成虫、原头蚴和生发层）的表达情况进行了验证。第四部分建立经腹腔感染两型棘球绦虫小鼠模型,观察E.granulosus s.s.和E.multilocularis感染组及对照组小鼠肝脏及腹腔病灶的大小和数量,分离肝脏、脾脏、腹腔灌洗液中的白细胞,流式细胞术检测三组小鼠感染不同时期肝脏、脾脏、腹腔中B细胞亚群分布（B1、B2和Breg）。磁珠分选获得B细胞并进行流式细胞术鉴定分选效果,于不同数量的两型棘球绦虫微囊共培养,观察两型棘球绦虫对B细胞增殖的影响。结果:第一部分,（1）HE染色结果显示泡型棘球蚴病（alveolar echinococcosis,AE）和囊型棘球蚴病（Cyst echinococcosis,CE）小鼠肝脏炎症细胞浸润和病理表现存在显著的差异。与CE相比,AE肝脏病灶生长速度很快,感染更严重。与对照组相比,E.multilocularis呈侵袭性生长,感染小鼠肝脏病灶呈蜂窝状,可见弥漫性的肉芽肿,病灶周围大量炎症细胞浸润,肝细胞肿大,部分发生坏死。在感染同期,E.granulosus s.s.感染后肝脏病灶可见典型的囊蚴结构（包括球状囊结构,有比较光滑的外囊、角质层和生发层,囊内充满透明囊液）,仅病灶周围少量炎症细胞浸润。（2）两型棘球绦虫感染小鼠中后期肝脏中免疫细胞浸润情况,对照组小鼠肝脏中巨噬细胞、T细胞和B细胞均在肝血窦和肝细胞索之间呈散在少量分布,在E.multilocularis和E.granulosus s.s.感染小鼠肝脏中巨噬细胞离病灶最近,在病灶周围呈环状散在分布,T细胞也在肉芽肿周围散在分布,B细胞则在病灶周围呈克隆性增殖分布。巨噬细胞、T细胞和B细胞的关键分子标志物分别是CD68、CD3和CD19。免疫组织化学结果显示E.multilocularis和E.granulosus s.s.感染小鼠肝脏病灶周围CD68、CD3和CD19的表达均显著高于对照组（P<0.05）。E.multilocularis感染组小鼠肝脏中CD68、CD3和CD19的表达略高于E.granulosus s.s.组,但差异均无统计学意义（P>0.05）。第二部分,（1）两型棘球绦虫微囊（cysts）和分泌物均具有丝氨酸蛋白酶（SP）的活性。E.granulosus s.s.cysts分泌上清液中SP的活性高于E.multilocularis（P<0.05）。（2）两型棘球绦虫微囊对小鼠Raw264.7和正常小鼠腹腔巨噬细胞均具有趋化作用,接近于细胞因子5ng/m L CCL5对巨噬细胞的趋化作用。E.multilocularis Cysts诱导巨噬细胞迁移的能力强于E.granulosus s.s.Cysts。两型棘球绦虫微囊对E.multilocularis感染致敏小鼠腹腔巨噬细胞和腹腔未贴壁细胞（嗜酸性粒细胞70%-80%）均具有诱导迁移的作用,并且分别在6-8h和24h内细胞迁移指数达峰值。（3）两型棘球绦虫囊液（Hydatid cyst fluid,HCF）和SP对Raw264.7巨噬细胞均具有趋化作用,但是对小鼠脾脏淋巴细胞趋化能力很弱。两型棘球绦虫E.multilocularis和E.granulosus s.s.SP对小鼠腹腔巨噬细胞均具有显著的趋化作用。两型棘球绦虫SP与C5a相互作用后诱导巨噬细胞迁移的能力均减弱,E.granulosus s.s.SP+C5a组低于E.multilocularis SP+C5a组（P<0.05）。SP与C5a共培养明显降低了C5a对巨噬细胞的趋化作用。第三部分,两型棘球绦虫KDPIs生物信息学结果显示共有19个E.multilocularis和23个E.granulosus s.s.的丝氨酸蛋白酶抑制蛋白（serpin,SPN）基因被预测为KDPIs,其中大多数包含一个Kunitz结构域。系统发育进化树结果表明E.granulosus s.s.和E.multilocularis KDPIs分为3个分支,包含9个聚类。多房棘球蚴和细粒棘球蚴的正电荷残基和中性残基的比例不同。研究发现,E.multilocularis KDPIs序列含有信号肽的百分比（17/19,89.47%）高于E.granulosus s.s.（14/23,60.87%）。Hi-seq转录组学分析结果表明所有E.granulosus s.s.KDPI基因在该虫的四个发育阶段都有差异表达,9个KDPIs（包括EGR＿07244、EGR＿08716和EGR＿10096）在成虫中显著上调,2个KDPIs（EGR＿09268和EGR＿09490）在生发层中高表达。采用q-PCR对这些基因的表达进行验证,结果显示EGR＿08716及其同源基因（Emu J＿001137000）在两种蠕虫的成虫中均高度特异表达。第四部分,（1）成功建立了两型棘球绦虫微囊腹腔感染小鼠模型,AE和CE病理差异显著,CE进展缓慢,AE生长速度快病灶面积大。（2）肝脏B细胞亚群的变化:在感染4周,E.granulosus s.s.感染小鼠肝脏中B细胞百分比高于E.multilocularis感染和对照组肝脏组织（P<0.05）,在感染2周和8周肝脏B细胞百分比无显著差异。在感染2周和4周,AE肝脏中B细胞分泌IL-10明显高于CE（P<0.05）,其中Breg发挥了重要作用,但在第8周分泌IL-10下降,AE和CE无显著差异（P>0.05）。两型棘球绦虫感染第8周肝脏浆细胞比例显著的升高,但脾脏未显著增加（P<0.05）。CE肝脏B1细胞无显著变化,在感染4到8周AE肝脏中B1细胞显著高于CE,B2细胞低于CE（P>0.05）。两型棘球绦虫感染后三组小鼠肝脏中边缘区B细胞（Marginal Zone B cells,MZB）和滤泡状B细胞（Follicular B cells,FOB）细胞比例无显著差异（P>0.05）。（3）脾脏B淋巴细胞亚群的变化:在感染2周后CE和AE脾脏B细胞均升高,但4周后无显著性差异。感染2周后AE脾脏中B1细胞高于对照组（P<0.05）,E.multilocularis感染小鼠肝脏组织Breg(CD19⁺CD1d^highCD5⁺)细胞的比例高于E.granulosus s.s.感染和对照组小鼠肝脏组织（P<0.05）。感染4周后AE和CE小鼠脾脏B1和Breg细胞无显著差异。在感染2周CE和AE小鼠脾脏B2细胞中MZB细胞均下降,4周后CE中MZB呈上升趋势。两型棘球绦虫感染早期对脾脏MZB的分化有抑制作用,其抑制率AE明显高于CE（P<0.05）。在感染8周AE中MZB显著低于CE,而FOB显著高于CE（P<0.05）,说明E.multilocularis感染抑制脾脏MZB细胞分化,促进FOB细胞分化。（4）腹腔灌洗液B细胞亚群的变化:在感染4周,E.multilocularis感染小鼠腹腔CD19⁺B细胞比例显著低于和E.granulosus s.s.感染和对照组小鼠腹腔,差异有统计学差异（E.multilocularis vs E.granulosus s.s vs control:32.77±6.66%vs61.43±3.43%vs 58.65±4.39%,P<0.05）。感染早期AE和CE腹腔中B1、Breg和MZB细胞均下降,AE中上述三群细胞显著低于CE;AE腹腔中FOB细胞明显高于CE（P<0.05）。感染中后期AE小鼠腹腔Breg和IL-10均显著高于CE（P<0.05）。CD19磁珠分选后流式细胞术鉴定B细胞分选情况（分选前vs分选后:54.4%vs 94%）,说明B细胞分选成功。与不同数量的两型棘球绦虫微囊共培养后B细胞表现出剂量依赖性效应,B细胞的数量随着共培养的两型棘球绦虫微囊数量的增加而增加,提示两型棘球感染能促进B细胞的增殖。结论:1.两型棘球绦虫感染后期小鼠肝脏病理表现出显著的差异,AE肝脏中可见弥漫性的肉芽肿,周围大量炎性细胞浸润,CE肝脏中可见典型的囊蚴结构,炎症反应弱。AE和CE虫体-宿主肝脏界面“炎症微环境”中CD19⁺B细胞则呈克隆性增殖分布,CD68⁺巨噬细胞和CD3⁺T细胞在肝脏病灶中呈散在分布。2.虫体抗原（cysts、HCF、SP）对巨噬细胞具有趋化作用,但对脾脏淋巴细胞趋化作用很弱。E.multilocularis cysts诱导巨噬细胞迁移的能力强于E.granulosus s.s.cysts。两型棘球绦虫微囊分泌物均具有SP活性,并且E.granulosus s.s.cysts分泌物SP的活性高于E.multilocularis cysts。SP与C5a共培养明显降低了C5a对巨噬细胞的趋化作用。3.生物信息学分析分析鉴定了在19个E.multilocularis和23个E.granulosus s.s.Kunitz结构域蛋白酶抑制剂。这些KDPIs在E.granulosus s.s.不同发育阶段的差异表达表明,它们在调节宿主免疫应答方面可能具有不同的功能。KDPIs表达的差异可能与这两种寄生虫的Metaestode阶段的不同病理学有关。4.两型棘球绦虫微囊可以促进B细胞的增殖。两型棘球绦虫微囊腹腔感染不同的脏器和部位B细胞亚群分布特点不同。（1）肝脏:两型棘球绦虫感染早期AE小鼠肝脏中Breg细胞和分泌IL-10高于CE,感染后期均下降。两型棘球蚴病在感染后在B1和B2细胞分化方面存在差异。两型棘球绦虫感染后期浆细胞显著的升高,但脾脏未显著升高,浆细胞的成熟场所可能不在脾脏,而在肝脏。（2）脾脏:两型棘球绦虫感染早期AE和CE中B1和Breg均升高,AE高于CE,中后期无显著差异。两型棘球绦虫感染早期均抑制脾脏MZB细胞分化,其抑制率AE明显高于CE,感染后期对脾脏MZB细胞分化存在差异。（3）腹腔:E.granulosus s.s.感染对腹腔B细胞无显著作用,E.multilocularis感染抑制腹腔B细胞募集。感染早期AE和CE均对腹腔中B1、Breg和MZB细胞分化有抑制作用,其抑制率AE明显高于CE;对腹腔B2主体细胞FOB的分化有促进作用。感染中后期两型棘球绦虫感染腹腔中Breg和IL-10均显著升高,AE高于CE,在感染后期负性调控分子IL10对免疫反应发挥一定的抑制作用。更多还原

【Abstract】 Objective:Echinococcosis is a near-cosmopolitan zoonosis caused by adult or larval stages of cestodes belonging to the genus Echinococcus.The two major species are Echinococcus granulosus sensu stricto（s.s.）（E.granulosus s.s.）and Echinococcus multilocularis（E.multilocularis）,which cause cystic echinococcosis（CE）and alveolar echinococcosis（AE）,respectively,which threating the global population health.The pathological phenotypes of the interface between the two types of Echinococcus and the host liver are distinct.The migration and infiltration of inflammatory cells are the main cause of different pathological reactions and granuloma around the two types of hydatid lesions.The aims for this study were to reveal the differences in inflammatory cell infiltration caused by two types of Echinococcus infection,to disclose whether the cysts of these two parasites and their major secreted antigens can attract these cells to the cysts and to study the mechanisms regulating recruitment of inflammatory cells in the two types of Echinococcus and host interface microenvironment.Methods:Part one,The mice model inoculated via the hepatic portal vein with E.granulosus s.s.and E.multilocularis protoscolex（PSC）were established,and the pathological changes were observed,including the formation of"parasitic infection inflammatory microenvironment",inflammatory cell infiltration and liver pathology;Immunohistochemistry were used to detect key immune cell subsets recruited around liver lesions in these two tapeworms infected mice.Part two,In vitro the two tapeworms microcysts were cultured to 0.5-1mm in size in cell culture flask.Then RTCA（real time cell analysis）platform（x CELLigence DP system）was used to monitor the migration of macrophages and other inflammatory cells induced by cytokine CCL5,E.granulosus s.s.and E.multilocularis cysts,complement component C5a and the major secreted antigens serine protease of the parasites and to investigate how E.granulosus s.s.and E.multilocularis cysts induced inflammatory cells migration.Part three,E.granulosus and E.multilocularis sequence data are accessible at Worm Base Para Site.To identify Kunitz-type domain protease inhibitors（KDPIs）present in the genomes of these two tapeworms and analyzed the gene sequences using computational,structural bioinformatics and phylogenetic approaches to evaluate the evolutionary relationships of these genes.Hi-seq transcriptome analysis showed that E.granulosus KDPIs were differentially expressed in the different developmental stages.Quantitative PCR was used to validate some of the genes expressed in adult worm,protoscolex and cyst germinal membrane of E.granulosus and E.multilocularis.Part four,To establish the mice model inoculated via intraperitoneal injection with E.granulosus s.s.and E.multilocularis protoscolex（PSC）,and to analyze the size on the surface of hepatic and abdominal cavity lesions in mice model.To isolate leukocytes in liver,spleen,peripheral blood,and peritoneal lavage fluid,and then the percentages of B cells subsets including plasma cells,B1,B2,and Breg type B cells were measured by flow cytometry.The ability of B cells subsets to secrete IL-10 were detected by flow cytometry,in vitro stimulation test.Results:Part one,（1）It showed that Alveolar echinococcosis（AE）that behaves like a malignant and progressive liver disease,is characterized by a tumor-like growth of the metacestode,a large number of inflammatory cells infiltration and tissue necrosis around the liver lesion.In the same period of infection,whereas CE is characterized by unilocular with mild cell infiltration which gradually gathered around the cyst.The germinal layer was formed and a thin and dense inflammatory zone was seen in CE liver lesions.（2）These two tapeworms infection inflammatory microenvironment was formed around the liver lesions in mice,which was composed of CD19⁺B cells,CD68⁺Macrophages,and CD3⁺T lymphocytes.Macrophages and T cells were scattered around the liver lesions in a ring shape,while B cells proliferated in clusters around the lesions.Immunohistochemistry results showed that the expression of CD68,CD3 and CD19 in the liver lesions of E.multilocularis and E.granulosus s.s.infected mice were significantly higher than those of the control group.The expression of CD68,CD3 and CD19 of E.multilcularis infected mice liver lesions was slightly higher than that of E.granulosus s.s.infected mice,but the difference was not statistically significant.Part two,（1）Two types of Echinococcus tapeworm microcysts（cysts）secretions have serine protease（Serine protease,SP）activity.The activity of SP in the secretion supernatant of E.granulosus s.s.cysts was higher than that of E.multilocularis.（2）E.multilocularis cysts and E.granulosus s.s.cysts have chemotactic effects on both mouse Raw264.7 and peritoneal macrophages which are close to the chemotaxis of cytokine 5ng/m L CCL5 to macrophages.Mouse peritoneal cells were elicited by 5000 E.multilcularis PSCs injection into the peritoneum of E.multilcularis PSC-sensitized infected mice.E.multilocularis cysts and E.granulosus s.s.cysts can induce migration of both peritoneal macrophages and peritoneal non-adherent cells（eosinophils 70%-80%）in E.multilcularis infection-sensitized mice.The cell migration index peaked within 6-8h for peritoneal macrophages and 24h for peritoneal non-adherent cells（eosinophils 70%-80%）respectively.It was showed that inflammatory cells,including Macrophages and Peritoneal lavage fluid（PLF）suspension cells,migrate significantly faster in the presence of E.multilocularis cysts than E.granulosus s.s.cysts.This indicate that E.multilocularis cysts have the bigger ability to increase the migratory and invasive properties of inflammatory cells than E.granulosus s.s.cysts.（3）Two types of Echinococcus hydatid cyst fluid（HCF）and SP also can recruit mouse Raw264.7macrophages,but the chemotaxis to mouse spleen lymphocytes is very weak.The ability of E.granulosus s.s.SP（EGR＿04862）and E.multilcularis SP（Emu J＿000184900）to induce macrophage migration was weakened after interaction with C5a,and the cell index of EGR＿04862+C5a group was lower than that of the Emu J＿000184900+C5a group.This indicate that SP of Echinococcus has serine activity and can reduce cell chemotaxis,suggesting that the activity of SP in the two species of Echinococcus are the key regulators in the interface pathological phenotypes between host and parasite.Part three,a total of19genes from E.multilocularis and 23genes from E.granulosus s.s.were predicted to be KDPIs with the most containing a single Kunitz-domain.A maximum likelihood method phylogenetic tree indicated that the E.granulosus s.s.and E.multilocularis Kunitz domain peptides were divided into three branches containing 9 clusters.The ratio of positively charged residues and neutral residues are different between E.multilocularis and E.granulosus KDPIs.We also found that E.multilocularis had higher percentage of sequences containing signal peptides（17/19,89.47%）than that of E.granulosus s.s.（14/23,60.87%）.Transcript analysis showed all the E.granulosus s.s.KDPI genes were expressed differentially in four developmental stages of the worm.Transcription analysis showed that 9 KDPIs（including EG＿07244,EGR＿08716 and EGR＿10096）were highly upregulated in adult worm,and 2 KDPIs（EG＿09268 and EG＿09490）were highly expressed in the cyst germinal membrane.Quantitative gene expression analysis（q PCR）of four genes confirmed the expression of these genes.EGR＿08716 and its homologous gene（Emu J＿001137000）were highly and specifically expressed in adult worms of the two worms.Part four,1.The mice model inoculated via intraperitoneal injection with E.granulosus ss and E.multilcularis PSC by were successfully established.There was significant difference in AE and CE pathological phenotypes.2.Liver B cell subsets:The percentage of B cells in the livers of E.granulosus s.s.-infected mice was higher than that in E.multilcularis-infected and control liver tissues in 4 weeks of infection（P<0.05）.But there was no significant difference in the percentage of B cells in the liver of these two tapeworms infected mice in second and eighth week.The percentage of Breg cells(CD19⁺CD1d^highCD5⁺)in liver tissue of the E.multilcularis infected mice was higher than that in control mice liver tissue,resulting in higher levels of IL-10 in the early stage.The ability of B cells in AE mice liver to secrete IL-10 was significantly higher than that in CE mice in second and fourth weeks（P<0.05）.But the B cells secretion of IL-10 decreased at the eighth week,and then there was no significant difference between AE and CE（P>0.05）.The percentage of CD45⁺CD19^intCD138⁺plasma cells in E.multilcularis infected mice liver tissue was significantly higher than that in control mice liver tissue in the middle and late stage（8 weeks）.The percentage of plasma cells in E.multilcularis and E.granulosus s.s.infected mice liver showed an increasing trend along with the prolongation of infection time.But there was no significant increase in spleen plasma cells.AE mice liver exhibited a higher percentage of B1 cells but lower percentage of B2 cells compared to CE mice in fourth and eighth week.There was no significant difference in the proportion of liver MZB and FOB cells among E.granulosus s.s.,E.multilcularis and control mice（P>0.05）.3.Spleen B cell subsets:In contrast to the control mice,AE and CE mice showed higher frequencies of B cells and the percentage of B1 cells and Breg cells of AE mice were higher than those of CE mice in second week（P<0.05）.There were no significant differences in spleen B cells,B1 cells and Breg cells of AE and CE mice after 4 weeks infection.AE and CE mice spleens exhibited lower percentage of MZB cells compared to the control mice in the early stage.After 4 weeks E.multilocularis infection inhibited the differentiation of spleen MZB cells and promoted the differentiation of FOB cells.The percentage of MZB and FOB cells of AE mice was significantly lower and higher than CE mice in eighth week respectively（P<0.05）.But E.granulosus s.s.infection had no significant effect on the differentiation of spleen MZB and FOB cells in the middle and late stage.These two tapeworms infection could inhibit the differentiation of spleen MZB,and the inhibition rate of E.multilcularis infection was significantly higher than that of E.granulosus s.s.infection（P<0.05）.It indicated that E.multilcularis infection inhibited the differentiation of spleen MZB cells and promoted the differentiation of FOB cells.4.Peritoneal cavity B cell subsets:The proportion of CD19⁺B cells in the peritoneal cavity of E.multilocularis-infected mice was significantly lower than that of E.granulosus s.s.-infected and control mice in fourth week,and the differences among three group mice were statistically significant（E.multilocularis vs E.granulosus s.s.vs control:32.77±6.66%vs 61.43±3.43%vs 58.65±4.39%,P<0.05）.In contrast to the control mice,E.multilcularis and E.granulosus s.s.infected mice showed decreased B cells.In the early stage of infection AE mice peritoneal cavity exhibited lower percentage of B1,Breg and MZB cells and relatively higher percentage of FOB cells compared to the CE mice in the early stage（P<0.05）.In middle and late stage of infection AE mice showed higher frequencies of Breg cells,and higher levels of IL-10 in peritoneal cavity than that of CE mice（P<0.05）.4.The percentage of B cells was 94%identified by Flow Cytometry after CD19 magnetic bead sorting,indicating that B cell sorting was successful.B cells proliferate as the number of cocultured E.multilocularis cysts and E.granulosus s.s.cysts increase,exhibiting a dose-dependent effect.Conclusion:1.The metacestodes of E.granulosus s.s.and E.multilocularis PSC cause remarkable difference in pathology with cell infiltration in the lesion of AE,whereas,little cell infiltration occurs in CE lesion.E.multilocularis elicited a severe hepatic granulomatous inflammation response characterized by attraction of various host immune cells around the metacestode lesion,whereas typical cysticercus structure was present in CE liver,with weak inflammatory response.These two tapeworms infection inflammatory microenvironment was formed around the liver lesions in mice,which was composed of CD19⁺B cells,CD68⁺Macrophages,and CD3⁺T lymphocytes.Macrophages and T cells were scattered around the liver lesions in a ring shape,while B cells proliferated in clusters around the lesions.2.E.multilocularis cysts and E.granulosus s.s.cysts can recruit mouse peritoneal macrophages and Peritoneal lavage fluid（PLF）suspension cells,which were close to chemotaxis of cytokine CCL5.But the recruitment of cysts to mouse lymphocytes is very weak.It was showed that inflammatory cells,including Macrophages and PLF suspension cells,migrate significantly faster in the presence of E.multilocularis cysts than E.granulosus s.s.cysts.E.multilocularis cysts have the stronger ability to increase the migratory and invasive properties of inflammatory cells than E.granulosus s.s.cysts.Two types of Echinococcus tapeworm microcysts（cysts）secretions have serine protease（Serine protease,SP）activity.The activity of SP in the secretion supernatant of E.granulosus s.s.cysts was higher than that of E.multilocularis.The ability of two tapeworms SP to induce macrophage migration was weakened after interaction with C5a.SP of Echinococcus has serine activity and can reduce cell chemotaxis,suggesting that the activity of SP in the two species of Echinococcus are the key regulators in the interface pathological phenotypes between host and parasite.3.A total 19 and 23 KDPIs were identified in the genomes of E.multilocularis and E.granulosus,respectively.The differential expression of these KDPIs in different stages may indicate their different roles in the different hosts.The difference in characterization of KDPIs may be associated with the different pathology of metacestode stage of these two parasites.4.E.multilocularis cysts and E.granulosus s.s.cysts can promote the proliferation of B cells.The distributions of B cell subsets have different characteristics in different organs from mice infected with two types of Echinococcus cysts.（1）Liver:E.multilocularis infected mice showed higher frequencies of Breg cells,and higher levels of IL-10 than that of E.granulosus s.s.infected mice in the early stage of infection.Breg cells an important cell group that secretes IL-10.AE mice liver showed higher frequencies of B1 cells and lower frequencies of B2 cells than CE mice in the middle and late stage（8 weeks）of infection,which may be the main cause of liver inflammatory cells migration.It shows that there were significant differences in B1 and B2 cell differentiation between the two types of echinococcosis after infection.The percentage of liver plasma cells in E.multilcularis and E.granulosus s.s.infected mice increased significantly in the middle and late stage.But there was no significant increase in spleen plasma cells.These results suggest that the maturation organ of plasma cells after infection with Echinococcos maybe is not in the spleen,but in the liver.（2）Spleen:E.multilocularis infected mice showed higher frequencies of B1 and Breg cells than that of E.granulosus s.s.infected mice in the early stage of infection.But there was no significant difference in the percentage of spleen B cells and B1 cells of E.granulosus s.s.and E.multilcularis infected mice in the middle and late stage.E.granulosus s.s.and E.multilcularis infected mice spleens exhibited a higher percentage of Breg cells but lower percentage of MZB cells compared to the control mice in the early stage,which might be an important mechanism of immune escape implemented by the parasite.The MZB cell frequencies in AE mice spleen were lower than that of CE mice.It shows that there were significant differences in MZB cell differentiation between the two types of echinococcosis after infection in the middle and late stage.（3）Peritoneal cavity:In contrast to E.granulosus s.s.,E.multilocularis infection inhibited the recruitment of peritoneal B cells.In the early stage of infection these two tapeworms significantly inhibited B cells in the peritoneal cavity,including B1,Breg and MZB cells,and the inhibition rate of E.multilocularis infected mice was significantly higher than that of E.granulosus s.s.infected mice.But they could promote the differentiation of B2 cells in peritoneal cavity,the percentages of B2 cells in AE mice were significantly higher than that of CE mice.In contrast to the early stage of infection,AE mice in middle and late stage of infection showed higher frequencies of Breg cells,and higher levels of IL-10 in peritoneal cavity than that of CE mice.There was no significant difference among E.granulosus s.s.,E.multilocularis infected and control mice with increased percentages of B1 cells and decreased percentages of B2 cells in middle and late stage of infection.更多还原