MHC不同基因型哈萨克绵羊人工感染细粒棘球绦虫虫卵后小肠、肝脏差异表达基因的筛选
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摘要
细粒棘球蚴病，俗称包虫病，是细粒棘球绦虫的幼虫寄生于人、绵羊等中间宿主所致的一种人畜共患病。绵羊是细粒棘球绦虫最适宜的中间宿主，感染包虫病后生长发育缓慢，其毛、肉和奶的产量均严重受到影响，且患病绵羊肝、肺不能食用。最新调查显示，新疆存栏绵羊4000多万只，饲养数达6000多万只，而包虫病患病率达到50%，这给畜牧业的健康发展以及广大农牧民的生产创收带来严重影响。
本研究前期做了大量的关于绵羊主要组织相容性复合体基因（MHC基因）与包虫病抗性、易感性的相关研究，对新疆三个主要绵羊品种：中国美利奴羊、多浪羊及哈萨克羊MHC-DRB1基因外显子2进行PCR-RFLP检测，成功筛选出哈萨克绵羊与包虫病抗性相关的单倍型MHC-DRB1MvaIbc-SacIIab-Hin1Iab，之后对带有该单倍型的绵羊（抗性组）和不带该单倍型的绵羊（非抗性组）进行人工攻虫，发现抗性组绵羊包虫病发病率比非抗性组绵羊低且差异显著。在此基础上，本文着重对前期筛选的具有包虫病抗性和非抗性的哈萨克绵羊的感染机制及其差异进行分析。
依据前期研究，本研究采用PCR-RFLP方法对250只哈萨克绵羊进行了相应抗性单倍型分析，共筛选出具有包虫病抗性的个体（基因单倍型为MHC-DRB1MvaIbc-SacIIab-Hin1Iab）14只,非抗性个体（基因单倍型为MHC-DRB1MvaIbb-SacIIaa-Hin1Iaa）22只。进一步采用B超和ELISA结合的方法对筛选出的绵羊进行包虫病检测，随访后购买17只健康的绵羊，其中包括用于人工感染实验的绵羊14只（抗性组和非抗性组各7只），以及健康对照的绵羊3只。MHC抗性组绵羊记为A组，MHC非抗性组绵羊记为B组，健康对照的绵羊记为C组。人工感染实验的14只绵羊每只饲喂约5000个成熟细粒棘球绦虫虫卵，建立人工感染细粒棘球绦虫模型后，开展了以下后续试验：
1.实验一旨在检测不同MHC基因型哈萨克绵羊感染细粒棘球绦虫虫卵后早期机体免疫指标的差异。本实验分别于感染前(0d)、感染后2h、3h、4h、9h、1d、2d、3d、7d采集绵羊颈静脉血，采用ELISA方法检测血清中抗体（IgM、IgE）、细胞因子（TNF-α、IFN-γ、IL-4、IL-10）及趋化因子（CXCL-9、CCL17）的水平，结果显示，MHC包虫病抗性组（A组）表现出以抗体IgE、IgM、Th1细胞因子（IFN-γ、TNF-α）和Th1趋化因子（CXCL-9）为优势的分泌水平，而非抗性组（B组）则表现出以Th2细胞因子（IL-4、IL-10）和Th2趋化因子（CCL17）为优势的分泌水平。且IgE、Th1细胞因子（IFN-γ、TNF-α）在虫卵感染后4h组间差异显著。提示，携带MHC抗性基因型的绵羊表现出强的包虫病抵抗力可能受其早期高水平的Th1细胞因子及IgE抗体的影响。
2.实验二旨在筛选出不同MHC基因型哈萨克绵羊感染细粒棘球绦虫后小肠组织差异表达的基因。本实验中，实验动物分为MHC包虫病抗性组（A）、MHC包虫病非抗性组(B)和健康对照组（C），A、B两组分别于感染后2h、3h、4h采集小肠组织，以C组健康绵羊作为健康对照组，采用基因芯片的方法分析不同MHC基因型哈萨克绵羊感染细粒棘球绦虫后小肠差异表达基因，其中感染组（A）、（B）与健康对照组（C）进行比较，结果发现:A与C之间差异表达的基因共4712个，其中表达量上调的有1959个，表达量下调的有2753个。B与C比较之后差异表达的基因4944个，其中2076个表达上调，2868个表达下调。从中选择出同C相比，A和B同为上调或下调，且表达量差异显著的基因共141个，其中上调的有32个基因，下调的有109个。上调的32个基因中，A组显著高于B组的有6个，低于B组的有26个；在下调的109个基因中，A组显著高于B组的35个，低于B组的74个。
3.实验三旨在筛选出不同MHC基因型哈萨克绵羊感染细粒棘球绦虫后不同时期（感染后8周、24周）肝脏组织差异表达基因。本实验中，实验动物分为MHC包虫病抗性组（A）、MHC包虫病非抗性组(B)和健康对照组（C），A、B两组分别于感染后8周、24周采集肝脏组织，以C组健康绵羊作为健康对照组，采用基因芯片的方法分析不同MHC基因型哈萨克绵羊感染细粒棘球绦虫后肝脏组织差异表达基因。
1）感染后8周，基因芯片结果显示:MHC抗性组（A）与对照组（C）之间差异表达的基因共3087个，其中表达量上调的有1768个，表达量下调的有1319个。MHC非抗性组（A）与对照组（C）比较之后差异表达的基因4839个，其中1888个表达上调，2951个表达下调。采用同样标准进一步筛选获得差异候选基因153个，其中上调的有87个基因，下调的有66个。上调的87个基因中，A组显著高于B组的有13个，低于B组的74个；在下调的66个基因中，A组显著高于B组的54个，低于B组的12个。
2）感染后24周，基因芯片结果显示:MHC抗性A组与C组之间差异表达的基因共3092个，其中表达量上调的有1374个，表达量下调的有1718个。MHC非抗性B组与对照C组比较之后差异表达的基因11717个，其中5381个表达上调，6336个表达下调。采用同样标准进一步筛选获得差异候选基因115个，其中上调的有40个基因，下调的有75个。上调的40个基因中，A组显著高于B组的有3个，低于B组的37个；在下调的75个基因中，A组显著高于B组的74个，低于B组的1个。对以上3组共8个样本的基因芯片结果进行荧光定量PCR检验，挑选8个基因进行验证，结果同芯片检测结果基本接近，说明检测结果真实可信。
3）对筛选到的差异表达基因进行GO富集和KEGG通路分析发现，差异基因主要的生化功能集中在代谢、细胞进程等方面，值得注意的是:KEGG分析结果显示参与补体凝集素途径的基因数量在小肠和肝脏24周结果中均为最多，同时在肝脏8周组中也相对较多，提示MHC同补体之间抵抗寄生虫感染可能存在关联。
4）结合相关文献报道和不同感染时期基因表达特点，本研究最终选择了杀伤细胞免疫球蛋白样受体（KIR2DS1）、补体凝集素C8、丝氨酸蛋白酶抑制剂、生长抑制DNA损伤基因（GADD45B）、PLA2G2A等基因作为绵羊抵抗包虫病的候选基因。
综上所述得出如下结论:高水平的Th1细胞因子及IgE抗体能够影响绵羊对包虫病的早期抵抗，补体凝集素代谢通路可能在小肠和肝脏后期起到一定的抵抗作用。总体来说，参与抵抗包虫病的相关基因数量较多，涉及到能量代谢、免疫、信号转导等多个方面，说明包虫病抵抗是一个复杂的生理过程。同时，同非抗性组绵羊检测结果相比，在3个检测点上（小肠、肝脏8周、肝脏24周）MHC抗性组基因表现出基因表达不活跃的特点，具体表现为大部分基因上调或者下调幅度均低于非抗性组，但是一些可能同包虫病抵抗相关的基因仍表现出抗性组高于非抗性组的特点。
Cystic echinococcosis (CE) is a chronic parasitic zoonosis caused by infection with the larval stage of the cestode Echinococcus granulosus (E. granulosus), resulting in the development of cysts in human and domestic animals. In domestic animals, especially in sheep, which appear to be highly susceptible to infection, CE causes considerable health problems and economical disadvantages because of production loss, as sheep infected with this disease often suffer from reductions in live weight gain, in milk yielding, in the fertility rates, in the value of wool or other products. In Xinjiang, there are more than60million sheep, with the high prevalence of CE reaching50%, which causes considerable health problems in sheep and thereby significantly influences the income of herdsman.
Previuosly, we carried a lot of studies to investigate the relationship between MHC-DRB1gene polymorphism and genetic resistance or susceptibility to CE in Chinese Merino, Duolang and Kazakh sheep. Genetic markers, MHC MvaⅠbc-SacⅡab-HinlIab haplotype, on CE resistance in Kazakh sheep have been divergently selected by us. The rates of Echinococcus granulosus (E. granulosus) infection in the internal organs of Kazakh sheep with this haplotype, is confirmed to be significantly lower than sheep without this haplotype, when exposure to the same level of parasites. CE resistant sheep therefore have an increased genetic capability to respond to and subsequently reject parasites when challenged. Carrying out a deep study in these sheep was, therefore, worth doing, since it could produce some interesting clues on the mechanism of resistance to E. granulosus infection both in immunology and genetics.
According to the previous methods stablished by us, we selected the genetic markers in250Kazakh sheep, again. Seventeen healthy two-year-old Kazakh sheep with different haplotype were selected and maintained during the course of the experiments. They were negative for antibodies to hydatid cyst fluid (HCF) antigen, assayed by a commercial ovine hydatidosis ELISA kit, and no hydatid cysts presented in internal organs detected by ultrasonography, prior to the experiment. The seven sheep with the MHC MvaⅠbc-SacⅡab-HinlⅠab haplotype (CE resistant haplotype) referred to group A, while the other seven sheep without this haplotype constitute group B. Each sheep in the two groups was infected perorally via a medical syringe with5000E. granulosus eggs suspended in1000ul physiological saline. Another three sheep choosed as healthy controls were named as group C. Then, the following studies were carried out:
1. The first experiment described here was designed to analyze the system immune response in the very early stage of E. granulosus infection in sheep with CE resistant haplotype. In this experiment, after experimentally infected with E. granulosus, blood samples from these sheep were collected on day0(prior to infection), hours2,3,4, and9post-infection as well as days1,2,3, and7 post-infection, respectively. ELISA assay was used to measure serum levels of antibodies (IgE and IgM), cytokines (Thl:TNF-α and IFN-γ; Th2:IL-4and IL-10) and chemokines (Thl:CXCL-9; Th2:CCL17) at different time points of E. granulosus infection in the two groups. Results showed that, in the early stage of E. granulosus infection, antibodies like IgM and IgE, Thl cytokines such as IFN-γ and TNF-a, as well as Thl chemokines CXCL-9were predominant in group A, especially for IgE and Thl cytokines, which were significantly higher, most were at or began from4h post-infection, as compared with group B. Our findings revealed that the influence of the host's genetic background on the immunopathology of E. granulosus infection in the early stage could be partially mediated by Thl-type cytokines and IgE.
2. The second experiment was designed to detect differential gene expression in intestine of Kazakh sheep with different MHC haplotype after perorally infected with E. granulosus eggs. In this experiment, three sheep of each group (groups A and groups B) were sacrificed on hours2,3and4post-infection, respectively. Besides, another3healthy Kzakh sheep, referred to group C, were choosed as healthy controls. The intestine tissues of the three groups were collected, and gene expression profiles were assessed using sheep DNA microarray analysis. Gene expression profiles in groups A and B were compared with that in group C, respectively. Results showed that,4712differentially expressed genes were identified between group A and C, with1959up-regulated and2753down-regulated. On the other hand,4944differentially expressed genes, with2076up-regulated and2868down-regulated, were also identified between group B and C. Then, we chose141significantly differential expression genes in the above two comparasions, which showed the corresponding upward or downward trend, including32up-regulated genes and109down-regulated genes. Among32up-regulated,6genes were significantly higher in group A than in group B, while26genes were significantly lower in group A than that in group B. Additionally, among109down-regulated genes,35genes were significantly higher in group A when compared with group B, while74genes were significantly lower in group A when compared with group B.
3. The third experiment was designed to detect differential gene expression in the liver of Kazakh sheep with different MHC haplotype after perorally infected with E. granulosus eggs, at different time points. In this experiment, one sheep of each group (groups A and groups B) were sacrificed at8and24weeks post-infection, respectively. Besides, another3healthy Kzakh sheep, referred to group C, were choosed as healthy controls. The liver tissues of the three groups were collected, and gene expression profiles were assessed using sheep DNA microarray analysis.
1) At8weeks post-infection, gene expression profiles in groups A and B were compared with that in group C, respectively. Results showed that,3087differentially expressed genes were identified between group A and C, with1768up-regulated and1319down-regulated. On the other hand,4839differentially expressed genes, with 1888up-regulated and2951down-regulated, were also identified between group B and C. Then, we chose153significantly differential expression genes in the above two comparasions, which showed the corresponding upward or downward trend, including87up-regulated genes and66down-regulated genes. Among87up-regulated,13genes were significantly higher in group A than in group B, while74genes were significantly lower in group A than that in group b. Additionally, among66down-regulated genes,54genes were significantly higher in group A when compared with group B, while12genes were significantly lower in group A when compared with group B.
2) At24weeks post-infection, gene expression profiles in groups A and B were compared with that in group C, respectively. Results showed that,3092differentially expressed genes were identified between group A and C, with1374up-regulated and1718down-regulated. On the other hand,11717differentially expressed genes, with5381up-regulated and6336down-regulated, were also identified between group B and C. Then, we chose115significantly differential expression genes in the above two comparasions, which showed the corresponding upward or downward trend, including40up-regulated genes and75down-regulated genes. Among40up-regulated,3genes were significantly higher in group A than in group B, while37genes were significantly lower in group A than that in group b. Additionally, among75down-regulated genes,74genes were significantly higher in group A when compared with group B, while1genes were significantly lower in group A when compared with group B.
3) The differentially expressed genes were classified with gene ontology (GO) analysis according to their functions. Results showed that most of them were related to the metabolism amd cellular processes. KEGG biological pathway analysis showed that most genes involved in the complement lectin pathway, especially in the intestine tissue and in the liver at24weeks post-infection, which indicated that different MHC genotype resistance to CE might be associated with complement levels.
4) Finally, the following genes:the killer cell immunoglobulin-like receptor (KIR2DS1), lectin complement C8, serine protease inhibitors, growth inhibition of DNA damage gene (GADD45B), PLA2G2A were choosed as candidate genes asscociated with CE resistance in sheep.
In conclusion, our studies revealed that the influence of the host's genetic background on the immunopathology of E. granulosus infection in the early stage could be partially mediated by Thl-type cytokines and IgE. On the other hand, genes involved in the complement lectin pathway might participate in the resistance of CE, after E. granulosus infection in the intestine tissue (the very early stage of infection) or in the liver tissue (the late stage of infection). In addition to that, microarray analysis showed that, a large number of genes involved in CE resistance in sheep, varying from energy metabolism, immunity, to signal transduction, indicating that CE resistance is a complex physiological process. Compared with healthy controls, sheep with MHC CE resistance haplotype (group A) displayed an inactivate gene expression trend at the three time points detected, showing that most of the genes upward or downward were lower than that in sheep without this haplotype (group B), but interestingly, some genes related to CE resistance in group A showed a high expression level than that in group B. It is, therefore, a further deep study on the relationship between these genes and CE resistance should be carried out, in future.
本研究前期做了大量的关于绵羊主要组织相容性复合体基因（MHC基因）与包虫病抗性、易感性的相关研究，对新疆三个主要绵羊品种：中国美利奴羊、多浪羊及哈萨克羊MHC-DRB1基因外显子2进行PCR-RFLP检测，成功筛选出哈萨克绵羊与包虫病抗性相关的单倍型MHC-DRB1MvaIbc-SacIIab-Hin1Iab，之后对带有该单倍型的绵羊（抗性组）和不带该单倍型的绵羊（非抗性组）进行人工攻虫，发现抗性组绵羊包虫病发病率比非抗性组绵羊低且差异显著。在此基础上，本文着重对前期筛选的具有包虫病抗性和非抗性的哈萨克绵羊的感染机制及其差异进行分析。
依据前期研究，本研究采用PCR-RFLP方法对250只哈萨克绵羊进行了相应抗性单倍型分析，共筛选出具有包虫病抗性的个体（基因单倍型为MHC-DRB1MvaIbc-SacIIab-Hin1Iab）14只,非抗性个体（基因单倍型为MHC-DRB1MvaIbb-SacIIaa-Hin1Iaa）22只。进一步采用B超和ELISA结合的方法对筛选出的绵羊进行包虫病检测，随访后购买17只健康的绵羊，其中包括用于人工感染实验的绵羊14只（抗性组和非抗性组各7只），以及健康对照的绵羊3只。MHC抗性组绵羊记为A组，MHC非抗性组绵羊记为B组，健康对照的绵羊记为C组。人工感染实验的14只绵羊每只饲喂约5000个成熟细粒棘球绦虫虫卵，建立人工感染细粒棘球绦虫模型后，开展了以下后续试验：
1.实验一旨在检测不同MHC基因型哈萨克绵羊感染细粒棘球绦虫虫卵后早期机体免疫指标的差异。本实验分别于感染前(0d)、感染后2h、3h、4h、9h、1d、2d、3d、7d采集绵羊颈静脉血，采用ELISA方法检测血清中抗体（IgM、IgE）、细胞因子（TNF-α、IFN-γ、IL-4、IL-10）及趋化因子（CXCL-9、CCL17）的水平，结果显示，MHC包虫病抗性组（A组）表现出以抗体IgE、IgM、Th1细胞因子（IFN-γ、TNF-α）和Th1趋化因子（CXCL-9）为优势的分泌水平，而非抗性组（B组）则表现出以Th2细胞因子（IL-4、IL-10）和Th2趋化因子（CCL17）为优势的分泌水平。且IgE、Th1细胞因子（IFN-γ、TNF-α）在虫卵感染后4h组间差异显著。提示，携带MHC抗性基因型的绵羊表现出强的包虫病抵抗力可能受其早期高水平的Th1细胞因子及IgE抗体的影响。
2.实验二旨在筛选出不同MHC基因型哈萨克绵羊感染细粒棘球绦虫后小肠组织差异表达的基因。本实验中，实验动物分为MHC包虫病抗性组（A）、MHC包虫病非抗性组(B)和健康对照组（C），A、B两组分别于感染后2h、3h、4h采集小肠组织，以C组健康绵羊作为健康对照组，采用基因芯片的方法分析不同MHC基因型哈萨克绵羊感染细粒棘球绦虫后小肠差异表达基因，其中感染组（A）、（B）与健康对照组（C）进行比较，结果发现:A与C之间差异表达的基因共4712个，其中表达量上调的有1959个，表达量下调的有2753个。B与C比较之后差异表达的基因4944个，其中2076个表达上调，2868个表达下调。从中选择出同C相比，A和B同为上调或下调，且表达量差异显著的基因共141个，其中上调的有32个基因，下调的有109个。上调的32个基因中，A组显著高于B组的有6个，低于B组的有26个；在下调的109个基因中，A组显著高于B组的35个，低于B组的74个。
3.实验三旨在筛选出不同MHC基因型哈萨克绵羊感染细粒棘球绦虫后不同时期（感染后8周、24周）肝脏组织差异表达基因。本实验中，实验动物分为MHC包虫病抗性组（A）、MHC包虫病非抗性组(B)和健康对照组（C），A、B两组分别于感染后8周、24周采集肝脏组织，以C组健康绵羊作为健康对照组，采用基因芯片的方法分析不同MHC基因型哈萨克绵羊感染细粒棘球绦虫后肝脏组织差异表达基因。
1）感染后8周，基因芯片结果显示:MHC抗性组（A）与对照组（C）之间差异表达的基因共3087个，其中表达量上调的有1768个，表达量下调的有1319个。MHC非抗性组（A）与对照组（C）比较之后差异表达的基因4839个，其中1888个表达上调，2951个表达下调。采用同样标准进一步筛选获得差异候选基因153个，其中上调的有87个基因，下调的有66个。上调的87个基因中，A组显著高于B组的有13个，低于B组的74个；在下调的66个基因中，A组显著高于B组的54个，低于B组的12个。
2）感染后24周，基因芯片结果显示:MHC抗性A组与C组之间差异表达的基因共3092个，其中表达量上调的有1374个，表达量下调的有1718个。MHC非抗性B组与对照C组比较之后差异表达的基因11717个，其中5381个表达上调，6336个表达下调。采用同样标准进一步筛选获得差异候选基因115个，其中上调的有40个基因，下调的有75个。上调的40个基因中，A组显著高于B组的有3个，低于B组的37个；在下调的75个基因中，A组显著高于B组的74个，低于B组的1个。对以上3组共8个样本的基因芯片结果进行荧光定量PCR检验，挑选8个基因进行验证，结果同芯片检测结果基本接近，说明检测结果真实可信。
3）对筛选到的差异表达基因进行GO富集和KEGG通路分析发现，差异基因主要的生化功能集中在代谢、细胞进程等方面，值得注意的是:KEGG分析结果显示参与补体凝集素途径的基因数量在小肠和肝脏24周结果中均为最多，同时在肝脏8周组中也相对较多，提示MHC同补体之间抵抗寄生虫感染可能存在关联。
4）结合相关文献报道和不同感染时期基因表达特点，本研究最终选择了杀伤细胞免疫球蛋白样受体（KIR2DS1）、补体凝集素C8、丝氨酸蛋白酶抑制剂、生长抑制DNA损伤基因（GADD45B）、PLA2G2A等基因作为绵羊抵抗包虫病的候选基因。
综上所述得出如下结论:高水平的Th1细胞因子及IgE抗体能够影响绵羊对包虫病的早期抵抗，补体凝集素代谢通路可能在小肠和肝脏后期起到一定的抵抗作用。总体来说，参与抵抗包虫病的相关基因数量较多，涉及到能量代谢、免疫、信号转导等多个方面，说明包虫病抵抗是一个复杂的生理过程。同时，同非抗性组绵羊检测结果相比，在3个检测点上（小肠、肝脏8周、肝脏24周）MHC抗性组基因表现出基因表达不活跃的特点，具体表现为大部分基因上调或者下调幅度均低于非抗性组，但是一些可能同包虫病抵抗相关的基因仍表现出抗性组高于非抗性组的特点。
Cystic echinococcosis (CE) is a chronic parasitic zoonosis caused by infection with the larval stage of the cestode Echinococcus granulosus (E. granulosus), resulting in the development of cysts in human and domestic animals. In domestic animals, especially in sheep, which appear to be highly susceptible to infection, CE causes considerable health problems and economical disadvantages because of production loss, as sheep infected with this disease often suffer from reductions in live weight gain, in milk yielding, in the fertility rates, in the value of wool or other products. In Xinjiang, there are more than60million sheep, with the high prevalence of CE reaching50%, which causes considerable health problems in sheep and thereby significantly influences the income of herdsman.
Previuosly, we carried a lot of studies to investigate the relationship between MHC-DRB1gene polymorphism and genetic resistance or susceptibility to CE in Chinese Merino, Duolang and Kazakh sheep. Genetic markers, MHC MvaⅠbc-SacⅡab-HinlIab haplotype, on CE resistance in Kazakh sheep have been divergently selected by us. The rates of Echinococcus granulosus (E. granulosus) infection in the internal organs of Kazakh sheep with this haplotype, is confirmed to be significantly lower than sheep without this haplotype, when exposure to the same level of parasites. CE resistant sheep therefore have an increased genetic capability to respond to and subsequently reject parasites when challenged. Carrying out a deep study in these sheep was, therefore, worth doing, since it could produce some interesting clues on the mechanism of resistance to E. granulosus infection both in immunology and genetics.
According to the previous methods stablished by us, we selected the genetic markers in250Kazakh sheep, again. Seventeen healthy two-year-old Kazakh sheep with different haplotype were selected and maintained during the course of the experiments. They were negative for antibodies to hydatid cyst fluid (HCF) antigen, assayed by a commercial ovine hydatidosis ELISA kit, and no hydatid cysts presented in internal organs detected by ultrasonography, prior to the experiment. The seven sheep with the MHC MvaⅠbc-SacⅡab-HinlⅠab haplotype (CE resistant haplotype) referred to group A, while the other seven sheep without this haplotype constitute group B. Each sheep in the two groups was infected perorally via a medical syringe with5000E. granulosus eggs suspended in1000ul physiological saline. Another three sheep choosed as healthy controls were named as group C. Then, the following studies were carried out:
1. The first experiment described here was designed to analyze the system immune response in the very early stage of E. granulosus infection in sheep with CE resistant haplotype. In this experiment, after experimentally infected with E. granulosus, blood samples from these sheep were collected on day0(prior to infection), hours2,3,4, and9post-infection as well as days1,2,3, and7 post-infection, respectively. ELISA assay was used to measure serum levels of antibodies (IgE and IgM), cytokines (Thl:TNF-α and IFN-γ; Th2:IL-4and IL-10) and chemokines (Thl:CXCL-9; Th2:CCL17) at different time points of E. granulosus infection in the two groups. Results showed that, in the early stage of E. granulosus infection, antibodies like IgM and IgE, Thl cytokines such as IFN-γ and TNF-a, as well as Thl chemokines CXCL-9were predominant in group A, especially for IgE and Thl cytokines, which were significantly higher, most were at or began from4h post-infection, as compared with group B. Our findings revealed that the influence of the host's genetic background on the immunopathology of E. granulosus infection in the early stage could be partially mediated by Thl-type cytokines and IgE.
2. The second experiment was designed to detect differential gene expression in intestine of Kazakh sheep with different MHC haplotype after perorally infected with E. granulosus eggs. In this experiment, three sheep of each group (groups A and groups B) were sacrificed on hours2,3and4post-infection, respectively. Besides, another3healthy Kzakh sheep, referred to group C, were choosed as healthy controls. The intestine tissues of the three groups were collected, and gene expression profiles were assessed using sheep DNA microarray analysis. Gene expression profiles in groups A and B were compared with that in group C, respectively. Results showed that,4712differentially expressed genes were identified between group A and C, with1959up-regulated and2753down-regulated. On the other hand,4944differentially expressed genes, with2076up-regulated and2868down-regulated, were also identified between group B and C. Then, we chose141significantly differential expression genes in the above two comparasions, which showed the corresponding upward or downward trend, including32up-regulated genes and109down-regulated genes. Among32up-regulated,6genes were significantly higher in group A than in group B, while26genes were significantly lower in group A than that in group B. Additionally, among109down-regulated genes,35genes were significantly higher in group A when compared with group B, while74genes were significantly lower in group A when compared with group B.
3. The third experiment was designed to detect differential gene expression in the liver of Kazakh sheep with different MHC haplotype after perorally infected with E. granulosus eggs, at different time points. In this experiment, one sheep of each group (groups A and groups B) were sacrificed at8and24weeks post-infection, respectively. Besides, another3healthy Kzakh sheep, referred to group C, were choosed as healthy controls. The liver tissues of the three groups were collected, and gene expression profiles were assessed using sheep DNA microarray analysis.
1) At8weeks post-infection, gene expression profiles in groups A and B were compared with that in group C, respectively. Results showed that,3087differentially expressed genes were identified between group A and C, with1768up-regulated and1319down-regulated. On the other hand,4839differentially expressed genes, with 1888up-regulated and2951down-regulated, were also identified between group B and C. Then, we chose153significantly differential expression genes in the above two comparasions, which showed the corresponding upward or downward trend, including87up-regulated genes and66down-regulated genes. Among87up-regulated,13genes were significantly higher in group A than in group B, while74genes were significantly lower in group A than that in group b. Additionally, among66down-regulated genes,54genes were significantly higher in group A when compared with group B, while12genes were significantly lower in group A when compared with group B.
2) At24weeks post-infection, gene expression profiles in groups A and B were compared with that in group C, respectively. Results showed that,3092differentially expressed genes were identified between group A and C, with1374up-regulated and1718down-regulated. On the other hand,11717differentially expressed genes, with5381up-regulated and6336down-regulated, were also identified between group B and C. Then, we chose115significantly differential expression genes in the above two comparasions, which showed the corresponding upward or downward trend, including40up-regulated genes and75down-regulated genes. Among40up-regulated,3genes were significantly higher in group A than in group B, while37genes were significantly lower in group A than that in group b. Additionally, among75down-regulated genes,74genes were significantly higher in group A when compared with group B, while1genes were significantly lower in group A when compared with group B.
3) The differentially expressed genes were classified with gene ontology (GO) analysis according to their functions. Results showed that most of them were related to the metabolism amd cellular processes. KEGG biological pathway analysis showed that most genes involved in the complement lectin pathway, especially in the intestine tissue and in the liver at24weeks post-infection, which indicated that different MHC genotype resistance to CE might be associated with complement levels.
4) Finally, the following genes:the killer cell immunoglobulin-like receptor (KIR2DS1), lectin complement C8, serine protease inhibitors, growth inhibition of DNA damage gene (GADD45B), PLA2G2A were choosed as candidate genes asscociated with CE resistance in sheep.
In conclusion, our studies revealed that the influence of the host's genetic background on the immunopathology of E. granulosus infection in the early stage could be partially mediated by Thl-type cytokines and IgE. On the other hand, genes involved in the complement lectin pathway might participate in the resistance of CE, after E. granulosus infection in the intestine tissue (the very early stage of infection) or in the liver tissue (the late stage of infection). In addition to that, microarray analysis showed that, a large number of genes involved in CE resistance in sheep, varying from energy metabolism, immunity, to signal transduction, indicating that CE resistance is a complex physiological process. Compared with healthy controls, sheep with MHC CE resistance haplotype (group A) displayed an inactivate gene expression trend at the three time points detected, showing that most of the genes upward or downward were lower than that in sheep without this haplotype (group B), but interestingly, some genes related to CE resistance in group A showed a high expression level than that in group B. It is, therefore, a further deep study on the relationship between these genes and CE resistance should be carried out, in future.
引文
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