【作者】 蒋松；

【导师】 贾斌；

【作者基本信息】 石河子大学 ， 动物遗传育种与繁殖， 2017， 博士

【摘要】 细粒棘球蚴病,又称囊型包虫病(CE),是细粒棘球绦虫的幼虫寄生于人、羊、牛等中间宿主所致的一种人畜共患寄生虫病。绵羊是其最适宜的中间宿主,感染该病后因营养不良,导致生长发育缓慢以及毛、肉和奶的产量和质量均会受到严重影响,患病羊肝、肺病变严重,不能食用。目前,新疆绵羊存栏数为3663.2万只,出栏数为3107.5多万只,而包虫病感染率9.8%,单就包虫病所造成的畜牧业经济损失近一亿元,给畜牧业的发展以及农牧民的健康带来严重影响。本研究前期成功筛选出抗包虫病哈萨克羊单倍型MHC-DRB1 Mva I bc–Sac II ab-Hin1 I ab,随后分别对带有和不带有该单倍型的绵羊进行人工感染攻虫,进一步验证其抗性和非抗性。由此,本论文着重研究抗性和非抗性的哈萨克羊六钩蚴侵染早期免疫因子和microRNA的变化,并对差异表达microRNA及其可能靶基因进行了分析。依据前期实验室研究,300只哈萨克羊采用PCR-RFLP方法进行抗性单倍型分析,筛选出具有包虫病抗性的个体(基因单倍型为MHC-DRB1 Mva I bc-Sac II ab-Hin1 I ab)和非抗性个体(基因单倍型为MHC-DRB1 Mva I bb-Sac II aa-Hin1 I aa),同时结合B超和ELISA的方法对筛选出的绵羊个体进行检测,回访后从中随机购买7只健康的绵羊,其中用于人工感染试验的抗性组绵羊3只,非抗性组3只,非抗性绵羊1只作为对照。抗性组和非抗性组绵羊每只喂约10000个成熟虫卵进行人工感染试验,随后进行以下试验:1.试验一检测抗性和非抗性哈萨克羊感染六钩蚴早期机体免疫指标的变化。人工感染后6h、8h、10h屠宰绵羊,采集绵羊各段小肠组织并匀浆取上清液,ELISA试剂盒检测小肠组织中抗体(IgM、IgG、IgE)、细胞因子(IL-2、TNF-α、IFN-γ、IL-4、sIgA)和趋化因子(CCL8)的水平。结果表明,IgE(P<0.05)在空肠后段、Ig M(P<0.01)在回肠、SIgA(P<0.05)在十二指肠和空肠以及IFN-γ(P<0.05)在十二指肠和空肠前段,抗性组显著高于非抗性组,IgG(P<0.05)在空肠前段、IL-2(P<0.05)在回肠抗性组显著低于非抗性组。而TNF-α、IL-4和CCL8在两组间没有显著差异。2.试验二研究抗性和非抗性哈萨克羊六钩蚴侵染早期(侵染6h、8h、10h)IgA、IgG、IgM在小肠组织(十二指肠、空肠前段、空肠中段、空肠后段、回肠)中的分泌和分布情况。抗性组、非抗性组于感染6h、8h、10h后采集小肠组织,对照组采集小肠组织,采用免疫组织化学的方法分析抗性和非抗性组哈萨克羊小肠组织中抗体的分泌和分布情况。结果表明,各试验组绵羊感染虫卵后各时间点,IgA、IgG、IgM主要分布在小肠各段基底部肠腺细胞,但IgM在小肠各段顶层腺体及固有膜也有分布,且随着攻虫后时间的增加,抗性组各肠段IgM分泌表达量高于非抗性组,在空肠及回肠最为明显。非抗性组IgA、IgG分泌表达量高于抗性组,其中非抗性组IgA在十二指肠和空肠中段最为明显,而Ig G则呈现出从基底部肠腺细胞向肠腔分泌的趋势,在固有膜有所表达。3.试验三筛选抗性和非抗性哈萨克羊六钩蚴侵染早期小肠组织差异表达的microRNA。采用高通量测序的方法分析抗性和非抗性哈萨克羊感染包虫虫卵后小肠差异表达microRNA,其中抗性组与非抗性组进行比较,结果发现:抗性组与非抗性组差异表达的microRNA共83个,其中表达量上调的有75个,下调的有8个;抗性组与对照组比较差异表达的microRNA有139个,其中10个表达上调,129个表达下调。非抗性组与对照组比较之后差异表达的microRNA有41个,其中10个表达上调,31个表达下调。通过实时定量PCR验证了部分差异表达microRNA((bta-mi R-93,bta-miR-192,bta-miR-92a,bta-miR-20a,bta-miR-145,bta-miR-181和bta-miR-101),发现microRNA表达与测序结果一致。差异表达microRNA的KEGG通路分析,发现排名前三的为代谢途径(Metabolic pathways),嗅觉转导途径(Olfactory transduction)和肿瘤通路(Pathways in cancer),而样本联立分析则发现KEGG通路涉及表皮细胞细菌入侵(Bacterial invasion of epithelial cells),焦点粘连(Focal adhesion)和肌动蛋白细胞骨架调节(Regulation of actin cytoskeleton)等途径。4.试验四通过双荧光素酶报告试验、实时定量PCR等就试验三筛选出的差异表达microRNA,miR-101和mi R-145分别对靶基因RAC1和CAV2的作用进行了验证。结果显示miR-101和miR-145分别下调了RAC1和CAV2的表达。从而证明miR-101和miR-145可能通过分别抑制RAC1和CAV2的表达,促进肠道细胞的免疫反应,参与包虫病的免疫应答。综上所述得出如下结论:携带MHC抗性基因型的绵羊在小肠组织表现出较强的包虫病抵抗力可能与其早期IFN-γ、SIgA和IgM分泌较高有关,免疫组织化学的方法进一步表明IgM可能发挥着更重要的作用。同时,结果提示miR-101和miR-145分别抑制RAC1和CAV2基因表达,参与了小肠抗包虫粘膜免疫。更多还原

【Abstract】 Echinococcosis granulosus(E.granulosus),also known as cystic echinococcosis(CE),is a parasitic zoonosis caused by giant echinococcus granulosus larvae parasitic in humans,sheep,cattle and other intermediates host.Sheep is the most suitable intermediate host of the disease,and has malnutrition,slow growth.The quality of hair,meat and milk production will be seriously affected,especially the affected sheep liver,lung with severe lesions,can not be edible due to infection with the disease.At present,Xinjiang sheep breeding stock is 36.623 million,the amount of slaughter is more than 3107.5 million,and hydatid disease infection rate is 9.8%,alone on the economic losses caused by hydatid disease nearly 100 million yuan,thereby has a serious impact on the health of animal husbandry development and the production of herdsmen.Previuosly,we successfully screened the kazakh sheep haplotype MHC-DRB1 Mva I bc-Sac II ab-Hin1 I ab,which was resistant to hydatid disease.Then,we carried out artificial infection to sheep with the haplotype or not,and verified the resistance and non-resistance.In this study,we focused on the changes of immune factors and microRNAs in the early stage of resistant and non-resistant kazakh sheep after infection with the E.granulosus eggs,and analyzed the differential expression of microRNA and its possible target genes.According to the previous study,the corresponding haplotype analysis of 300 Kazak sheep was carried out by PCR-RFLP method.The healthy individuals with resistance(genotype haplotype MHC-DRB1 Mva I bc-Sac II ab-Hin1 I ab)and non-resistant healthy individuals(genotype haplotype MHC-DRB1 Mva I bb-Sac II aa-Hin1 I aa)were selected and purchased after follow-up.Seven sheep(resistant and non-resistant groups)were used for artificial infection experiments,both resistant and non-resistant group were three sheep and a non-resistant control of the sheep.Resistant and non-resistant sheep were fed with about 10,000 mature eggs for artificial infection,followed by the below experiments:The experiment one,the changes of immune parameters were detected in the early stage of oncospheres infection Kazak sheep with resistance or not.The levels of antibody(IgM,IgG,IgE),cytokines(TNF-α,IFN-γ,IL-4,sIgA)and chemokine(CCL8)in small intestine were detected by sheep-related ELISA kits,and the supernatant of small intestine homogenate was collected at 6h,8h and 10 h after artificial infection.The results showed that the expression level of IgE(P<0.05)in the segmenta posterius of jejunum,IgM(P<0.01)in the ileum,SIgA(P<0.05)in the duodenum and jejunum,IFN-γ(P<0.05)in the duodenum and segmenta posterius of resistance group were higher than that in the resistance group,howerer the IgG(P<0.05)in the segmenta posterius of jejunum,IL-2(P<0.05)in the ileum of was resistance group were lower than that in the resistance group.The TNF-α,IL-4,and CCL-8 were not significantly different between the two groups.The experiment two,was supposed to study the secretion and distribution of IgA,IgG,IgM in the small intestine tissue(duodenum,jejunum,ileum)of resistace and non-resistance group.the small intestine tissue was collected from Kazak sheep after echinococcus infection after 6 h,8 h,10 h in the resistace and non-resistance group.The results showed that IgA,IgG and IgM were mainly distributed in the basal gland cells of the small intestine at each time point,but IgM was also distributed in the upper gland and the intrinsic membrane of the small intestine.The expression of IgM in the resistance group was higher than that in the non-resistance group with the increase of time after the artificial infection,and the most obvious display was in the jejunum and ileum.The expression of IgA and IgG in the non-resistant group was higher than that in the resistant group,and IgA was the most obvious in the middle of the duodenum and jejunum,while the IgG showed a tendency that secreted from the intestinal gland cells to the intestinal cavity,expressed in the intrinsic membrane.The experiment three,to screen out the differentially expressed microRNAs of the small intestine in the early stage of E.granulosus oncospheres infection in the Kazakh sheep with resistance or non-resistance to CE.The differential expression microRNAs were analysed by high-throughput sequencing in the small intestine of Kazakh sheep.There were 83 differentially expressed microRNAs,75 were up-regulated,and 8 were down-regulated in the resistance group,compared with the non-resistant group.There were 139 differentially expressed microRNAs,of which 10 were up-regulated and 129 were down-regulated in the resistant group,compared with the control group.There were 41 differentially expressed microRNAs,of which 10 were up-regulated and 31 were down-regulated in the non-resistant group,compared with the control group.Some microRNAs(bta-miR-93,bta-miR-192,bta-miR-92 a,bta-miR-20 a,bta-miR-145,bta-miR-181 and bta-miR-101)were verified by real-time quantitative PCR and found that the expression of these microRNAs was consistent with the sequencing results.KEGG analysis of differentially expressed microRNAs revealed that pathway focused on metabolic pathway,olfactory transduction and pathways in cancer,while the sample of simultaneous analysis revealed that the KEGG pathway involved in bacterial invasion of epithelial cells,focal adhesion and regulation of actin cytoskeleton.The experiment four,the expression of RAC1 and CAV2 were tested by double luciferase reporter assay,RT-PCR.The effects of mi R-101 and mi R-145 on target RAC1 and CAV2 were verified.The results showed that miR-101 and miR-145 downregulated the expression of RAC1 and CAV2,respectively.It has been shown that miR-101 and miR-145 may promote the immune response of intestinal cells by inhibiting the expression of RAC1 and CAV2,respectively,and participated in the immune response of hydatid disease.In conclusion,the sheep with of the MHC-resistant genotype showed a stronger resistance to hydatid disease in the small intestine,which may be associated with higher levels of early IFN-γ,SIgA and IgM secretion.The immunohistochemical method further confirmed that IgM may play a more important role.At the same time,the result suggested that miR-101 and miR-145 inhibit the expression of RAC1 and CAV2,respectively,and participated in mucosal immunity of small intestine.更多还原