| E.granulosus parasites of human beings and cloven hooves etc,resulting in the compression of liver,spleen,lung and other organs,which is known as cystic echinococcosis and endangeres people’s health and husbandry production.Cystic echinococcosis is a major zoonotic diseases in China.There were 368 endemic counties and 60 million people in China,ranking first in the world.According to the survey of Tibet in 2016,the infection rate of domestic animals(28.82%),the rate of household self-slaughter(90.6%)and odds ratio(56.28)were high in Ali region.However,the gene polymorphism and population amplification of E.granulosus in Ali region are still unclear.After infecting the host,the mechanism that E.granulosus causes immune response is complex and unclear,which becomes the bottleneck of the development of diagnostic reagents and drugs for echinococcosis.This study was carried out from pathogen and host.First,PCR method was used to amplify mitochondrial gene,and Contig,BLAST and Clustalxl.83 software were used to analyze gene polymorphism of E.granulosus isolated from patients,so as to understand the dominant genotype in Ali region.Secondly,the expression of PD-1 on the surface of lymphocytes and the proportion of lymphocytes in peripheral blood of mice infected with Echinococcus granulosus were detected.Thl,Th2.proinflammatory,immunosuppressive cytokines in mice and human were detected.To explore the functional state of lymphocytes in intermediate host after infection of E.granulosus.This study laid a good experimental foundation for the traceability of E.granulosus and the mechanism of interaction between pathogen and host.Part 1.Investigation and analysis of genetic polymorphisms of E.granulosus derived human in Ali region of TibetObjectiveTo preliminarily understand the genetic polymorphisms and population explation of E.granulosus in Ali region.To provide support for tracing the origin of cystic echinococcosis in Ali region.MethodsThe specimens of cystic echinococcosis were collected in a hospital of Ali area.Mitochondrial cytochrome c oxidase subunit 1(cox1)and cytochrome b(cob)gene were amplified by PCR.The products were jointed by Contig,analyzed phylogenetic trees by BLAST,ClustalX1.83 and MEGA7.0 software.The haplotype networks of E.granulosus based on coxl and cob were analyzed and made by DnaSP6 and NetWork software.The cox1 gene sequences of E.granulosus in China were downloaded and the haplotype networks were analyzed and made.ResultsA total of 79 cases of hydatidosis were collected and aged from 6 to 76 years.There were 49 female surgical patients and 30 male patients.According to the sample distribution,the number of surgical cases in Ali area from high to low:Geji(29),Gaize(14),Ritou(10),Pulan(9),Cuoqin(7),Zhada(6),geer(4).The genotypes of E.granulosus from human isolates in Ali area were G1,G3,G6 and G7,including 1 case of G3,4 cases of G6 and 1 case of G7,the rest of cases belonged to G1 genotype of E.granulosus.Eight haplotypes of cob gene were detected,the main haplotype was Hap-4,the diversity of haplotype was 0.64,and the diversity of nucleic acid was 0.00154.Tajima’s D value of cob gene of E.granulosus in Ali area was-1.89,P<0.05,Fu’s FS statistic value was-5.101,P<0.05.Six haplotypes of cox1 gene were detected,the main haplotype was Hap-1,the diversity of haplotype was 0.36,and the diversity of nucleic acid was 0.00116.Tajima’s D value of cox1 gene of E.granulosus in Ali area was-1.90,P<0.05,Fu’s FS statistic value was-3.53,P<0.05.There are 40 haplotypes of cox1 gene of E.granulosus in China,the main haplotype was Hap-4,the diversity of haplotype was 0.78,and the diversity of nucleic acid was 0.03997.ConclusionThere are abundant genotypes and haplotypes of E.granulosus in Ali region.The genotypes of E.granulosus from human isolates in Ali region were G1,G3,G6 and G7,G1 was the most.Eight and six haplotypes of cob and cox1 mainly were Hap-4 and Hap-1,respectively.The neutral test results of E.granulosus in Ali area showed that E.granulosus experienced the effects of population expansion and natural selection.Part 2.The functional status of lymphocytes in the intermediate host of E.granulosus infectionObjectiveTo understand the functional status of lymphocytes of the intermediate host and lay foundation for further study on the interaction between E.granulosus and its intermediate host.MethodIn this study,the infection model of BALB/c mices were established by intraperitoneal injection E.granulosus.The dynamic changes of T,B lymphocytes.CD4+T,CD8+T and its surface inhibitory receptor PD-1 of BALB/c mice were detected at 4th,6th,8th,12th and 24th weeks,the dynamic changes of cytokine of BALB/c mice were detected at 2nd,3rd,4th,6th,8th,12th and 24th weeks,the expression pattern of cytokines in cystic echinococcosis were detected,in order to study the functional status of lymphocytes in the intermediate host of E.granulosus infection.ResultsIn the peripheral blood of BALB/c infected with E.granulosus,the percentage of T lymphocytes was significantly higher at the 4th after infection(P<0.05).The percentage of inhibitory receptor PD-1 of T lymphocyte was significantly higher at the 6th and 8th week after infection(P<0.05).The percentage of B lymphocytes was significantly higher at the 6th week after infection(P<0.05).The percentage of inhibitory receptor PD-1 of B lymphocyte was significantly higher at the 6th,8th,12th week after infection(P<0.05).In the peripheral blood of BALB/c infected with E.granulosus,the percentage of CD4+T cells was significantly higher at the 6th,8th,12th and 24th week after infection(P<0.05).The percentage of PD-1 of CD4+T was significantly higher at the 6th,8th and 12th week after infection(P<0.05).The percentage of CD8+T cells was significantly lower at the 6th and 8th week after infection(P<0.05).The percentage of PD-1 of CD8+T lymphocyte was significantly higher at the 6th week(P<0.05),The expression of IL-2 and IL-1β was significantly higher at the 2nd week after infection(P<0.05).The expression of IFN-γ,GM-CSF and TNF-α was significantly higher at the 2nd,3rd week and lower at the 4th week after infection(P<0.05).The expression of IL-4 was significantly higher at the 3rd,12th and 24th week after infection(P<0.05).The expression of IL-13 was significantly higher at the 24th week after infection(P<0.05).The expression of IL-10 was significantly higher at the 8th,12th week after infection(P<0.05).In the serum of patients,the expression of IL-17A and IFN-y was significantly lower than uninfected control group(P<0.05).The expression of IL-4 and TGF-β was significantly higher than uninfected control group(P<0.05)ConclusionThe BALB/c mices were in the stage of inflammatory response at the 20d-3rd weeks after infection.The BALB/c mice started specific immune response at the 4th-6th weeks after infection.The BALB/c mices were in the stage of immunosuppressive at the 6th-12th weeks after infection.Th2 polarization and immunosuppression were observed in patients with cystic echinococcosis. |
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