| Objective:Alveolar echinococcosis(AE)is a multilocular Echinococcus(Echinococcus multilocularis,E.m)larvae of E.m parasitic disease caused by a deadly,a diffuse infiltration growth within the liver parenchyma,known as the "cancer".Studies have shown that E.m is popular in western provinces in our country is very serious,AE infection patients account for more than 90% of cases in the world.However,there is currently no effective vaccines and drugs to control the disease,infection and host bubble E.m cause liver damage mechanism is also unknown.Research has shown that different bubble ball larva source,its pathogenicity differences for people and animals;it is not clear that different source of E.m infected host liver damage.Therefore,this study intend to apply E.m of different 5 strains to research,including Alaska strains(EM-1),Xinjiang(EM-2),Japanese(EM-4),Ningxia(EM-5)and Qinghai strain(EM-6),compare the different geographic strains of mice pathogenic differences,the differences between the induced liver injury in mice and the difference of mitochondrial genome,trying to illustrate the different geographic strains pathogenic difference and evolution law of understanding in order to further lay a foundation to find the pathogenic mechanism of echinococcosis.To find out these problems,this study through three experiments to complete:1.Establish a model of abdominal infection of EM-1,EM-2,EM-4,EM-5 and EM-6 strains,which explore the methods of species preservation of E.m,as well as the changes in body weight,liver and spleen of different strains of protoscoleces(PSCs)and the differences in the number of PSCs produced by EM-1,EM-2,EM-4,EM-5 and EM-6 strains in the mice after abdominal infection.2.Establishing animal models of hepatic portal vein infection of E.m thatcame from different locations and analyzing the differences in pathological changes of liver after infection in experimental animals,also,the correlation between the balance of Th1/Th2/Th17 in peripheral blood and anti-infection and pathogenicity.The analysis of transcriptional expression profiles from thepathogenicity of strongest and weakest.3.Applyingthe method of bioinformatics and comparative genomics analysis of 5 strains of different classification of EM mitogenome classification genes,coding protein gene differentiation base composition,sequence,single protein gene codon usage,mutation rate and other characteristics.To exploring E.m genetic evolution model and providing the certain reference value for the next step of functional genomics research.Methods:1.Ten Kunming(KM)mice were intraperitoneally inoculated with 5000 PSCs from different sources.After 120 days,the mice were weighed,sacrificed and the infection rate was calculated.The cysts were weighed to separate PSCs and calculate the volume.After weighing the liver and spleen,the infected liver and other infected organs were fixed in 4% paraformaldehyde,stained with HE,and examined under a microscope for the PSCs average value under five different visual fields of 100-fold.The linear relationship between the average PSCs and the obtained PSCs was calculated for the cyst weight and liver HE staining at 100-fold at 5 different visual fields.2.The laboratory mice were inoculated 2000 PSCs by the hepatic portal vein,which were weighted in 120 days after sacrificed,then the eyeballs were removed for blood collection.Anatomy laboratory mice,observe infection situation a part of the infection of the liver fixed in 4% paraformaldehyde,another part of the save to-80℃.Analysis of liver pathological changes: the using of HE and Masson staining and immunohistochemical analysis mice actin(α-SMA),COL3A1,ERK(ExtraCell ular signal-regulated kinase)and the number of lesions in the pathological process,and analyzes the differences between different strains.Applying(Cytometric Bead Array,CBA)CBA method to detect peripheral blood Th1/Th2/Th17 cytokine expression quantity in anti-infection effect and the differences between five strains.The expression differences of key immune molecules in the liver in EM-1 and EM-4 were screened by high-throughput technology todetect part of the differentially expressed genes screened and verified by qRT-PCR method.3.PSCs DNA were extracted,digested and purified,and Illumina sequencing was completed to splice mitochondrial rings after the library was established.Using DnaSPv5,DNAMAN5.2.2,Mega6.0,Network 5.0 and software analysis the differences of mitogenome sequences: 12 protein gene Codon Usage and Relative Synonymous Codon Usage(RSCU)has carried on the statistics,and 12 PCGs nucleotide and amino acid of genetic distance analysis,clustering analysis,nucleotide and amino acid and the reference gene mutation rates and homology analysis.Results:1.The infection rate of EM-1 reached the highest of 87.5% by abdominal infection and the lowest infection was EM-4,reached 50%.The PSCs volume showed that EM-1(280±100)>EM-5(183.33±83.33)>EM-2(90.56±55)>EM-6(70±36)>EM-4(40±32).The average PSCs of liver HE staining under 5 visual fields of 100-fold showed that EM-1(11.52±2.22)>EM-5(8.16±0.83)>EM-2(7.8±0.96)>EM-4(3.8±0.07)>EM-6(1.5±0.45).There was a positive correlation between cyst weight and PSCs volume(r=0.4178,P=0.0241),and cyst weight was positively correlated with PSCs in 5 different fields of liver HE staining(r=0.4946,P=0.0102),it is suggesting that cyst weight was positively correlated with the generation of PSCs volume.2.Fifty percent of the infected mice in the EM-1 group of became "bubbles",followed by EM-2.The weakest infection was EM-4 with no“bubbles” infection.The order of infected ability were EM-1>EM-2>EM-5>EM-6>EM-4.HE staining showed that the number of hepatic lesions was the highest in EM-1 and the lowest in EM-4,EM-1(27.58±7.85>EM-2(24.64±7.97>EM-5(21.4±5.68)> EM-6(15.36±3.19>EM-4(15±3).The results of Masson staining showed that the degree of liver fibrosis of EM-1 and EM-2 infection was significantly higher than the control group(P<0.001,P<0.0001).The occurrence of fibrosis was mainly dominated by collagen deposition,and obvious fibrosis lesions were observed,followed by EM-5 and EM-6.Immunohistochemical expression and localization of α-SMA and ERK were similar,which expression of EM-1 and EM-2 was the highest,followed by EM-5 and EM-6,and the expression of EM-4 was the weakest.The degree of infection of EM-1 and EM-2 with alpha-sma and ERK was significantly higher than that of the control group(P<0.001,P<0.01).CBA results showed that IL-2 was the highest in em-1 group and the lowest in CON group,and there was no statistical difference between other groups and CON group(P>0.05).Serum IL-4 was the highest in EM-4 group and the lowest in CON group,and there was no statistical difference between other groups and CON group(P>0.05).Serum IL-6 in CON group was the highest,and EM-5 group was the lowest.The CON group was significantly different from em-1,EM-2,EM-5 and EM-6(P<0.05,P<0.05,P<0.01,P<0.05),but no significant difference was found between the two groups(P>0.05).Em-4 serum ifn-gamma group was the highest and CON group was the lowest.The difference of EM-2 in CON group was significant(P<0.05),but there was no significant difference with other groups(P>0.05).Serum TNF-αscore was the highest in EM-2 group and the lowest in EM-5 group,but there was no statistical difference between other groups and CON group(P>0.05).Serum IL-17 A was the highest in EM-4 group and the lowest in EM-2 group,but there was no statistical difference between other groups and CON group(P>0.05).The serum IL-10 in EM-5 group was the highest,and the EM-4 group was the lowest.The difference between EM-4 and CON group was significant(P<0.05),but there was no statistical difference between other groups and CON group(P>0.05).The gene expression profiles of EM-1 and EM-4 showed that 231 genes were up-regulated and 350 genes were down-regulated,Which raised expressions factor CD163 is a typical M2 reaction,increase the pathological changes,another increase factor of ATP expression of atpase prompts the body to release more of inflammatory factor,aggravating the host inflammatory symptoms;SPN cut factor due to the low activity,cannot effectively degrade the complement chemokines,is the number of inflammatory factors and inflammatory cells gathered around the lesions,aggravating the host’s immune response.3.The sequencing results showed that the lengths of EM-1,EM-2,EM-4,EM-5 and EM-6 were 13740 bp,13737bp,13737 bp,13738bp and 13740 bp,respectively.The contents of leucine,valine,phenylalanine,serine and other four amino acids in the genome of 5 strains of mitogenome were four top,which were 14.88%,13.12%,12.41%,10.32%,respectively.Among the 12 PCGs of mitogenome,the genetic distances of ATP6,Cox1,Cox3,Nad1 and Nad5 amino acids are greater than the genetic distances of nucleotides,and Cox2 and Nad4 l are basically equal.At the level of nucleotides and amino acids,Cox2 and Nad4 l are the most conservative and the slowest to evolve.The nucleotide genetic distance of Cob,Nad2,Nad3,Nad4 and Nad6 is greater than that of amino acid.The analysis of genetic distance and Network 5.0 showed that EM-1 was significantly different from the other four strains.Compared with the reference genes,the variation rate of EM-1 nucleotide and amino acid was higher than the other four strains,and the homology was lower than the other four strains.Conclusion:1.We have established the animal models of KM mice with species preservation of E.m in Alaska,Xinjiang,Japan,Ningxia and Qinghai,The PSCs productionof were different in relation to locations(EM-1>EM-5>EM-2>EM-6>EM-4).The weight of cysts and the number of PSCs produced after liver infection were positively correlated with the production of PSCs by cysts.2.It is the first time to establish the animal models via hepatic portal vein infection by different locations PSCs,which EM-1was the most pathogenic and EM-4 was the least pathogenic.Th1 /Th2 /Th17 related cytokines were expressed differently during the infection process.IFN-γ was associated with anti-fibrosis,which was beneficial for the host to clear the infection.The Th2 factor IL-4 is beneficial to parasite parasitism.During the later infection process,the decrease of Th17 factor IL-17 A promoted the parasitization of E.m in the host.Up-regulation factor CD163 is the M2 response of typical by macrophages,which aggravates the inflammatory response in the liver and is beneficial to parasite.Another important up-regulated expression factor ATP prompts the body to release more inflammatory factors,aggravating host inflammatory symptoms.The down-regulated factor SPN cannot effectively degrade the complement chemokines,so that a large number of inflammatory factors and inflammatory cells gather around the lesion,aggravating the immune response of the body.3.It is the first time to apply the method of bioinformatics and comparative genomics to study the detailed gene structure and sequence evolution of the mitogenome of five strains from different origins and analyze the relationship between the structural characteristics and pathogenicity of the mitogenome of E.m from the molecular level of the mitogenome.The distance inTwelve PCGs of EM-1with other 4 strains from far away;10 PCGs clustering analysis found that EM-1 with other 4 strains from far away,the EM-1 nucleotide mutation rate is highest,lowest compared with the reference gene homology,the strongest pathogenicity;EM-4 nucleotide mutation rate is the lowest,compared with the reference gene homology is highest,the most weak pathogenicity.In 12 PCGs,ATP6,Cox1,Cox3 and Nad1 Nad5 evolve faster,E.m in pathogenicity may be associated with these five quick PCGs evolution. |
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