| Part Ⅰ:The correlation between macrophage polarization and Hepatic Alveolar EchinococcosisObjective:To investigate the correlation between macrophage polarization and hepatic alveolar echinococcosis.Methods:Liver samples(marginal liver tissue and normal liver tissue)and serum samples from patients with hepatic alveolar echinococcosis were collected,and serum samples of healthy people were collected.The liver tissues were detected by immunohistochemistry for analysing infiltration of M1/M2 macrophages.Serum samples were detected by ELISA for analysing the expression of M1/M2macrophages-associated cytokines.Results:1.Clinical data suggested that there was no significant difference in gender,age and occupation between HAE patients and healthy controls(P>0.05).However,there were significant differences in antibodies,PT,ALT,AST,ALP and ALB between the two groups.(P<0.05).2.The results of HE staining suggested that there was a large amount of inflammatory cells infiltrating in the marginal liver tissue.The results of Immunohistochemistry staining of liver tissue showed:(1)The density(cells/mm~2)of macrophages in normal liver tissue and marginal liver tissue were(10.91±0.42,N=20),(38.76±1.02,N=20),P<0.05,the difference was statistically significant.(2)In normal liver tissue and marginal liver tissue,the density of M1macrophages were(5=3.97±0.23,N=20),(4.54±0.28,N=20)respectively,P>0.05,the difference was not statistically significant;In normal liver tissue and marginal livertissue,thedensityofM2macrophageswere(5.38±0.23,N=20),(26.33±1.09,N=20)respectively,P<0.05,the difference was statistically significant;(3)The ratio of M2/M1 in marginal liver tissues of HAE patients was higher than that of normal liver tissues,which was(6.25±0.45)in the experimental group and(1.48±0.13)in the control group.There was a significant difference between the two groups(P<0.05).2.The results of Serum ELISA showed that the levels of IL-6,TNF-α,TGF-β1 and IL-10 in serum of the patients increased to some extent compared with the normal person.The expressions of IL-6(pg/mL)were(16.56±10.79)in the experimental group and(0.54±0.39)in the control group;The expressions of TNF-α(pg/mL)were(17.92±8.89)in the experimental group and(3.04±1.123);The expressions of TGF-β1(ng/mL)were(4.84±0.73)in the experimental group and(3.91±0.41)in the control group;The expressions of IL-10(pg/mL)were(9.74±3.36)in the experimental group and(8.68±4.60)in the control group.Among the four interleukins,IL-6,TNF-αand TGF-β1,P<0.05,the difference was statistically significant.Conclusion:There is a large number of inflammatory cells in the marginal live tissue of hepatic alveolar echinococcosis patients,in which the number of macrophages increased significantly,mainly M2 macrophages.The results of ELISA showed that the levels of M1/M2 macrophages-associated cytokines such as IL-6,TNF-αand TGF-β1 increased to some extent,suggesting that these inflammatory factors may be involved in the progression of the disease.Part Ⅱ: Dynamic changes of macrophage polarization in different stages of Echinococcosis multilocularis infectionObjective: The changes of M1/M2 macrophages in mice infected with Echinococcus multilocularis were observed at different time points to investigate the role of macrophage polarization in the development of this disease.Methods: Mouse hepatic alveolar echinococcosis model was established in vitro.Mice were sacrificed at 1 month,2 months,3 months and 6 months after infection. The peritoneal macrophages and blood samples were collected.And flow cytometry and ELISA were applied to detect the number of M1 / M2 macrophages and expression of related interleukins respectively.Results: 1.The results of flow cytometry showed:(1)Compared with normal mice(0.25%±0.13%),the percentages of CD16/32+ macrophages in peritoneal macrophages were(5.00%±0.50%),(7.00%±0.45%),22.55%±3.61%)and(33.38%±4.83%)respectively at 1 month,2 months,3 months and 6 months after infection with Echinococcus multilocularis;(2)Compared with normal mice(0.30%±0.14%),the percentages of CD16/32+ macrophages in peritoneal macrophages were(3.93%± 0.98%),(4.88%±0.45%),(5.73%±0.51%)and(3.4%±0.62%)respectively at 1 month,2 months,3 months and 6 months after infection with Echinococcus multilocularis.(3)The percentages of CD32/16+macrophages(M1)and CD206+macrophages(M2)at 1 month,2 months,3 months and 6 months after infection with Echinococcus multilocularis were increase in varying degrees.P<0.05,the difference was statistically significant.The percentage of CD 16/32+macrophages(M1)was highest at 6 months post-infection,and the percentage of CD206+ macrophages(M2)was highest at 3 months post-infection.2.The results of ELISA showed that :(1)The levels of IL-6,TNF-α and TGF-β1increased to different degrees at 1 month,2 months,3 months and 6 months post-infection compared with the normal control group,P <0.05,The difference was statistically significant;while the difference of IL-10 was not significant,P> 0.05,the difference was not statistically significant.The levels of IL-6 were(0.351±0.052)at normal control group,(28.642±4.705)at 1 month post-infection,(82.484±18.684)at 2months post-infection,(172.910±27.336)at 3 months post-infection and(112.725±165.099)at 6 months post-infection respectively.The levels of TNF-αwere(0.338±0.210)at normal control group,(0.861±0.586)at 1 month post-infection,(1.495±0.253)at 2 months post-infection,(8.908±1.080)at 3 months post-infection and(16.607±4.650)at 6 months post-infection respectively.The levels of TGF-β1were(43.817±16.702)at normal control group,(78.988±5.407)at 1 month post-infection,(87.872±8.707)at 2 months post-infection,(94.362±14.965)at 3months post-infection and(120.875±20.624)at 6 months post-infection respectively. The levels of IL-10 were(0.024±0.006)at normal control group,(0.027±0.009)at 1month post-infection,(0.029±0.006)at 2 months post-infection,(0.027±0.009)at 3months post-infection and(0.028±0.012)at 6 months post-infection respectively.(2)IL-6 increased in the first 3 months of infection and then decreased slightly.TNF-αincreased with the time of infection,and it was highest at 6 months after infection.TGF-β1 increased with the time of infection.It was highest at 6 months after infection;IL-10 also increased slightly,but the trend was not obvious.Conclusion: We observed the dynamic changes of the macrophage polarization in mice at different time points after Echinococcus multilocularis infection.Our results suggested that macrophage polarization is involved in the whole process of Echinococcus multilocularis infection.M1/M2 macrophages were also constantly changing during infection.When they were embalanced,they may cause opposite effects: killing or protecting pathogens. |
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