| Objectives:To separate and identify Blastocystis hominis(B.hominis)from human-being,and use them to establish B.hominis-infected rat models,to observe the intestinal pathological damage and investigate the expressions of TLR2/TLR4 and inflammation-related cytokines in intestines of infected rats.Methods:1.The separation and identification of B.hominis:A total of 187 fecal specimens were collected from the outpatient department of the Cancer Hospital of Guangxi Medical University.Two strains of the B.hominis were obtained by LES culture.Morphology of B.hominis by HE staining was observed,and DNA of the B.hominis were extracted for genotyping.2.The axenic culture of B.hominis and establishment of B.hominis-infected rat models:combine LES medium with amphotericin B(2.5?g/ml),penicillin(1000 U/ml),streptomycin(the final concentration of 1000?g/ml),amoxicillin(31.25?g/ml),chloramphenicol(20?g/ml)and fosfomycin(20?g/ml)for axenic culture.For monoclone of B.hominis by using eggs supernatant-horse serum-Bacto agar mixed agar plate was tested.32 SD rats were divided into 4groups randomly and 8 rats for each group.One group was the control group,and the other three groups were infected with B.hominis at 7 day,14 day,and 21day as the treatment groups.After immunosuppression by dexamethasone,the treatment groups were infected with B.hominis at 1×10~8/rat.The rats in the control group were treated with the same volume of PBS.The rats were sacrificed at 0 day,7 day,14 day and 21 day.B.hominis strains were obtained from rat feces of treatment groups.HE staining of cell was used to observe the morphology while DNA of B.hominis while strains was extracted and genotyped by PCR.The pathological damage of intestinal tissue and mesenteric lymph nodes was observed by HE staining of tissue.3.The detection and analysis of TLR2/TLR4 and inflammation-related cytokines:The expression of TLR2,TLR4,TNF-?,IFN-?,IL-4,IL-6,IL-10mRNA in intestines were detected by fluorescence quantitative real time PCR.Results:1.The separation and identification of B.hominis:two strains of B.hominis were detected in fecal samples taken from the hospital while the DNA was extracted by PCR method,both of which were 3 types.2.The establishment of animal model:The rats'feces of the treatment groups in 7 day,14 day,21 day were soft,and B.hominis was detected after 48h in vitro,the morphology was consistent with the pre-infection B.hominis.The genotyping results of PCR were consistent with that of B.hominis genotyping before infection.The rats'feces of the control group were normal while no B.hominis was found.3.Intestinal histopathological examination:B.hominis was observed in the cecum of treatment groups at 14 day and 21 day.The complete structure of the intestinal tissue in the rest groups were observed without inflammatory cell infiltration,mucosal erosion or other pathological damage.The other intestinal tissues were normal.4.The detection of TLR2/TLR4 and inflammation-related cytokines:1)Duodenum:(1)duodenum:There were no significant changes in mRNA expression of TLR2,TLR4,TNF-?,IFN-?,IL-4,IL-6 and IL-10.(2)duodenum Peyer's patches:There were no significant changes in mRNA expression of TLR2,TLR4,TNF-?,IFN-?,IL-4,IL-6 and IL-10.2)Jejunum:(1)jejunum:There were no significant changes in mRNA expression of TLR2,TLR4,TNF-?,IFN-?,IL-4,IL-6 and IL-10.(2)Jejunal Peyer's patches:The expression levels of TLR2 mRNA in the7 day and 14 day groups were significantly decreased(P<0.05).The expression level of TLR4 mRNA was significantly increased in the 7 day group(P<0.05).The expression of IL-4 mRNA was significantly increased in the 14day and 21 day groups(P<0.05).The level of IL-6 mRNA expression was significantly increased in the 7 day group(P<0.05).The expression level of IL-10 mRNA was significantly decreased in the 14 day group(P<0.05).There was no significant change in the expression of TNF-?and IFN-?mRNA in each group.3)Ileum:(1)ileum:The expression level of IFN-?mRNA was significantly increased in the 7 day group and the 21 day groups(P<0.05).The expression levels of IL-4 mRNA was decreased in the 7 day group and the 21 day groups(P<0.05).There was no significant change in the expression of TLR2,TLR4,TNF-?,IL-6 and IL-10 mRNA in each group.(2)ileal Peyer's patches:The expression level of TLR2 mRNA in the 21 day group was significantly increased(P<0.05)in the ileal Peyer's patches of rats.The expression level of TNF-?mRNA was significantly increased in the 14 day group(P<0.05).There was no significant change in the mRNA expression of TLR4,IFN-?,IL-4,IL-6,IL-10 in each group.4)Cecum:The expression of TLR2 mRNA was significantly increased in the 7 day group,14 day group,and 21 day group(P<0.05).The expression of TNF-?mRNA was significantly increased in the 14 day and 21 day groups(P<0.05).The expression level of IFN-?mRNA was significantly decreased(P<0.05).The expression level of IL-6 mRNA in the 21 day group was significantly decreased(P<0.05).There was no significant change in the mRNA expression of TLR4,IL-4 and IL-10 in each group.5)Colon:The expression level of IL-4 mRNA was significantly increased in the 14 day and 21 day groups.There was no significant change in the expression of TLR2,TLR4,TNF-?,IFN-?,IL-6 and IL-10 mRNA in each group.Conclusion:1.B.hominis can be isolated and cultured from human feces successfully.After DNA extraction,both strains were identified as type 3 by PCR genotyping.After oral infection of SD rats with axenic type 3 B.hominis,rat infection model was successfully established.2.There was no obvious pathological changes in intestinal tissue in SD rats infected with B.hominis.Scattered B.hominis was found in the cecum of SD rats,suggesting that the cecum may be the parasitic site of B.hominis.3.The infection of B.hominis can cause changes in mRNA expression of TLR2,TLR4 and related cytokines TNF-?,IFN-?,IL-4,IL-6 and IL-10 in jejunal peyer's patches,ileal peyer's patches,cecum,colon,and the innate immunity system can generate an immune response to infection in these parts of the intestinal tissue to produce an inflammatory response suggesting that it may not be parasitic site of B.hominis.There was no significant change in the mRNA expression levels of TLR2,TLR4 and inflammation-related cytokines TNF-?,IFN-?,IL-4,IL-6 and IL-10 in the duodenum,duodenum Peyer's patches.This portion of intestinal tissue did not produce an inflammatory response to B.hominis infection suggesting that it may not be parasitic site of B.hominis. |
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