| Background and Objective:Chlamydia trachomatis(C.trachomatis)is a Gram-negative obligatory intracellular pathogen and one of the most common pathogens of sexually transmitted diseases.In female,C.trachomatis can infect the upper genital tract,causing salpingitis,pelvic inflammatory disease,ectopic pregnancy and infertility.Chlamydia muridarum(C.muridarum)and C.trachomatis simultaneously sharing more than 99%of all predicted open-reading frames.So C.muridarum is often used as an effective alternative strain to study the pathogenic mechanism and control measures of C.trachomatis.Research shows that Chlamydia can be detected in the intestines of humans and animals.Moreover,the C.muridarum pathogenic strain that can cause fallopian tube disease can establish long-term and stable colonization in the intestinal tract,while the C.muridarum attenuated strain that can not cause significant fallopian tube disease can not effectively colonize in the intestinal tract.Intestinal colonization of Chlamydia may play an important role in the fallopian tube lesions caused by genital tract infection,but the mechanism of its colonization remains unclear.Intestinal epithelial cell barrier is a crucial physical barrier in the intestinal mucosal barrier,which has the function of selective permeability and involves the regulation of a variety of extraintestinal diseases.However,it is unknown whether the intestinal epithelial cell barrier can play a role in the intestinal colonization caused by Chlamydia genital tract infection.Therefore,this thesis studied whether the intestinal epithelial cell barrier was involved in regulating the mechanism of intestinal colonization caused by Chlamydia genital tract infection.It lays a foundation for the study of the role and preliminary mechanism of the intestinal epithelial cell barrier in fallopian tube disease caused by C.muridarum genital tract infection and provides a new idea for the prevention and treatment of clinical C.trachomatis infectious infertility.Methods:1.The genital tract infection model of C.muridarum pathogenic strain(G13.32.1)and C.muridarum attenuated strain(G28.51.1)in C57BL/6J mice were established.The degree of tissue inflammation was evaluated by measuring the length of the colon and detecting tissue inflammatory factors.2.Established and identified the intestinal epithelial monolayer cell barrier model of mouse colon cancer CT26.WT cells,detect the expression level of tight junction protein,and detected the effects of different Cm infection on intestinal epithelial barrier integrity and permeability at the cellular and molecular level.3.The genital tract infection model of C57BL/6J mice was established.Fluorescein thiocyanate dextran(FITC-dextran)was used to detect the changes of intestinal permeability and the expression level of tight junction protein in intestinal tissue at different time points before and after infection.Proved the effect of C.muridarum genital tract infection on the integrity and permeability of intestinal epithelial cell barrier in mice with the animal experiments in vivo.4.Established a mice colitis model induced by dextran sulfate sodium(Dextran Sulfate Sodium,DSS).Two groups of strains were used to infect colitis model mice through the rectum,and the intestinal colonization of C.muridarum in mice with damaged intestinal wall was detected.Results:1.Female mice at the age of 6 and 8 weeks were inoculated with 2×10~5IFUs/mice.The vaginal secretions of the mice were taken every 3 or7 days to detect the Chlamydia load in the vagina and intestines.The results showed that compared with the attenuated strain,in the C.muridarum infection model,the vaginal Chlamydia load had similar level,while the intestinal Chlamydia load was significantly increased,and the disease rate of oviduct edema was also significantly increased.2.The length of colon tissue from the experimental groups that infected with C.muridarum and its attenuated strain and the negative control group were measured and compared.It was found that the colon length of mice infected with attenuated C.muridarum strain was not significantly different from that of uninfected mice,but it was significantly longer than that of mice infected with C.muridarum pathogenic strain(Wilcoxon rank sum test,P<0.05).Pathological section observation also showed that the intestinal mucosal barrier was intact and the morphology of epithelial cells was normal in the negative control group and C.muridarum attenuated strain infection group,while the intestinal mucosal barrier in the C.muridarum pathogenic strain infection group was destroyed,goblet cells decreased and inflammatory cells infiltrated.The results of q RT-PCR showed that the levels of inflammatory factors IL-1,IL-6,IL-10 and CXCL-2(homology of human IL-8)in colon tissue of mice infected with Cm pathogenic strain were significantly higher than those of mice infected with Cm attenuated strain(Two-tailed Student’s t test,P<0.05).while there was no significant difference between Cm attenuated strain infected group and non-infected group.3.In the constructed CT26.WT monolayer cell barrier model,the pathogenic strain and attenuated strain of C.muridarum were inoculated respectively,and the uninfected negative control group was set up.The sodium fluorescein permeability of the monolayer cell model was detected and compared.The results showed that the transmission rate of sodium fluorescein in C.muridarum pathogenic strain infection group was significantly higher than that in negative control group and C.muridarum attenuated strain infection group at 24 hours after infection with C.muridarum(Independent-Sample Test,P<0 05).The results of q RT-PCR showed that C.muridarum pathogenic strain could down-regulate the m RNA level of ZO-1,Occludin,Claudin-1 protein in CT26.WT monolayer cells,compared with negative control group and Cm attenuated strain infection group,the difference was statistically significant(Two-tailed Student’s t test,P<0.05).Compared with attenuated strains,pathogenic strain can significantly increase the permeability of CT26.WT monolayer cell model and destroy its integrity by regulating tight junction protein,which affects its colonization ability in intestinal tract.4.In the mouse reproductive tract infection model constructed byC.muridarum pathogenic strain and attenuated strain,the intestinal wall permeability of mice in each group was compared by the amount of FITC-dextran permeability.The FITC-dextran permeability of intestinal wall in C.muridarum pathogenic strain infection group was significantly higher than that in negative control group and C.muridarum attenuated strain infection group,and the difference was statistically significant(Independent-Sample Test,P<0.05).The q RT-PCR results showed that the C.muridarum pathogenic strain could down-regulate the m RNA level of ZO-1,Occludin,Claudin-1 protein in in mouse colonic tissue,compared with the negative control group and C.muridarum attenuated strain infection group,the difference was statistically significant(Two-tailed Student’s t test,P<0.05);WB results were consistent with q RT-PCR results.Compared with the mice infected with attenuated strain,the mice infected with C.muridarum pathogenic strain could significantly increase the intestinal wall permeability and destroy the integrity of intestinal wall by regulating the tight junction protein in the intestine of mice.5.In the colitis mouse model of rectal infection with C.muridarum pathogenic strain and attenuated strain,the rectal secretions of mice were taken every 3 or 7 days to detect the infection of C.muridarum in the intestine.The results showed that compared with the normal mice infected with C.muridarum,the intestinal colonization load and time of colitis mice infected with C.muridarum were significantly increased,and the intestinal colonization of attenuated C.muridarum strain was prolonged from 7 day to the 28 day.Conclusions:1.Intestinal epithelial cell barrier is involved in the regulation of intestinal colonization of Chlamydia reproductive tract infection.2.Compared with the attenuated C.muridarum strain,the pathogenic strain of C.muridarum may affect the permeability of intestinal epithelial cells and enhance its colonization ability by significantly destroying the tight junction protein that destroys the barrier of intestinal epithelial cells. |
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