| The mucosal immune system (mucosal immune system, MIS)-----that is widely distributed in the respiratory tract, gastrointestinal tract, urogenital tract mucosa and some of the lymphoid tissue of the exocrine glands, are different from those on the structure and function of the system immune system. Mucosa-associated lymphoid tissue in the mucosal immune system (mucosal associated lymphoid tissues, MALT), including:the gut-associated lymphoid tissue (GALT), bronchus-associated lymphoid tissue (BALT), nasal-associated lymphoid tissue (NALT) and urogenital mucosa-associated lymphoid tissue and so on. Lymphocytes of MALT are recyling in the mucosa within the lymphatic system, which is related to their expression of homing molecule.MIS can be divided into induced parts and effect sites according to different functions. The induced parts and effect sites are linked mainly by lymphocyte homing. Homing from the mucosa induced parts to effect site is collectively referred to as the common mucosal immune system (common mucosal immune system). Common mucosal immune system has the common mucosal immune effects, but on different parts of the mucosa, the immune mechanism has its own particularity based on the anatomy and function of the various organs. The research about GALT is more in-depth study. While the female reproductive tract is a special mucosal immune system because of that endocrine function, so immunity of the reproductive tract mucosal is more complex. Now the role of the immune system of the genital tract also slowly is concerned. In our previous work, a recombinant Lactobacilli live vaccine, Lb.hCGβ-C3d3were successfully constructed and could induce a high level of systemic and local mucosal immune response. But there are little report about the immune response of lymphocytes homing and residence in the reproductive tract. Therefore, our experiments focus on the problem that adhesion molecule which had been proved to been related to the lymphocyte homing to the other MALT. Our concern is the expression of adhesion molecular and local micro-environment on lymphocyte homing to reproductive tract. Then our studies might provide some basis for embryo implantation and reproductive tract immunity against infection.Part Ⅰ. The expression of lymphocyte homing molecules on reproductive tract mucosa via vagina immunizationObjective:Explore mucosal lymphocyte homing adhesion molecule expression in BALB/c mice after inoculation by wild-type and recombinant Lactobacillus vaccine via vaginaMethods:Female BALB/c mice of6-8weeks were immunized with1010Lb, Lb.hCGβ or Lb.hCGβ-C3d3and1×PBS as ctrl in50μl respectively, via vagina, and boosted three weeks later with same dosage. Two weeks later after booster immunization, the immunized mice were sacrificed to get the uterus and vagina tissues which was carried out by using standard techniques of fixation (for48h Paraformaldehyde at4%in phosphate buffer at0.1M) inclusion of tissue in paraffin and sectioned (5μm thick). Part of the specimens were placed in liquid nitrogen storage until the tissue RNA extraction used real-time quantitative PCR detecting adhesion molecule gene expression and tissue protein extraction used Western Blot experiments quantitating the protein level.Results:After booster immunization, reproductive tract mucosal adhesion molecule expression were changed, adhesion molecules expression of E-selectin, ICAM, VCAM, VAP-1and E-cadherin can be detected in the gene and protein level. And the expression of the most of the adhesion molecules were increased after recombinant Lactobacillus immunization..Conclusion Adhesion molecules of E-selectin, ICAM, VCAM, VAP-1and E-cadherin were involved in local lymphocyte homing to the reproductive tract.Part II.The surface integrin expression of mucosal homing lymphocytes in the reproductive tract mucosa after immunization via vaginaObjective:Explore related adhesion molecule ligand expression by wild-type and recombinant Lactobacillus vaccine through the vaginal mucosa after inoculationMethods:The methods are same as the first part. Then the uterus and vagina tissues of the immunized groups were pooled and digested to get primary cells such as spleen cells and local immune cells in FRT. These primary cells were used for flow cytometry to detecte the integrin expression. The amount of TGF-β1expression was measured by ELISA in endometrial stromal cell culture supernatant of each experimental group.Results:The molecular adjuvant C3d3can significantly increase the ligands of homing-related adhesion molecules ΩEβ7, α4β7, LFA-1, VLA-4and PSGL-1expression, and can significantly promote the expression of TGF-β1in recombinant Lactobacillus immunized groupConclusion:After booster immunization, molecular adjuvant C3d3can increase adhesion molecule ligands αEβ7, α4β7, LFA-1, VLA-4and PSGL-1expression and thus participate to increase local T and B lymphocyte homing to the reproductive tract. The recombinant Lactobacillus can significantly strengthen the expression of TGF-β1by endometrial stromal cells.Part Ⅲ. Reproductive tract microenvironment can affect lymphocyte homing after booster immunization.Objective:To observe the reproductive tract microenvironment influence on the lymphocyte homing.Methods:The immunized pattern is same as the first part. The primary separation of endometrial stromal cells was cultured and its supernatants were collected. Using transwell migration assay to collected the cells which had migrated to the lower chamber. The migrated lymphocytes were detected the proportion of lymphocytes by flow cytometry. In addition, T lymphocytes phenotypes were detected by flow cytometry in spleen and reproductive tract.Results:After immunization, the experimental group compared to culture medium can significantly increase the total lymphocyte numbers, especially our recombinant Lactobacilli immunized groups. The micro-environment of the reproductive tract is more likely to increase the proportion of T cells, and promote T cell to migrate toward the lower chamber, especially γδT cells.Conclusion:the local micro-environment can significantly increase the proportion of T lymphocytes, especially γδ positive T cells. |
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