Construction Of Adenovirus Harboured Galectin-9 And It's Effects On The Differentiation And Proliferation Of CD4~+CD25~+ Regulatory T Cells In Mice

In recent decades,knowledge about genes and proteins have been paid much attentions.But little is known about the sugar-conjugates on the surface of the cells which has been found in early researches.Galectins constitute a family of evolutionarily conserved animal lectins, which are defined by their affinity for poly-N-acetyllactosamine-enriched glycoconjugates and sequence similarities in the carbohydrate recognition domain(CRD). During the past decade, attempts to dissect the functional role for galectins in vivo have been unsuccessful in comparison to the overwhelming information reached at the biochemical and molecular levels. Novel implications of galectins in cell adhesion, cell growth regulation, immunomodulation, apoptosis, inflammation, embryogenesis, metastasis and pre-mRNA splicing .Elucidation of the molecular mechanisms involved in galectin functions will certainly open new avenues not only in biomedical research, but also at the level of clinical intervention, attempting to delineate new therapeutic strategies in autoimmune diseases, inflammatory processes, allergic reactions and tumor spreading. To date, 15 different galectins have been characterized; they are numbered according to the chronology of discovery and are widely distributed from lower to higher vertebrates . They are cytosolic proteins from the cytosol, galectins may be targeted for secretion by unusual mechanisms; they may also be translocated into the nucleus, into vesicles, or accumulate at sub-cytosolic sites. Galectin-1 and Galectin-3 have been investigated by researchers much more among diversity aspects,such as Galectin-3 has some apparent effects on anti-cancer contribution. Galectin-9 is a member of galectin family members , belonging to animal lectins.Galectin-9 is isolated as a tandem-repeat type galectin having two CRD with selective affinity toβ-galactoside.Like other galectins,galeetin-9 exhibits lactose-binding activity and is believed to be involved in cell-cell or cell-matrix interactions.Galectin-9 exhibits a variety of biological functions,such as cell aggregation,adhesion, proliferation and cell apoptosis and modulation of inflammation.We recently found that Gal-9 induces the maturation of human dendritic cells (DC) from immature DCs, and that it also stimulates innate immune cells, such as monocytes and DCs, to secrete a small level of TNF-αin mouse and human systems. The research of the relation between galectin-9 and carcinoma has just started,it is reported that galectin-9 induces breast carcinoma cell apoptosis and aggregation in vitro.And the expression of galectin-9 reduces adhesion of breast cancer cell to extracellular matrix proteins and the expression of galectin-9 in breast cancer tissue is related to prognosis.The same conclusion also been found in human melanoma cell,galectin-9 protein induce the melanoma cell apoptosis and aggregation in vitro.T cell immunoglobulin domain and mucin domain 3(Tim-3), a Th1 specific type membrane protein, is not expressed on the surfaces of naive T cells but emerges on the cell surface of fully differentiated CD4~+ Th1 cells. Recently, galectin-9 has been identified as a ligand for Tim-3, binding of galectin-9 to Tim-3 causes an inhibitory signal, resulting in apoptosis of Th1 cells and negatively regulates Th1 type immunity. galectin-9 also can promote the induction of regulatory T cells. Furthermore,our previous results showed that activation of Tim-3-Galectin-9 pathway negatively regulated CD4~+ and CD8+ alloreactive T cells and resulted in prolonged survival of allogeneic skin graft.Immunosuppression play a role of suppression of the immune system by negative adjustment of regulatory T cells. Treg cells is a kind of immune regulatory function T-cell subsets differentiating type of Th1 and Th2 ,having function of Immune incompetence and immune suppression. It mainly play immunosuppressive effects through two mechanisms ,one is Cell contact: TGF-βof membrane surface binding receptor of TGF-βof the target cell surface after Treg cell activation, suppressing proliferation and activation of effector T-cell and mature of dendritic cells ;meantime, Its surface expression CTLA-4 can also play a suppressing function by binding the CD80 and CD86 .the another is secretion Cytokine : Treg cell suppress antigen-presenting of APC and activation of the effector T cells by release cytokine,such as IL-10,TGF-βand so on. Treg cells come from the differentiation of T cells in thymus,also can come from differentiation of periph resting T cells. Transcription factor Foxp3 gene participate the central and peripheral formation process of Treg cells ,and impact function of Treg cells.Treg cells also surpress the effect of NK cells,B cells and other immunological cells.there are evidences that Treg cells play an important role in the immunological tolerance. The raise of Treg cells is parallel with the good state of graft organs and immunological tolerance.Galectin-9 is belong to the family of galectins. It activates the biochemical effects such as cell aggregation,adhesion, proliferation and cell apoptosis and modulation of inflammation, and we predict that Galectin-9 whether it may induce the Treg cells`s differentiate and proliferation, in that case, it will rise the enlightenment that enlightenment play a role in the immunological tolerance of transplantation .so that we have chosen the AdEasy ? Adenoviral Vector System to construct Galectin-9 adenovirus vector, and the recombinant high expression adenovirus vector, pAdeasy- Galectin-9, was constructed and prepared for transfection. Then, we injected the Galectin-9 adenovirus vector in mice heart transplantation model ,and analyzed clinical common immunological effects that adenovirus vector and Galectin-9 adenovirus vector affect the proliferation of CD4 + CD25 + T cells and expression of Treg cell transcription factor Foxp3 in vivo,and affect the immune tolerance, which can provide some novel ideas about immunological suppression and immunological tolerance brought by Galectin-9. Part I Construction of Adenovirus Harboured Galectin-9Since there are some advantages of the adenovirus vector, such as simple structure, a wide range of host cells, high-titer, high efficiency of infection and no mutational risk of insertion, we have chosen the AdEasy ? Adenoviral Vector System to construct Galectin-9 adenovirus vector. First, LPS was injected into C57BL/6 mice to stimulate the mice for 4 hours. Then Galectin-9 gene was obtained from mouse spleen by RT-PCR. The amplified cDNAs were linked and inserted into pShuttle-CMV plasmid (pShuttle-CMV- Galectin-9). The homologous recombination took place between pShuttle-CMV- Galectin-9 and Adeasy-1.So the Galectin-9 adenovirus was packed in 293A cells. After a series of molecular cloning technologies, the recombinant high expression adenovirus vector, pAdeasy- Galectin-9, was constructed and prepared for transfection.Part II Experimental Study on the effect of Galectin-9 on regulatory CD4~+CD25~+T cells in mice heart transplantation modelExperimental Study on the effect of Galectin-9 on regulatory CD4~+CD25~+T cells in mice heart transplantation model.Objective: To explore the feasibility and possible mechanism of induction of immune tolerance with Galectin-9 by investigating effect of survival time of heart CD4~+CD25~+Treg cells value and the expression of Foxp3 under pAdeasyTM Galectin-9 or pAdeasyTM to heterotopic heart transplantation model in mice. Methods: Establish male BALB / C→C57BL/10 mice neck heterotopic heart transplantation model by Cuff method, in accordance with postoperative medication, receptor divided into 3 groups (n = 10) :control group (with NS ), pAdeasyTM group, pAdeasyTM Galectin-9 treatment group. Records of survival time of graft heart, Take spleen when graft heart don't beat or 14 d after operation, isolated mononuclear cells. detect CD4~+CD25~+ Treg cells value on flow cytometry and expression level of Foxp3 mRNA by Semi-quantitative RT-PCR. Results: Compared with the control group, adenovirus vector Galectin-9 group were significantly improved survival time of cardiac allograft (p <0.01), Compared with the control group, adenovirus vector group were not significantly improved survival time of cardiac allograft (p> 0.05), Compared with the control group. the proportion of CD4~+CD25~+ Treg cells in CD4 + T cells of adenovirus vector Galectin-9 group significantly increased (12.20% vs 7.35% , p <0.01); the proportion of CD4~+CD25~+ Treg cells in CD4 + T cells of adenovirus vector group was not obviously different (6.90% vs 7.35% , p> 0.05);expression of Foxp3 mRNA of adenovirus vector Galectin-9 group was significantly higher than that of the control group (P <0.01); expression of Foxp3 mRNA of adenovirus vector group compared with the control group was not obviously different (p> 0.05);Conclusion: adenovirus vector Galectin-9 was significantly improved survival time of cardiac allograft after heart transplantation in mice , there are no obvious gap in survival time between adenovirus vector and control group promote the proliferation and growth of CD4~+CD25~+ Treg cells in vivo. Foxp3 positively correlated with CD4~+CD25~+Treg cells in vivo.