The Biological Characteristics And The Proliferative Effect On Dendritic Cells Of Decidual Stroma Cells In Mice

【Objective】1.To develop a simple and satisfactory method to culture mice decidual stromal cell(DSC). The construction of a co-coltured system of decidual cells will lay a good foundation to study pathological pregnancy.2. To establish method of expanding dendritic cells from mice bone marrow in vitro. The cells morphological changes were observed by inverted microscope . Finally,we studied on its Biological Characteristic and Identification the cells.3. A Preliminary Investigation on effect of DC proliferation in DSC/mDC co-culture system.【Methods】1.Decidual tissues of pregnancy on days 6 through 8 from C57BL/6 mice were digested with trypsin,hyalace and collagenase,then cultured of composition of decidual cells. Erythrocytes,lymphocytes and non-adherent components were removed by changing medium to overnight primary cells.After being passaged for three times,the DSCwere isolated and purified.After purification,the growth features of the cultured cells were observed and finally idencified by immunohistochemistry using prolaction(PRL)kit. Cytokine and chemokine secreted by DSC was tested by RT-PCR.2. Cells isolated from mice bone marrow were cultured in GM-CSF and IL-4, morphological change was observed by optical phase contrast microscopy, T cell proliferation was detected by 3H-TdR incorporting into assay, and surface molecular was detected by FACS, and cytokines were assayed by ELISA.3.At last,we sorted mature DC from murine bone marrow,and culured them with DSC for at least 5 days.Then we observed proliferated changes of DC. 【Results】1. Almost the cultured DSC were adhered Twenty four hours after implantion,and the adhered cells showed the shape of fibroblast.Monolayer cells were formed on days 5 through 7.Primary cultured DSC were amplified for several weeks,passaged one time on days 4 through 5.The shape of passaged cells was not changed,but the polarity of passaged DSC was not obvious gradually.The immunohistochemistry revealed that PRLpositivity was 95%,the shape and size of cells were approximately similar.There were fine brown-yellow particles in the cytoplasm and the deeper color was close to nucleus.Some cytokines and chemkines were assayed by semi-quantitative RT-PCR.It implied that some stimulating factors have been produced by the stroma.2.Bone marrow mononeuclear cells,prepared from the mouse femurs by depletion of red cells,were cultured in the presence of 10ng/ml recombinant murine GM-CSF and 1ng/ml murine IL-4.The nonadherent cells were gently washed off after 3 day,s culture,the remained clusters were cultured foe 4 days;on day 8,CD11c+ mature DC were positively selected using CD11c magnetic microbeads.These mature DCs express high levels of MHC-II,CD80,CD86,CD40 molecules and had potent ability of stimulating auto and allogenic T cell proliferation. LPS promoted DCs to be mature and secrete higher level of IL-12(p70),IL-6,TNF-α和IL-1β.3.The matured DCs were seeded on the monolayer when the stromal cells teached 50% confluence,and can be reversedly turned to proliferative progenitors in the presence of DSC.【Conclution】1. Abortive decidual tissues were digested with trypsin,hyalace and collagenase.All components of the decidual cells were cultured together.The present method is simple,economical and practical.As a result,high purity DSC can be obtained with the isolation procedures. The construction of a co-coltured system of decidual cells will lay a good foundation to study pathological pregnancy.2. A large amount of mature DCs could be generated by in vitro culture of mice bone marrow cells. Biological Characteristic of DC was indentificated by morphological observation , Relatively Specific surface phenotype and potent ability of stimulating auto and allogenic T cell proliferation.Then it will lay a foundation for Related Fundamental Research and clinical application of DC.3.The mature DC can be reversedly turned to proliferative progenitors in the presence of DSC,and DSC can protect DCs from activation-induted apoptosis or cell death. The results show that stromal microenvironment composition of DSC effected natural outcome of mature DCs and which indicated that regulation of immune cells by Maternal-Fetal Interface immune microenvironment played an important role in inducing Maternal-Fetal immune tolerance.