| [Objective] Establishing a practical, simple and efficient culture method to obtain high purity and large numbers of different mature state of dentritic cells(DC) from rat bone marrow. It also could control its mature phase to get different phenotypes of DC, such as imDC, smDC and mDC. Then, we analyzed its differences in mediating immune tolerance among the three phenotypes of DC to provide the experimental basis for further study of smDC in immune tolerance of organ transplantation.[Methods] We used rrGM-CSF and rrIL-4to induce differentiation of DC of rat bone marrow derived mononuclear cells. Then adding TNF-a and LPS induced different differentiation phases of DC. The difference of cell morphology, expression pattern of cytokine, active ability for allogeneic lymphocyte of different phenotypes of DC were analyzed and compared. Then it could conclude the difference of different phenotypes of DC in immune tolerance of transplantation, and provide a basis for further experimental study.[Results] It could get different phenotypes of DC,. such as imDC, smDC and mDC1. smDC morphology appeared to be at an intermediate developmental stage between imDC and mDC. ImDC. smDC and mDC could express OX62highly. The percentage was69%~89.3%,82%~89.6%and93.8%~97.7%respectively. The expressions of MHC-II, CD80. CD86for imDC were68.788%,53.847%.54.58%; for smDC were83.188%,73.05%,62.017%; for mDC were90.005%,83.065%,73.373%, respectively. Among the phenotypic expression of the three kinds DC. the mDC was the highest, followed the smDC. and the imDC was the lowest.2. The secretion of IL-1β. IL-6and IL-12of mDC were higher than the imDC and smDC. The secretion of IL-6of smDC was slightly lower than imDC, but the secretion of IL-12is slightly higher than imDC and the secretion of IL-1β without difference. The secretion of IL-10of imDC. smDC and mDC were all low-level.3. In mixed lymphocyte reaction (MLR), when the number of stimulating cells were same, the ability of T cell proliferation were stimulated by imDC and smDC similarly, but the ability for mDC was the strongest, followed by positive control group, next was the smDC intervention group, the last were imDC and smDC group. As the stimulating cells were the same, if the number was for20X103(scilicet R:S equal to1:10), the ability of T cell proliferation was the strongest, followed by5X103and10X103groups(there is no statistically significant difference between the two groups),1X103,3X103and5X103was lowest. When the R:S equal to1:10. the CCK-8absorbance of imDC was0.355+0.033; smDC was0.412±0.018: mDC was2.283±0.222; smDC intervention group was1.418±0.057: positive control group was1.887±0.07. Therefore, stimulating index for imDC(1.18) and smDC (1.37) was significantly lower than mDC (7.61); smDC intervention group(4.73) was lower than mDC and positive control group (6.29).4. The ratio of CD4+CD25+Tr cell (23.97±2.13) was higher than control group(1.89±0.36) after stimulated by smDC.[Conlusions]1. smDC was a independent subset of dendritic cells depending on its morphology and functions. It could express the OX-62and MHC-II highly, CD80and CD86moderately:it could secret IL-1β. IL-6and IL-12lowly. This was the basis of immune tolerance by inducing.2. imDC could express MHC-II lowly and moderately. expressCD80and CD86lowly; it could secret inflammatory cytokine lowly, such as, IL-lβ, IL-6and IL-12. Accordingly, it leaded to immune tolerance by inducing inefficiency or apoptosis of effector T cell.3. mDC could express MHC-II. CD80and CD86highly, the secretion of IL-1β, lL-6and IL-12which belong to inflammatory cytokine were high, mediating the immune response. |
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