| Part 1 Isolation, cultivation and identification of bone marrowme derived dentritic cells, spleen derived T cells and metanephric mesenchymal cellsAbstract Objective To isolate and culture rat spleen T cells, embryonic metanep-hric mesenchymal cells, and bone marrow-derived dendritic cells in vitro as the cell source for the following research including the immune regulation and transplantation.Methods (1)The mononuclear cells obtained from the bone marrow of SD rats after anesthetized by 10% chloral hydrate, were cultured in with complete RPMI1640 medium containing GM-CSF, IL-4 and TNF-a in 37℃and 5% CO2 incubator and cellular immunochemistry was performed to test the special antigens. (2) The spleen was obtained from SD rats under sterile conditions after anesthesia and was grinded into the suspension followed by density gradient centrifugation to get the T cells which were cultured at 37℃,0.05% CO2 incubator. The flow cytometry was performed to determine the cells antigen expression. (3) Rats were anaesthetized on gestation day 14 and metanephric balstemata were separated from ureteric buds by microdissection. Cells were grown in DMEM containing 10% fetal calf serum at 37℃in a 5% CO2 atmosphere. The morphology of MMS were observed by inverted phase contrast microscope, HE staining, and electronic microscope. The surface antigen phenotypes were identified by immunocytochemistry, and the purification was identified by flow cytometry. A growth curve to be used for studying the characteristics of growing. The morphology and the surface antigen phenotypes (vimentin, cytometry) of MMS at passage 12 and resused MMS at passage 5 were also observed.Results (1) the bone marrow mononuclear cells showed a typical dendritic cell morphology, such as burr-like protrusions, semi-adherent growth, and expressed CD11C, CD86, MHC-II after the induction (2) T-lymphocytes isolated from the spleen showed that CD3-positive rate was 75%, CD4-positive rate was 48%, CD8-positive rate was 31% by flow cytometry,(3) MMS grew adherencely as fibroblast-like, positive for vimentin, nestin, and CD 133. Instead, they were negative for keratin, CD34 and Dolichos Biflorus (DB). The flow cytometry showed a single peak of MMS. The morphology and the surface antigen phenotypes (vimentin, cytometry) of MMS at passage 12 and resused MMS at passage 5 had no changes.Conclusion dendritic cells cultured showed a typical morphology of dendritic cells in vitro, spleen-derived T cell got a considerate purity to meets the test requirements, matenephric mesenchymal cells hold a qualified purity, stability and showed stem cell characteristics. Objective To observe the effect of metanephric mesenchymal stem cells and anti-IL-10 antibody on the proliferation of T cells, dendritic cells and T cells induced by dentric cells and the expression of IL-4, INF-γ, perforin and granzyme B in T cells. To explore the mechanism.Methods (1) Culture the bone marrow derived dendritic cells, spleen derived T cells and the metanephric mesenchymal stem cells in vitro. (2) The establishment of mixed-culture system was performed and the influence of metanephric mesenchymal cells and anti-IL-10 antibody to the proliferation rate of spleen-derived T cells, dendritic cells and T cells induced after dendritic cell was recorded. (3) The establishment of Transwell culture system was performed and the influence of metanephric mesenchymal cells and anti-IL-10 antibody to the proliferation rate of spleen-derived T cells, dendritic cells and T cells induced after dendritic cell was recorded. (4) The establishment of mixed-culture system was performed and the influence of metanephric mesenchymal cells and anti-IL-10 antibody to the expression of IL-4, INF-γ, perforin and granzyme B in T cells after induced by dendritic cells were recorded. (5) The establishment of mixed-culture system was performed and the influence of metanephric mesenchymal cells and anti-IL-10 antibody to the expression of IL-4, INF-γ, perforin and granzyme B in T cells after induced by dendritic cells were recorded.Results (1) Compared with that in control, the proliferation rate of T lymphocytes, dendritic cells, T cells after dendritic cells stimulation in mixed culture system decreased in the intervention group with a dose-dependent pattern within the R:S ratio1:20-1:80(P<0.05). The difference of cell proliferation rate in the 1:80 andl:160 groups held no obvious significance (P> 0.05). Compared with R:S (1:80) group, the cell proliferation rate in anti-IL-10 group increased significantly (P＜0.05), while decreased significantly when compared with that in control group (P<0.05);(2) Compared with that in control, the proliferation rate of T lymphocytes, dendritic cells, T cells after dendritic cells stimulation in Transwell culture system decreased in the intervention group with a dose-dependent pattern within the R:S ratio1:20-1:80(P<0.05). The difference of cell proliferation rate in the 1:80 and1:160 groups were comparable(P> 0.05). Compared with R:S (1:80) group, the cell proliferation rate in anti-IL-10 group increased significantly (P<0.05), while decreased significantly when compared with that in control group (P<0.05);(3) Compared with that in control, the expression of IL-4, INF-y, perforin and granzyme B mRNA and protein T lymphocytes after dendritic cells stimulation in mixed culture system decreased in the intervention group with a dose-dependent pattern within the R:S ratio1:20-1:80(P＜0.05). The difference of cell proliferation rate in the 1:80 andl:160 groups held no obvious significance (P> 0.05). Compared with R:S (1:80) group, the cell proliferation rate in anti-IL-10 group increased significantly (P<0.05), while decreased significantly when compared with that in control group (P<0.05);(4) Compared with that in control, the expression of IL-4, INF-y, perforin and granzyme B mRNA and protein T lymphocytes after dendritic cells stimulation in transwell culture system decreased in the intervention group with a dose-dependent pattern within the R:S ratio1:20-1:80(P<0.05). The difference of cell proliferation rate in the 1:80 andl:160 groups held no obvious significance (P> 0.05). Compared with R:S (1:80) group, the cell proliferation rate in anti-IL-10 group increased significantly (P<0.05), while decreased significantly when compared with that in control group (P <0.05).Conclusion Metanephric mesenchymal stem cells inhibited the proliferation rate of dendritic cells, T lymphocytes and T lymphocytes after dendritic cell stimulation, inhibited IL-4, INF-y, Perforin and granzyme B mRNA and protein expression in T lymphocytes after dendritic cells stimulation. And the direct contact depression and paracrine could be responsible for the inhibition influence. Objective To investigate the immunomodulation of metanephric mesenchymal stem cells in chronic serum sickness nephritis rats as well as the therapeutic effects on renal injury.Methods (1) The metanephric mesenchymal stem cells were cultured and labelled by GFP in vitro, (2) The chronic serum sickness nephritis establishment in rats was performed, (3) Rats were divided into 5 groups: blank control, negative control, model, MMS preconditioning group and MMS transplantation group; (4) The localization of MMS labeled by GFP in rats was determined by fluorescence microscope, (5) Renal pathological changes in rats was observed by conventional pathological staining, (6) T cell subsets of rats from five groups were detected by flow cytometry, (7) ELISA assay was employed to detect serum IL-4, INF-y concentration; (8) RT-PCR assay was employed to detected IL-4, INF-y, perforin and granzyme B mRNA expression of mononuclear cells from rats perpheral blood of five groups.Results (1) Compared with that from blank control and negative control rats, rats from model and MMS preconditioning group showed increased urinary protein and cholesterol level and decreased total protein and albumin concentration when the chronic serum sickess nrphritis model was finished. Compared wth the model rats, MMS preconditioning rats displayed a lower urinary protein(P<0.05). (2) Compared with that in the model rats, a decreased urinary protein level was found in MMS transplantation rats showed (P＜0.05) and MMS preconditioning rats(P<0.01). Compared with the MMS transplantion rats, MMS preconditioning rats showed a decreased urinary protein levels(P<0.05). (3) GFP labeled MMS were found in the liver, spleen and kidney, without heart and the brain tissue. (4) Compared with model rats, MMS transplantation rats displayed a slight kidney damage and even slighter in MMS preconditioning rats. (5) Compared with the blank control and negative control rats, model rats, MMS preconditioning rats and MMS transplantation rats showed a decreased Th cells and Th/Ts ratio, increased Ts cell ratio in peripheral blood(P<0.05). Compared with model rats, the decreased Th cells and increased Ts cell ratio in MMS and MMS preconditioning rats did not holded significance(P＞0.05), while Th/Ts ratio decreased significantly(P<0.05). Compared with the MMS preconditioning rats, MMS transplantion rats held a decreased Th/Ts ratio(P<0.05). (6) Compared with that in the blank control rats and and negative control rats, an increased IL-4 and decreased INF-γconcentration in peripheral blood were found in model rats (P＜0.01) and MMS preconditioning rats and MMS transplantation rats(P<0.05). Compared with the model rats, MMS preconditioning rats and MMS transplantation rats got a reduced level of IL-4 and increased INF-y levels in peripheral blood(P<0.05). Compared with MMS preconditioning rats, MMS transplantation rats held an higher IL-4 and lower INF-γconcentration in peripheral blood(P<0.05). (7) Compared with that in the blank control rats and and negative control rats, an increased IL-4, perforin, granzyme B mRNA expression and reduced INF-γmRNA expression was abtained in mononuclear cells of peripheral blood were found in model rats(P<0.01) and in MMS preconditioning rats and MMS transplantation rats(P<0.05). Compared with the model rats, MMS preconditioning rats and MMS transplantation rats got a reduced level of IL-4 perforin, granzyme B mRNA expression and increased INF-ymRNA expression in peripheral blood(P<0.05). Compared with MMS preconditioning rats, MMS transplantation rats held an higher IL-4, perforin, granzyme B mRNA expression in a lower level of expression,and lower INF-γconcentration in peripheral blood(P<0.05).Conclusion Metanephric mesenchymal cells could perform the immunomodulation in serum sickness nephritis rats and reduce the renal damage.
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