| Objective: To establish a stable method of rat bone marrow-derived dendritic cells culture in vitro, immature dendritic cells transfected by IKK2dn and loaded by donor antigen. Regulatory T cells were induced by imDC and recipient T cells that mixed lymphocyte reaction in vitro, from which the high activity and high purity CD4~+CD25~-T cells were screened, immune of CD4~+CD25~-T cells were identificated in the initial, which laid a foundation for the induction of Treg in the field of transplantation tolerance.Methods: This study was divided into two parts. In the first part, bone marrow cells were removed from the femur and tibia of Lewis rats in sterile, in which erythrocyte was lysed by Tris-NH4Cl, most of the granulocytes, platelets and their precursors were removed by density gradient centrifugation, the residual granulocyte was gradually elutinged by the adhesion method, bone marrow mononuclear cells were obtained. Bone marrow mononuclear cells were cultured with GM-CSF and IL-4, we collected 5th, 7th, 11th, 13th day cultured cells. Cell morphology were observed through Light microscopy and transmission electron microscopy, cell phenotype were watched by flow cytometry, cell function were identificated by mixed lymphocyte reaction. In the second part, immature dendritic cells transfected by IKK2dn and loaded by antigen of donor BN rats, regulatory T cells were induced by imDC and recipient Lewis rats T cells that mixed lymphocyte reaction in vitro. CD4~+CD25~-T cells were screened by MACS, cell morphology were watched through light microscopy and transmission electron microscopy, cell purity were detected by flow cytometry. Two groups were involed in the experiment: which the control group: BN rat spleen cells inactivated by mitomycin C stimulated Lewis rat T cells; and the experimental group: BN rat spleen cells inactivated by mitomycin C stimulated Lewis rat T cells, while adding CD4~+CD25~-T cells. MTT assaied CD4~+CD25~-T cells immune function to Lewis rat T cells in MLR.Results: 1. Each rat bilateral femur and tibia obtained (3.5±0.75)×107 mononuclear cells. MDC and imDC that observed by light microscopy and transmission electron microscopy were consistent with its structural features. Flow cytometry detected cell phenotyp: DC cultured for 7th day of their CD80, CD86, MHC-II expression positive rate were about (39.35±1.58)%, (31.22±1.28)% and (33.34±5.55)%. DC cultured for 11th day of their CD80, CD86, MHC-II expression positive rate were about (72.12±5.15)%, (59.44±3.02)% and (61.35±1.56)%. The 11th day'the phenotypic test results was significantly higher than the 7th day'(P <0.05). DC cultured for 7th day and 11th day in the MLR absorbance value was statistically significant difference (P <0.05). 2. Regulatory T cells were induced by imDC transfected by IKK2dn and loaded by antigen of donor BN rats and recipient Lewis rats T cells that mixed lymphocyte reaction in vitro. Activity and purity about more than 95% CD4~+CD25~-T cells can be obtained by MACS screening, absorption scale value of proliferative response about recipien T cells was assaied by MTT in the MLR: which the control group was 0.259±0.057, and the experimental group was 0.064±0.016. There were significant differences between the two groups (P <0.05).Conclusions: 1. We established a stable method of rat bone marrow-derived dendritic cells culture in vitro, with GM-CSF and IL-4 in cultured rat bone marrow-derived mononuclear cells of 5 to 7 days, we gained sufficient quantity and purity imDC, which were suitable for transplantation research in the field of immune tolerance. 2. Regulatory T cells were induced by imDC transfected by IKK2dn and loaded by antigen, from which CD4~+CD25~-T cells with high purity and high activity can be obtained by MACS screening. 3. CD4~+CD25~-T cells in the MLR had immunosuppressive effects in the reactive T cells. 4. Isolation and function identification of CD4~+CD25~-T cells provided a experimental and theoretical basis for transplantation tolerance study in vivo and vitro, which may laid a foundation for further establishing clinical transplantation tolerance. |
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