Objective:To detect the inhibiting co-stimulating molecule CTLA4 and cytokines secreted by Treg cells, and explore the immunology mechanism of T regulatory cells acting on effector T cells in co-cultured system(CCS) a...Objective:To detect the inhibiting co-stimulating molecule CTLA4 and cytokines secreted by Treg cells, and explore the immunology mechanism of T regulatory cells acting on effector T cells in co-cultured system(CCS) and separating-cultured system(SCS). Methods: Detecting the percentage of CTLA4 and CD28 expressed on the Treg cells and effector T cells, and then adding Treg cells to mixed lymphocyte reaction(MLR) system in CCS and TransWell Millicell-PCF SCS, at the same time, adding or not adding anti-IL-10 or anti-TGF-β1 to the reacting systems, examining the inhibitory capacity of Treg cells exerting on the MLR. Results: Compared with effector T cells, Treg cells expressed higher level CTLA4 and secreted much more IL-10 and TGF-β1(P<0.01). The inhibitory capacity of Treg cells co-cultured with effector T cells is much stronger than that in separating cultured group(P<0.01). Moreover, the inhibiting rate of Treg cells exerting on effector T cells through secreting IL-10 was more powerful than that through secreting TGF-β1(P<0.01). Conclusion: Both cell-to-cell contact and cytokines secretion mechanisms are involved in CD4 +CD25 + Treg cells operating function. However, the former is more important. Intrestingly, we for the first time point found that IL-10 plays more powerful roles than TGF-β1 in the cytokines secretion mechanism.展开更多
AIM: To clone and express mouse peroxiredoxin Ⅰ in IEC-6 cells.METHODS: Total RNAs were isolated from cultured IEC-6 cells, and the coding region of peroxiredoxin I was amplified by RT-PCR. After it was cloned into T...AIM: To clone and express mouse peroxiredoxin Ⅰ in IEC-6 cells.METHODS: Total RNAs were isolated from cultured IEC-6 cells, and the coding region of peroxiredoxin I was amplified by RT-PCR. After it was cloned into T-vector and sequenced,pSG5 was used to transiently express peroxiredoxin I in IEC-6 by liposome-mediated transfection, and the expression of peroxiredoxin I was evaluated by RT-PCRand Western blot. RESULTS: A DNA fragment about 750 bp was amplified from total RNAs of IEC-6 cells using specific primers of peroxiredoxin Ⅰ. The sequencing confirmed the coding region was successfully cloned into T-vector, which was completely coincident with the sequence in GeneBank. After the EcoRI-BamHI fragment of T-vector containing peroxiredoxin I was inserted into pSG5, the recombinant plasmid was transferred to IEC-6 cells. RT-PCR assay showed that a DNA fragment of 930 bp could be amplified,which indicated the transcription of pSG5-Prx. Western blot confirmed the expression of peroxiredoxin Ⅰ in IEC-6 cells.CONCLUSION: Mouse peroxiredoxin I can be successfully expressed in IEC-6 cells.展开更多
文摘Objective:To detect the inhibiting co-stimulating molecule CTLA4 and cytokines secreted by Treg cells, and explore the immunology mechanism of T regulatory cells acting on effector T cells in co-cultured system(CCS) and separating-cultured system(SCS). Methods: Detecting the percentage of CTLA4 and CD28 expressed on the Treg cells and effector T cells, and then adding Treg cells to mixed lymphocyte reaction(MLR) system in CCS and TransWell Millicell-PCF SCS, at the same time, adding or not adding anti-IL-10 or anti-TGF-β1 to the reacting systems, examining the inhibitory capacity of Treg cells exerting on the MLR. Results: Compared with effector T cells, Treg cells expressed higher level CTLA4 and secreted much more IL-10 and TGF-β1(P<0.01). The inhibitory capacity of Treg cells co-cultured with effector T cells is much stronger than that in separating cultured group(P<0.01). Moreover, the inhibiting rate of Treg cells exerting on effector T cells through secreting IL-10 was more powerful than that through secreting TGF-β1(P<0.01). Conclusion: Both cell-to-cell contact and cytokines secretion mechanisms are involved in CD4 +CD25 + Treg cells operating function. However, the former is more important. Intrestingly, we for the first time point found that IL-10 plays more powerful roles than TGF-β1 in the cytokines secretion mechanism.
基金Supported by the National Natural Science Foundation of China,No.30230360
文摘AIM: To clone and express mouse peroxiredoxin Ⅰ in IEC-6 cells.METHODS: Total RNAs were isolated from cultured IEC-6 cells, and the coding region of peroxiredoxin I was amplified by RT-PCR. After it was cloned into T-vector and sequenced,pSG5 was used to transiently express peroxiredoxin I in IEC-6 by liposome-mediated transfection, and the expression of peroxiredoxin I was evaluated by RT-PCRand Western blot. RESULTS: A DNA fragment about 750 bp was amplified from total RNAs of IEC-6 cells using specific primers of peroxiredoxin Ⅰ. The sequencing confirmed the coding region was successfully cloned into T-vector, which was completely coincident with the sequence in GeneBank. After the EcoRI-BamHI fragment of T-vector containing peroxiredoxin I was inserted into pSG5, the recombinant plasmid was transferred to IEC-6 cells. RT-PCR assay showed that a DNA fragment of 930 bp could be amplified,which indicated the transcription of pSG5-Prx. Western blot confirmed the expression of peroxiredoxin Ⅰ in IEC-6 cells.CONCLUSION: Mouse peroxiredoxin I can be successfully expressed in IEC-6 cells.
