ABSTRACT
To investigate the effect of co-expression of sleeping beauty (SB) transposon and IL-10 mediated by adenovirus on the balance of Th17/Treg cells in the splenocytes of the non-obese diabetes (NOD) mice, and to illuminate the possible mechanism of IL-10 in the treatment of NOD mice. Methods: The splenocytes were extracted from spleen of the C57BL6 mice and cultured. The splenocytes were divided into control group, empty vector group and therapy group. The cells in control group didn't receive any treatment, the cells in empty vector group were co-infected with the adenovirus vector without IL-10 gene containing transposon sequence and the adenovirus vector without transposase, and the cells in therapy cells were co-infected with the adenovirus vector containing IL-10 gene and transposon sequence and the adenovirus vector with transposase. After 48 h of infection, the expression leves of IL-10 mRNA in the splenocytes of the mice in various groups were assessed by RT-PCR. The cells of above three groups were subcutaneously injected into popliteal space in right hind leg of the NOD mice in control group, empty vector group and therapy group; there were six mice in each group; once a week and six times. The mice were killed 4 weeks after injection, the expression levels of IL-10 in serum of the NOD mice in various groups were assessed by ELISA, and the proportions of CD4 +IL-10+, CD4 + IFN-γ+, Th17 and Treg cells in the splenocytes were detected by flow cytometry. Results: Compared with control group and empty vector group, the expression level of IL-10 mRNA in splenocytes of the C57BL6 mice in therapy group was increased (F=72.71, P0.05). Conclusion: The co-expression of SB transposon and IL-10 can significantly increase the IL-10 level in serum of the NOD mice, increase the proportion of CD4 + IL-10+ cells, decrease the proportion of Th17 cells and increase the proportion of Treg cells in the splenocytes, which can regulate the balance of Thl/Th2 and Th17/Treg cells and play a therapeutic effect in the NOD mice.
ABSTRACT
Objective:To investigate the effect of co-expression of sleeping beauty(SB)transposon and IL-10 mediated by adenovirus on the balance of Th17/Treg cells in the splenocytes of the non-obese diabetes(NOD)mice, and to illuminate the possible mechanism of IL-10 in the treatment of NOD mice.Methods:The splenocytes were extracted from spleen of the C57BL6 mice and cultured.The splenocytes were divided into control group,empty vector group and therapy group.The cells in control group didn't receive any treatment,the cells in empty vector group were co-infected with the adenovirus vector without IL-10 gene containing transposon sequence and the adenovirus vector without transposase,and the cells in therapy cells were co-infected with the adenovirus vector containing IL-10 gene and transposon sequence and the adenovirus vector with transposase.After 48 h of infection, the expression leves of IL-10 mRNA in the splenocytes of the mice in various groups were assessed by RT-PCR. The cells of above three groups were subcutaneously injected into popliteal space in right hind leg of the NOD mice in control group,empty vector group and therapy group;there were six mice in each group;once a week and six times.The mice were killed 4 weeks after injection,the expression levels of IL-10 in serum of the NOD mice in various groups were assessed by ELISA,and the proportions of CD4+IL-10+,CD4+IFN-γ+,Th17 and Treg cells in the splenocytes were detected by flow cytometry.Results:Compared with control group and empty vector group,the expression level of IL-10 mRNA in splenocytes of the C57BL6 mice in therapy group was increased (F=72.71,P<0.05);the expression level of IL-10 in serum of the NOD mice was increased(F=8.89,P<0.05);the proportions of CD4+IL-10+ cells and Treg cells were significantly increased(F=72.09,P<0.05;F=12.98,P<0.05);the proportion of Th17 cells was decreased(F=6.39,P<0.05).The proportion of CD4+IFN-γ+ cells had no significant differences between various groups(F=2.72,P>0.05).Conclusion:The co-expression of SB transposon and IL-10 can significantly increase the IL-10 level in serum of the NOD mice,increase the proportion of CD4+IL-10+ cells,decrease the proportion of Th17 cells and increase the proportion of Treg cells in the splenocytes,which can regulate the balance of Th1/Th2 and Th17/Treg cells and play a therapeutic effect in the NOD mice.
ABSTRACT
Objective To establish the sperm specific Sleeping Beauty ( SB ) transposase-expression transgenic mouse for the study of the genetic modification mediated by transposon system in mouse .Methods Prm1 promoter was cloned from mouse genomic DNA to drive the expression of SB transposase .The transgenic mice were generated by microinjection .The gene type of transgenic line was identified by PCR .The expressing level in testis was determined by western blot and immunohistochemistry (IHC) staining.Results Five lines of transposase transgenic mice were obtained by microinjection and three can be germline .One mouse line with higher expression level of transposase in the testis was obtained.Conclusion One transgenic mouse model with Sleeping Beauty transposase - expression was successfully established .This model will greatly contribute to the research of genetic modification mediated by transposon in mouse.