The Research On Functional Difference Among Different Dose And Frequency Transplantation Of Human Bone Marrow Mesenchymal Stem Cell In Treatment Of NOD Mice

Objective:1、To confirm whether the curative effect and the maintain time of efficacy could be increased by increasing the dose and frequency of hBMSCs transplantation in the treatment of type 1 diabetes model (NOD/Ltj);2、Further confirm if hBMSCs could differentiate cross mesoderm into insulin-secreting cells.3、Observation the Immunomodulatory effect difference among different dose and frequency transplantation of human bone marrow mesenchymal stem c ell in treatment on NOD/Ltj mice.4、Observation of hBMSCs transplanted into mice can cause tumors occur.Methods:Mice were divided into four groups:Low-dose hBMSC transplantation injection group(LMSC) (0.5×106); High-dose hBMSC transplantation injection group(HMSC)(1×106);Low dose of repeat hBMSC transplantation injection group(LRMSC)(0.5 X 106, Infusion twice a week); Control group. The mice were injected hBMSC via tail vein. Blood glucose and weight of the mice were measured every week Mice were executed at corresponding stage and blood from orbital venous was collected(.Mice in all transplantation groups were executed respectively 2,4,6 weeks after established T1D model, control group were executed respectively 0,2,4,6 weeks after established TID model). The contents of mouse insulin and human insulin in the mouse peripheral serum were measured by ELISA method. The proportions of CD4+CD25+Foxp3+Treg and CD4+/CD8+T lymphocyte in the spleen mononuclear cell suspension were measured by flow cytometry. The pancreases were dyed by HE to observe the pancreatic structure and the content of insulin in the islets was measured using IHC.Results:1、Functional difference: ①compared with control group, the blood glucose level of HMSC group declined on 1w,2w,3w,4w and 5w (p<0.05); the level of LRMSC group declined on 1w,2w,3w,4w,5w and 6w (p<0.05); the level of LMSC group declined only on 1w,2w,3w and 4w (p<0.05); the content of mouse insulin of HMSC group and LRMSC group in peripheral serum was higher on 2w,4w(p<0.05), LMSC group was higher only on 2w(p<0.05); ②compared with LMSC group, the blood glucose level in LRMSC group declined only on 6w (p<0.05).2、The expression of human insulin:no human insulin was detected in peripheral serum of the mice in different dose and frequency transplantation groups.3、Immunomodulatory effect difference:①compared with the control group, the proportion of Treg in HMSC and LRMSC group rised on 2W,4W and6W (p<0.05), MMSC group rised on 4W(p<0.05);the proportion of CD8+T lymphocytes in HMSC group and LRMSC group declined on 4W,6W(p<0.05); the level of IFN-γ in LMSC group declined on 4W(p<0.05), the level of HMSC and LRMSC declined on 2W,4W and 6W(p<0.05); the level of TNF-α in the three transplantation groups declined on 2W,4W and 6W((p<0.05),the level of IL-10 in LMSC group rised on 2W (p<0.05),the level in HMSC and LRMSC group rised on 2W,4W and 6W(p<0.05); the level of TGF-β in the three transplantation groups rised on 4W and 6W(p<0.05);the insulitis score in HMSC group declined on 2W and 6W(p<0.05),the score in LRMSC group only declined on 6W(p<0.05); the IOD values of Insulin positive area were increased in HMSC and LRMSC group on 2W,4W and 6W(p<0.05),the IOD values in LMSC group were increased on 6W(p<0.05).②Compared with the LMSC group, the proportion of Treg in HMSC group were increased on 2W,4W and 6W(p<0.05), the proportion in LRMSC group were increased on 6W(p<0.05); the proportion of CD8+T lymphocytes in HMSC group declined on 2W,6W(p<0.05), the proportion in LRMSC group declined on2W,4W(p<0.05); the level of IFN-y in HMSC and LRMSC group declined on 6W(p<0.05); the level of IL-10 in HMSCand LRMSC group rised on 4W and 6W (p<0.05);the insulitis score in HMSC group declined on 2W and 6W(p<0.05), the IOD values of Insulin positive area were increased in HMSC group on 2W,4W and 6W(p<0.05),the IOD values were increased in LRMSC group on 2W,4W(p<0.05).③ Compared with the HMSC group, the proportion of Treg in LRMSC group were increased on4W (p<0.05), the proportion of CD8+T lymphocytes in LRMSC group declined on 4W (p<0.05), the level of IFN-γ,TNF-a and IL-10 were increased on 4W(p<0.05),and the level of TGF-β were increased on 6W(p<0.05).4、Security of transplantation:Bone or cartilage tumor has not been found in mice of All transplantation groups in this research. Only a inflammatory tuber has been found at the lower jaw in the LRMSC group.Conclusion:1、The blood glucose of NOD/Ltj mice can be reduced in varying degrees after transplantation with different doses and frequencies of hBMSC, and serum insulin levels can be elevated to different degree, amongst the repeat infusion of high-dose and low-dose group has a longer duration of effect.2、The immunomodulatory effects of hBMSC on circulating T lymphocytes (increased Treg proportion, decreased pathogenic CD8+T/CD4+T cells, reduced concentration of pro-inflammatory cytokines, increased anti-inflammatory cytokines) and the repair on damaged islets in NOD/Ltj mice are positively correlated with the amount and frequency of reinfusion of hBMSC.3、Although tumor-like change in mice has not been found in this study, potentially malignant MSCs can not be excluded. The safety remains to be further investigated.