| Objectives: To explore the preventive effect of mesenchymal stem cells on murine type 1 diabetes.This experiment can provide a new theoretical and experimental basis for the prevention and treatment of T1 DM by MSC.Methods: 1.Isolation,culture,and identification of MSCs from bone parenchyma of mice.Femur and tibia of 7-day-old C57BL/6 mice were collected,and MSCs derived from bone parenchyma were isolated and cultured.Cell surface markers were detected by fluorescence-activated cell sorter(FACS),and further differentiation of MSCs into osteoblasts or adipocytes was detected by induction of MSCs.2.Experimental study on the preventive effect of MSCs on T1 DM.The mice were randomly divided into four groups: control group(NC group),T1 DM group,MSC prevention group(P group),and MSC treatment group(C group).Mice in the T1 DM group,P group,and C group were intraperitoneally injected with Streptozotocin(STZ)solution at a dose of 50mg/kg,while mice in the NC group were injected with sodium citrate buffer at the same volume for 5 days consecutively.Further,MSC was injected into the tail vein of mice in group P and group C on the 0.5 and 15 days after the first injection of STZ.The changes in body weight and blood glucose of mice were detected weekly.On the 56 th day after the first dose,the pancreatic tissue and renal tissue of mice were separated,and the pathological changes were analyzed by hematoxylineosin staining(HE)staining,immunohistochemistry,Masson staining,and other methods.3.Preliminary study on the preventive mechanism of MSCs against T1 DM.At the first 10 days and 56 days after STZ injection,peripheral blood and spleen in mice were taken and analyzed by using fluorescent quantitative PCR(quantitative real-time polymerase chain reaction,q-PCR)to detect the expression of inflammation factor TNF-α,IFN-γ in the spleen tissue;Enzyme-linked immunosorbent assay was used to detect the expression of TNF-α,IFN-γ in peripheral blood.In addition,splenic lymphocytes were isolated and FACS were used to inspect changes in the proportion of Th1(type 1 helper T)lymphocyte subsets.Results: 1.MSCs derived from bone parenchyma of femur and tibia of grade SPF 7-day-old C57BL/6 mice were successfully isolated and cultured.The FACS results showed that surface molecular markers CD29,CD90 and Sca-1 were highly expressed,while CD11 b,CD31,CD34,CD45,and MHCII were not.MSCs were induced to differentiate into adipocytes,and the results of oil red O staining showed that a large number of red lipid droplets can be observed under the microscope in the induction group.Meanwhile,the key transcription factors of lipids PPAR-γ,C/EBP-α were highly expressed.The differentiation of MSCs into osteoblasts was induced by alkaline phosphatase staining,and the results showed that the induced group presented a significant positive result of alkaline phosphatase staining and high expression of Runx2 and Osteocalcin genes related to osteoblasts.The mentioned results indicated that the cells we isolated and cultured had the ability to differentiate into adipocytes and osteoblasts,which confirmed that the cells were MSCs.2.The results of the study on the preventive effect of MSCs on T1 DM showed that the typical symptoms of diabetes in mice in the prevention group(group P)and the treatment group(group C)were significantly more relieved than those in the T1 DM group.The body weight of group P was higher than that of group T1 DM and group C(P<0.05).On the 14 th day after the first injection of STZ,the blood glucose of group P increased slowly,which was significantly lower than that of group T1 DM and group C(P<0.05).On the 35 th day,blood glucose of group C showed a downward trend,which was lower than that of the T1 DM group(P<0.05).HE staining results of islet tissue showed that the islet damage in group P was between group C and T1 DM group,and the islet damage was the most serious in the T1 DM group.The results of insulin immunohistochemistry showed that the islet area and immunohistochemical cumulative optical density of group P were between group C and T1 DM group,and significantly greater than that of T1 DM group(P<0.05).The results of Masson staining of kidney showed that the degree of the renal lesion in the T1 DM group was significantly higher than that in P group and C group.q-PCR results show that the Collagen fibrosis-related gene Collagen Ⅰ and TGF-β1 expression of P group were significantly lower than T1 DM group(P < 0.05).The results above suggest MSCs have the effect to prevent the occurrence and development of T1 DM.3.The preliminary study on the mechanism of MSCs in preventing T1 DM in mice.qPCR was used to detect the expression of inflammatory factors in the spleen tissues of mice in the P group at 10 days and 56 days after STZ treatment,and the results showed that the expression levels of inflammatory factors in the spleen,IFN-γ and TNF-α,were significantly lower than those in the T1 DM group(P<0.05).The expression of these two inflammatory factors in peripheral blood was detected by ELISA.The results showed that the levels of IFN-γ and TNF-α in peripheral blood serum of the P group were significantly lower than those of the T1 DM group(P<0.05).FACS was further used to detect the proportion of Th1 subgroup in mouse spleen lymphocytes.The results indicated that the proportion of Th1 cells in group P was lower than that in group T1 DM on day 10 and 56 after STZ treatment.These results suggest that MSCs can delay T1 DM by inhibiting the expression of Th1 cells and inflammatory cytokines TNF-α and IFN-γ.The specific mechanism of this phenomena remains to be further explored.Conclusion: Early infusion of MSCs can effectively delay the occurrence and development of T1 DM.This provides a theoretical and experimental basis for further research on the prevention of diabetes by MSC. |
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