Distribution And Homing Efficiency Of Different Dosages Or Frequencies Of Human Bone-marrow Mesenchymal Stem Cells In NOD Mice

Part One Distribution and homing efficiency of different dosages or frequencies of human bone-marrow mesenchymal stem cells in NOD miceObjective:1. Detect the distribution of hMSCs and compare the differences of MSCs quantities among organs where MSCs distribute.2. Evaluate effects on islet inflammation of T1DM model (NOD Mice) by different doses and frequency of hMSCs infusion.3. Compare differences of the protective effect on β-cells by different dosage and frequency infusion of MSCs.4. Confirm the correlation between the level of homing efficiency of hMSCs in pancreas and the local effect (immunity function and anti-inflammation) of hMSCs.Methods:1.The NOD mice were divided into four groups:control group (saline infusion), small dose group (0.5x106MSCs infusion), high dose group (1x106MSCs infusion), and small dose double times Group (0.5x106 MSCs Infusion twice a week), observation points are 2,4 and 6 weeks after infusion. Using real-time quantitative PCR method employing fluorescent TaqMan probes to quantify the number of MSCs resident in tissues of transplant recipients.2.Insulitis scores of each groups in different observation points were assessed under microscope after HE staining.3. Analyse the proportion of insulin-positive cells in the islet by immunohistochemical.Results:1.(1) Compare levels of hMSCs biodistribution in different groups:① In two weeks after transplantation, Intestines were found to harbor highest amounts of hMSCs DNA (all P< 0.05) in all groups.② In four weeks after transplantation, Pancreases and intestines had higher percentages of human genomic DNA than kidneys,livers and lungs in low-dose group (all P<0.05).Pancreases had higher percentages of human genomic DNA than other organs (including kidneys,livers,lungs and intestines) in high-dose hMSCs administration group (all P<0.05). In repeated hMSCs administration group, the intestines had the highest percentages of human genomic DNA (all P<0.05)③ In six weeks after transplantation, Pancreases were found to harbor highest amounts of hMSCs DNA in repeated hMSCs administration group (all P<0.05). In single-dose hMSCs administration groups (including low-dose hMSCs administration group and high-dose hMSCs administration group), the intestines and kidneys had the highest percentages of human genomic DNA respectively(all P<0.05).(2) Compare levels of hMSCs’s engraftment in pancreas between different groups:① In two weeks after transplantation, there was no significant difference between all groups;② In four weeks after transplantation, percentages of human genomic DNA in pancreas were higher in high-dose hMSCs administration group and repeated hMSCs administration group than that in low-dose hMSCs administration group, and the difference in the percentage level was significant between high-dose hMSCs administration group and low-dose hMSCs administration group (P<0.05)③ In six weeks after transplantation, percentages of human genomic DNA in pancreas were higher in high-dose hMSCs administration group and repeated hMSCs administration group than that in low-dose hMSCs administration group, and the difference in the percentage level was significant between repeated hMSCs administration group and low-dose hMSCs administration group (P<0.05)2. Compare insulitis scores between groups in different observation points:① The islets in control groups showed more severe islet lymphocyte infiltration than each transplantation groups(P<0.05).② In two and four weeks after transplantation, the islets in high-dose hMSCs administration group and repeated hMSCs administration group showed less severe islet lymphocyte infiltration than that in low-dose hMSCs administration group, and the difference in the infiltration level between high-dose hMSCs administration group and low-dose hMSCs administration group was significant(P<0.05).③ In six weeks after transplantation, the islets in high-dose hMSCs administration group and repeated hMSCs administration group showed less severe islet lymphocyte infiltration than that in low-dose hMSCs administration group, and the difference in the infiltration level between repeated hMSCs administration group and low-dose hMSCs administration group was significant(P<0.05).3.Compare the proportion of insulin-positive cells in the islet between groups in different observation points:① In each observation points, the proportion of insulin-positive cells in the islet was higher in high-dose hMSCs administration group than that in control group,and for low-dose and repeated hMSCs administration group, the proportion of insulin-positive cells in the islet was not significant higher than that in control group.② In two and six weeks after transplantation, the proportion of insulin-positive cells in the islet was higher in high-dose hMSCs administration group and repeated hMSCs administration group than that in low-dose hMSCs administration group.③ In four weeks after transplantation, he proportion of insulin-positive cells in the islet was higher in high-dose hMSCs administration group and repeated hMSCs administration group than that in low-dose hMSCs administration group, the difference in the proportion was significant between high-dose hMSCs administration group and low-dose hMSCs administration group was significant(P<0.05).Part Two Preliminary study on single-dose delivery of allogenic mesenchymal stem cells in the treatement of ketosis-onset patients with type 1 diabetesConclusion:1. Following systemic infusion of hMSCs into NOD mice, these cells can be detected in a wide tissues distribution among 2,4 and 6 weeks, and they are more likely to home to injury tissue.2. The number of hMSCs detected in the pancreas of NOD mice reached its peak at 4 week after transplantation and was decreased there after;3. hMSCs increase homing efficiency and local effect (immunity function and anti-inflammation) in pancreas in dose-dependent and frequency-dependent manner.4. There was a positive correlation between the increase in homing efficiency of hMSCs and local effects.Part Two Preliminary study on single-dose delivery of allogenic mesenchymal stem cells in the treatment of ketosis-onset patients with type 1 diabetesObjective:Explore clinical efficacy of single infusion of MSCs in the treatment of ketosis-onset patients with type 1 diabetes.Methods:This was a historical control study.All cases belonged to two groups:MSCs transplantation group(MSCs transplantation+basal insulin treatment) and control group(only basal insulin treatment).9 patients were included in each group, and they all completed 1 year follow-up intervals. There were no significant difference between values in the two groups at the baseline. The differences in HbAlc, FCP, PCP120’compared between the two groups in the follow-up period.Results:The differences in HbAlc, FCP, PCP120’were not significant between the two groups in the follow-up period (P> 0.05)Conclusion:There was no obvious clinical efficacy of single infusion of MSCs in the treatment of ketosis-onset patients with type 1 diabetes.