| Objective:By exploring the differences in the number and function of Treg cells in type 1 diabetes mellitus(T1DM)mice and normal mice,T1DM was treated with Treg cells by induced expansion cultured in vitro,the changes in the number,subtype and function of Treg cells in mice after treatment were observed,as well as the effect and safe and effective dose of Treg treatment,and the pancreas of normal control group,T1DM untreated group and T1DM treatment group(three therapeutic doses)were subjected to HE staining and immunohistochemistry.The repair effect of Treg cells on islets in T1DM mice after transplantation was studied,TNF-α,IFN-γ,IL-4 and IL-17 were differentially analyzed,and the molecular mechanism of T1DM therapy by Treg cells was explored.To study the repair effect of Treg cells on islets in T1DM mice after transplantation.Methods:1.Healthy normal mouse spleen CD4~+CD25~+Treg cells were obtained,and the contents of IL-10,IL-35 and TGF-βsupernatant of Treg cells on day3,day5,day 7,day9,day11,day 13 and day15 were detected after expansion and culture,and cell activity was detected;2.Forty-five male mouse with an 8-week-old weight of 18-22 g were randomly divided into 2 groups:9 in the normal control group(Control);T1DM group 36 pcs.On the day of successful modeling,T1DM mice were recorded as Day 0,and 3 mice were necked and sacrificed on Day0 to measure blood glucose,body weight,water intake,urine glucose,insulin,C-peptide,Treg cell changes,pancreatic HE staining and immunohistochemical staining.Day1 Control group 6 mice,24 T1DM mice according to different therapeutic doses of Treg cell number,randomly divided into 4groups:T1DM(6,PBS),T1DM+Treg(6,1×10~6/kg),T1DM+Treg(6,5×10~6/kg),T1DM+Treg(6,10×10~6/kg).Tail vein injections of PBS and Treg cells are performed on Day 1 and Day 14,respectively.On Day0,Day4,Day7,Day10,Day14,Day 18,Day25,Day32,Day34 days,fasting blood sugar,weight,water intake,urine sugar.In Day0,Day14,Day34,the content of insulin and C peptide in serum was detected;The levels of Th1,Th2,Th17 and Treg in spleen cells were detected by flow cytometry.Mouse pancreatic paraffin tissue sections were HE stained and immunohistochemical sections were performed and pancreatic inflammation scores were performed.Adverse events and complications were recorded,and the efficacy,safety and possible immune mechanism of Treg cells on T1DM were observed.3.The experimental results were analyzed by SPSS16.0 software.Oneway ANOVA was used for analysis between multiple groups,and two sample t-test was used for statistical analysis between the two groups:P<0.05 was considered to be statistically significant.Results:1.Detection of IL-10,IL-35 and TGF-βcontent of Treg cell supernatant:CD4~+CD25~+Treg cells were successfully obtained,and the cells grew well.2.Comparison of indicators of various groups:(1)Comparison of fasting blood glucose(mmol/l),urine output(ml),water intake(ml),and body weight(g)between groups:compared with the control group:fasting blood glucose in the T1DM group was significantly increased,urine output and water intake were significantly increased,and weight loss was obvious,and the differences were statistically significant;Compared with the T1DM group:after injection of Treg cells,the blood glucose of the treatment group decreased significantly,polyuria and polydipsia were less than that of the untreated group,and the weight loss was small,and the Treg medium-dose group(5×10~6/kg)and high-dose group(10×10~6/kg)were effective,and the effect was more obvious in the high-dose group.(2)Detection of insulin and C peptide content in serum:At Day 0,compared with the control group,the content of insulin and C peptide in the T1DM group decreased significantly(P<0.001).After Treg cell injection,insulin and C peptide decreased significantly in the T1DM group and Treg cell therapy group at Day14,and the difference was statistically significant,P<0.001;compared with the T1DM group,the levels of insulin and C peptide in the Treg cell therapy group increased slightly,but the difference was not statistically significant.At Day34,compared with the control group,the levels of insulin and C-peptide decreased significantly in T1DM group and Treg cell therapy group(P<0.01);after injection of Treg cells,compared with the untreated T1DM group,the C-peptide levels in the Treg medium dose group and the high dose group were statistically significant.The P<0.05;the C-peptide level in the high-dose group was the highest,close to the normal group,P<0.001;after injection of Treg cells,the insulin level in the high-dose group of Treg was higher than that in the T1DM group(P<0.05);the insulin level in the Treg low and medium-dose groups was slightly higher,the difference was not statistically significant,Treg cell therapy could improve islet cell function,and the high-dose group had the best effect.(3)Pancreatic HE staining and pancreatic inflammatory score results of mice in each group:Compared with the normal group,the pancreatic area of T1DM mice gradually decreased with the extension of disease course;After injection of Treg cell therapy,the pancreatic area increased to varying degrees,and the high-dose group(10×10~6)had a good therapeutic effect for 14 days,and the HE stained isletitis score was low.(4)Immunohistochemistry:analysis of anti-glucagon antibody(αcells)expression and anti-insulin antibody(βcells)expression of islet cells:compared with the normal control group,the proportion ofαcell area in the T1DM group gradually increased and the proportion ofβcell area gradually decreased as the disease progressed;After Treg cell injection,the proportion ofβcell area was higher than that of the untreated group,the proportion ofαcell area was lower than that of the untreated group,and the high-dose group(10×10~6)recovered the best.(5)Changes in Th1,Th2,Th17 and Treg levels in mice in each group:At 0 on Day,the T1DM group was analyzed with the normal control group,and the levels of Th1,Th2 and Th17 in the spleen cells of mice in the T1DM group were lower than those in the normal control group,and the Treg was higher than that of the normal control group,but there was no significant difference in the changes.At Day 14,compared with the control group,the levels of Th1 and Th2 in splenocytes of mice in the T1DM group were significantly regulated,with a statistically significant difference of P<0.05,Th17 level was down,and Treg level was raised,but the difference was not statistically significant.After injection of Treg cells,compared with the T1DM group,the levels of Th1 and Th2 cells were restored,the level of Th17 was raised,the level of Treg was adjusted at low and medium doses,and the levels of Th1 and Treg in the medium dose group were high,the Th2 level in the low dose group was high,and the Th17 level in the high dose group was high.At Day 34,compared with the normal control group,the T1DM group had higher levels of Th2,Th17 and Treg than the normal control group,and the level of Th1 was lower than that of the normal control group,but the difference was not statistically significant.After injection of Treg cells,compared with the T1DM group,the levels of Th1 in the low-dose treatment group were lower than those in the T1DM group,and the levels of Th2 and Th17 were higher than those in the T1DM group.The levels of Th1,Th2 and Th17 in the medium-dose treatment group were higher than those in the T1DM group.The levels of Th1,Th2 and Th17 in the high-dose group were lower than those in the T1DM group.The levels of Treg in the low,medium and high dose groups were lower than those in the T1DM group,and the differences were not statistically significant.Treg cells had irregular effects on Th1 cells in mice.To a certain extent,Treg cells can regulate the proportion of Th2 cells,and the low-dose group is better at Day14;There were no significant differences in the effect of Treg cells on Th17 cells.Treg cells can increase the proportion of Treg in mice,and the medium-dose group is preferred at Day14.Conclusions:1.Treg cells play a key role in regulating the occurrence and development of T1DM;2.Treg cells can effectively reduce blood glucose,improve polydipsia,polyuria,and weight loss symptoms in T1DM mice,and the treatment effect of high-dose group is better,which explored a new way for T1DM treatment;3.Compared with the untreated group,the levels of insulin and C-peptide in T1DM mice in the Treg cell therapy group were higher than those in the untreated group,which had a repair and protective effect on the islets of T1DM mice;4.Compared with the untreated group,the islet area of the Treg cell therapy group was larger than that of the untreated group,the severity of pancreatitis score was lower than that of the untreated group,the proportion ofβcell area was high,the proportion ofαcell area was low,Treg cells could slow down the area shrinkage ofβcells and the decline of islet function,and the degree of islet inflammation and damage was reduced after Treg treatment;5.After Treg cell therapy,the ratio level of Th1/Th2 cells can be adjusted,and the proportion of Treg cells in mice can be upregulated. |
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