Immunomodulatory Effects Of Hyaluronic Acid Combined With Human Amniotic Epithelial Cells On Human Peripheral Blood Mononuclear Cells And Type 1 Diabetic Rats After Transplantation

Objective:To investigate the regulatory effects of hyaluronic acid(HA)combined with human amniotic epithelial cells(hAECs)on allogeneic T lymphocytes.Then their immunomodulatory and therapeutic characteristics was further investigated by the orthotopic transplantation of pancreatic subcapsule in the streptozotocin(STZ)-induced type 1 diabetes mellitus(T1DM)rat model.Methods:hAECs were isolated by enzymatic digestion and then phenotypically identified by immunohistochemical staining and flow cytometry(FCM).Peripheral blood mononuclear cells(PBMCs)were isolated by Ficoll-hypaque density gradient centrifugation.The co-cultured cell ratio screening experiment was divided into 3 groups of 2:1,10:1 and 50:1 according to the ratio of PBMCs to hAECs,and the control group was PBMCs.Each group was stimulated with 10?g/mL phytohemagglutinin(PHA-P)and cultured for 72 h,the transcription level of Foxp3 was then detected by RT-qPCR.In addition,the OD values of each group were detected by MTT assay to screen the optimal cell ratio of hAECs and PBMCs after 96 h of co-culture.HA concentration screening experiments were divided into a total of 10 groups,including PBMCs,different concentrations of HA+PBMCs,hAECs+PBMCs(hAECs:PBMCs=1:2),and different concentrations of HA+hAECs+PBMCs,in with the relative molecular mass of HA was300 KD and the concentrations were 0.001,0.01,0.1 and 1 mg/mL,respectively.Likewise,the transcription level of Foxp3 was detected after 72 h of co-culture,and the optimal HA concentration was obtained by cell proliferation assay after 96 h.The effects of combined treatment on T cell subsets in PBMCs were performed in 4 groups:PBMCs,HA+PBMCs,hAECs+PBMCs,HA+hAECs+PBMCs,of which HA concentration was 0.1 mg/L. After 72 h of culture,the proportion of Th1,Th2,Tc1,Tc2,Th17 and Treg subpopulation in T cells was detected by FCM.For in vivo experiments,T1DM model rats were constructed by intraperitoneal injection of streptozotocin(STZ,30 mg/Kg for 4consecutive days),and after successful modeling,they were randomly divided into 5groups(n=6):model group,insulin treatment group(intraperitoneal injection of 3 U protamine zinc insulin,biw),HA group(0.1 mg/L),hAECs group(2×10~6only),and HA+hAECs group,and treated through orthotopic transplantation into the subcapsule of pancreas,and the normal group was used as the control(n=6).Blood was taken from the tail vein every week to measure blood sugar.After 30 days of treatment,blood was taken from the abdominal aortic vein,and FCM was used to detect the proportion of venous blood T lymphocyte subsets.Islet tissue structure and function were evaluated by HE staining and immunohistochemical staining.Meanwhile,serum C-peptide,IFN-?,TNF-?,IL-4,IL-17 and TGF-?1 levels were determined by ELISA.Results:The isolated and cultured P2 hAECs expressed high level of CD29,CD166,CD73 and low level of CD44,and did not express hematopoietic stem cell markers CD45,CD34 andHLA-DR,a MHC-II molecule.They also expressed high level of epithelial cell marker CK19,but did not express the mesenchymal cell marker vimentin.The results of MTT assay indicated that hAECs inhibited the proliferation of allogeneic PBMCs stimulated by PHA-P in a dose-dependent manner.When PBMCs:hAECs was 2:1,the proliferation inhibition rate reached the maximum(32.67±3.918)%.In the co-culture system,the addition of HA at doses of 0.001,0.01 and 0.1 mg/mL significantly promoted the inhibition of proliferation of PBMCs by hAECs(P<0.05),and the inhibition rates were(40.91±4.387)%,(46.58±2.785)%,and(40.24±5.671)%,respectively.However,1mg/mL HA had no obvious promoting effect,and HA alone had no effect on the proliferation of PBMCs.Further studies suggested that hAECs could promote the expression of Foxp3 by PHA-P-stimulated allogeneic PBMCs.Moreover,at the concentrations of 0.01 and 0.1 mg/mL,HA could synergize hAECs to promote the expression of Foxp3 in PBMCs(P<0.01),while at the doses of 0.001 and 1 mg/mL,HA had no significant such synergistic effect,and HA alone had no significant effect on the expression of Foxp3 gene in PBMCs as well.The FCM detection of T lymphocyte subsets suggested that hAECs could reduce Tc1 subpopulation in T lymphocytes compared with PBMCs group(P<0.05),but had no significant effect on Treg,Th17,Th1,Th2,Tc2subpopulations;HA alone had no significant effect on the subpopulation of PBMCs;compared with the PBMCs group and the HA+PBMCs group,HA combined with hAECs reduced the ratio of Th1 and Tc1 subpopulations,but had no significant effect on the proportion of Treg,Th17,Th2,and Tc2 subpopulations.The results of in vivo experiments showed that after 30 days of treatment,compared with the model group,there was no significant change on blood glucose levels in the insulin-treated group,the blood glucose levels of the T1DM rats in the hAECs-treated group and the HA+hAECs-treated group were significantly lower(P<0.01),and there was no significant therapeutic effect in the HA group.Compared with the insulin-treated group,the blood glucose levels of the T1DM rats in the hAECs-treated group and the HA+hAECs-treated group were significantly lower(P<0.05 or P<0.01).The C-peptide content in the serum of the rats corresponded to the blood glucose levels of the rats.HE staining and fluorescent staining of pancreatic pathological sections showed that compared with the model group,the number and volume of islets in the hAECs and HA+hAECs treatment group were larger,the number of insulin-positive cells in the islets was significantly higher,and a small number of hAECs homed into the islet site and were partially differentiated into insulin-positive cells.Further T cell subsets and immune-related cytokine analysis suggested that hAECs treatment can significantly improve the Th17/Treg ratio,and the combination of HA can significantly enhance the regulation capacity of hAECs on Th17/Treg.Conclusion:HA can enhance the inhibitory effect of hAECs on the proliferation of PHA-P-stimulated allogeneic human PBMCs and their regulatory ability on T lymphocyte subsets.In vivo,HA can enhance the therapeutic efficacy of hAECs on STZ-induced T1DM in rats.The mechanism may be related to reversing the ratio of Th17/Treg in T1DM rats,restoring the body's immune balance and reducing the damage of is let ? cells.