The Immunological Regulation Effects Of Human Umbilical Cord Mesenchymal Stem Cells On RF/6A Cultured In High Glucose

Objectives:To observe the cytology and immunological regulation effects of human umbilical cord mesenchymal stem cells on glucose-damaged RF/6A influenced. Studying and comparing whether MSCs can reduce the early DR microvascular damage and retinal capillary blood-retinal barrier damage,delaying the progression of DR.Methods:In our research, there were three groups:RF/6A blank control group?high glucose induced RF/6A group ? h UCMSCs and high glucose induced RF/6A co-cultured group. 1.Incubating RF/6A cells.h UCMSCs cells were added to upper chamber, the lower chamber containing 25mmol/L glucose and RF/6A by using composite culture system(Transwell plates), h UCMSCs will co-culture according to 1:1 mixing with RF/ 6A.The large protein can interact with each other and two kinds of cells do not contact each other within the culture plates, the membrane pore size is 0.4?m. 2.Using MTT assay to determine RF/6A cell viability of each group.Calculating the cell viability in different conditions, repeated five times to obtain the average. Formula: cell viability( survival rate) = experimental group/control group A value×100%. 3.Using wound healing experiment to test the effect of each intervention factor on RF/6A,to examine the effect of h UCMSCs influce the RF/6A cell migration in high glucose. 4.Using the cell adhesion test to measure adhesion ability in each group. Adhesion rate =(A value of experimental group / control A value-1) x100%. 5.Using Matrigel experiment to test lumen formaton ability in each group,to detect lumen forming ability of h UMSCs on RF/6A cells in glucose environment. 6.Using Western blot to detect protein expression of Foxp3 in each experimental group. 7.Using enzyme-linked immunosorbent assay to detect the concentration of IL-1?, IL-17 and ICAM-1 in supernatant in each group.Results:1.MSCs was successfully established with the RF/6A co-cultured in high glucose system. 2.MTT assay to determine the RF/6A cell viability in high glucose cultured,in 3,7d.Three groups of single factor analysis of variance were compared, the first 1D, the survival rate of the three groups had no significant difference(F=2.03, P>0.05). 7d, the survival rate of high glucose group and high glucose co-culture group of was compared, the difference was statistically significant(P<0.05)., shows that high glucose can reduce RF/6A cell viability and inhibit RF/6A cell proliferation, but h UCMSCs can significantly improve RF/6A cell viability in high glucose environment. 3.Wound healing experiment to test the effect of each intervention factor on RF/6A, the results showed that, through single factor variance analysis, the difference of three groups was statistically significant(F=189.7?369.4,P<0.05) and LSD-t test shows that high glucose co-cultured group compared. High glucose group significantly inhibited the RF/6A cell migration(P<0.05), the difference was statistically significant. 4.Cell adhesion assay to test the effece of RF/6A adhesion in different conditions,the results showed that Three groups of single factor analysis of variance, the difference was statistically significant(F=160.7, p<0.05), after LSD-t test, high sugar co culture group compared with the control group, the difference was not statistically significant(p>0.05),suggesting that high glucose can reduce the adhesion of RF/6A cells, h UCMSCs can increase RF/6A cells adhesive capacity in high glucose environment. 5.Cell tube formation Experimental results show that three groups of single factor analysis of variance, the difference was statistically significant(F=113.60?350.71, p<0.05)compared with the control group, high glucose can inhibit the lumen formation, inhibition rate: 55.36%2 d, 76.71%1 w; the inhibition rate of lumen formation in high glucose co-cultured group and the control group as compared: 17.01%2d, 12.61%1 w, compared with high glucose group,the lumen formation rate in high glucose co-cultured group significantly increased(t=-12.79,-11.12, p<0.05) 6.Western blot to test Foxp3 expression.the results showed that,after 1 week of high glucose cultured, foxp3 protein expression was significantly decreased compared with the control group, while foxp3 protein was significantly higher in h UCMSCs co-cultured in high glucose group(p<0.05). 7.Enzyme-linked immunosorbent assay to test culture supernatant was collected in three groups of IL-1?, IL-17, ICAM-1 concentration. Compared with the control group, the concentrations of IL-1?, IL-17, ICAM-1 were significantly increased in high glucose group and high glucose co-cultured group, the difference was significant(p<0.05).Conclusions: High sugar can inhibit RF/6A cell proliferation, migration, adhesion, angiogenic ability, h UCMSCs can increase its biological capacity all above, It might be that h UCMSCs can be able to regulate Treg/Th17 cell imbalance to repair RF/6A,and have immunomodulatory effects on early diabetic retinopathy.