| Objective To investigate the effects of human umbilical cord mesenchymal stem cells(hUCMSCs)on the proliferation of human retinal vascular endothelial cells(HREC)and the expression of IL-8 and IL-10 in human high-glucose cultured human retinal vascular endothelial cells(HREC).Materials and methods:1:Take the complete medium HREC into the 96-well plate,about 4000 cells per well,wait for the cells to adhere to the wall,starve for 24 hours,add the high-sugar medium and incubate for 12,24,36,48 hours,then add in each well.20 ul of CCK-8 reagent was placed in a 37°C incubator for 2-4 hours.The absorbance(OD)at 450 nm in each well was measured by an enzyme-linked device and recorded and analyzed.2:Take HREC in complete medium and add it into 96-well plate,about 4000 cells per well,wait for cells to adhere,and starve for 24 hours,add respectively(1)Experimental group containing DMEM/30mmol/L D-glucose base.(The supernatant containing the umbilical cord mesenchymal stem cells),(2)The control group contained DMEM/30 mmol/L D-glucose complete medium.Theremaining wells were filled with cell-free DMEM/30mmol/LD-glucose complete medium alone and cells without DMEM/30mmol/L D-glucoseconditioned supernatant.(containing the supernatant of umbilical cord mesenchymal stem cells),after incubation for 12,24,36,and 48 hours,respectively,20 ul of CCK-8 reagent was added to each well,incubated in a37°C incubator for 2-4 hours,and each well was examined by an enzyme-linked device.Absorbance(OD)at 450 nm was recorded and analyzed.3:HUCMSCs were added to the upper chamber of the Transwell system using a complex culture system,and 30 mmol/L glucose and HREC were added to the lower chamber;hUCMSCs were co-cultured with HREC at a ratio of 1:1.The experiment was divided into control group(complete culture medium),high glucose group and hUCMSCs co-cultured with HREC group.The concentration of interleukin(IL)-8 and interleukin(IL)-10 in the cell culture supernatant was detected by ELISA.4:The HRECs were divided into three groups and added to the normal control group(N)cell culture medium containing 5.5 mmol/L glucose,30 mmol/L glucose,and 30 mmol/L mannitol,respectively,after intervention for 12,24,36,and 48 hours respectively.Cellular RNA was extracted to examine the effect of high glucose on the expression of IL-8 and IL-10 in HREC.5:HUCMSCs were added to the upper chamber of the Transwell system using a complex culture system,and 30 mmol/L glucose and HREC were added to the lower chamber;hUCMSCs were co-cultured with HREC at a ratio of 1:1.The experiment was divided into control group(complete culture medium),high glucose group and hUCMSCs co-cultured with HREC group.RT-PCR was used to detect the mRNA expression of IL-8 and IL-10 in HREC of high glucose group and co-culture group,respectively.Result:1:CCK-8 results show:At 36 hours and 48 hours,there was a statistically significant difference in the cell survival rate between the high glucose group and the high glucose hUCMSCs(P<0.05),and the difference was statistically significant between the high glucose group and the control group(P<0.05).<0.05).2:ELISA test results show:(1)Under high glucose stimulation,IL-8 levels in the supernatant of the high glucose group were significantly lower than those in the control group,and the IL-8 expression in the supernatant gradually increased with time;At the same time,at 12 h and 24 h,compared with the control group,IL-8 expression in the co-cultured normal group and co-cultured high-sugar group was decreased,and the difference was statistically significant(P=0.000,P<0.01);Under high glucose stimulation,the level of IL-8 in the culture supernatant of co-cultured high-glucose group was significantly lower in the higher glucose group(P=0.000).There was a positive correlation between the concentration of IL-8 in the supernatant of the four groups and the time.(2)Under high glucose stimulation,the IL-10 level in the supernatant of the high glucose group was significantly lower than that of the control group,and the IL-10 expression in the supernatant gradually increased with time;At the same time,at 12 h and 24 h,the expression of IL-10 in the high-glucose co-culture group was significantly lower than that in the normal co-culture group(P<0.05);the high glucose group was co-cultured with high glucose.The level of IL-10 in the culture supernatant was significantly higher in the glucose group,and the expression level of IL-10 was positively correlated with time.The difference between the two was statistically significant.(P=0.000)3: The PCR result shows:(1)The expression of IL-8 mRNA was significantly increased in high glucose-stimulated HREC,which was higher than that in the normal and mannitol groups(P<0.05).The expression of IL-8 mRNA in the high glucosegroup was significantly higher than that in the control group.Control group,mannitol group,co-cultured normal group,co-cultured high glucose group;Compared with high glucose group and control group,the expression of IL-8mRNA in the high-glucose group was significantly decreased,and the difference was statistically significant(P<0.05).(2)High glucose-stimulated HREC significantly decreased the expression of IL-10 mRNA,which was lower than that in the normal group and the mannitol group(P<0.05).At the 36 h and 48 h time points,the co-culture was higher than the high glucose group.The level of IL-10 mRNA in the glucose group was significantly higher(P<0.05).With the passage of time,the expression of IL-10 mRNA in the high glucose group gradually decreased.(P<0.05)Conclusion:1:hUCMSCs can increase the activity of human retinal vascular endothelial cells that are inhibited by high glucose.2:hUCMSCs can inhibit the expression of inflammatory factor IL-8 in human retinal vascular endothelial cells under high glucose,and increase the expression of anti-inflammatory cytokine IL-10. |
PDF Full Text Request