| Objective To analyze the influence of local application of rhGM-CSF combine with insulin on the expression of TGF-β1,FGF-2 and CD34 in wound healing with II degree burn in diabetic rats and explore the possible mechanisms.Methods After 1 week of adaptive feeding,100 male SD rats were randomly divided into the normal control group(group A,n=15)and the diabetes model group(n=85).The latter was fasted for 12 h,and treated with intraperitoneal injection of a single dose of 1%STZ(55 mg/kg)to induce type I diabetic model.After the blood glucose of the diabetic model group was stabilized for 4 weeks,each group of rats was anesthetized with pentobarbital sodium,and their back was depilated.The next day after each group of rats was anesthetized,2 circular II degree deep burns which was about 2.5cm in diameter were prepared on the skin of their back,with a self-made scald device(keep injuries temperature of 100°C).According to the preliminary experiment,the burn time of normal rats was 10 s,and that of diabetic rats was 8s.60 diabetic rats with depth II scald were randomly selected from 69 successful models and divided into diabetes diabetic control group(group B),insulin group(group C),rhGM-CSF group(group D)and GM-CSF mixed insulin group(group E),with 15 in each group.The rats were treated with corresponding wound treatment once a day for 21 days.The wound area was measured in 3,7,15 and 21 days after burn to calculate the rate of wound healing.Morphology of skin tissues of wounds were observed by H-E staining and Masson’s special staining.The expression of TGF-β1、FGF-2 and CD34 in wound tissues was detected by immunohistochemistry method.Results(1)the calculation of wound healing rate showed that compared to other groups,group A and E had higher rates of wound healing,while group B had lower rates of wound healing with significant difference(P < 0.05)in 7,11,15 and 21 days after scald.At day 7 after injury,the wound healing rate of group A was higher than that of group E,while it was lower than that of group E at other time points,but the difference was not statistically significant(P > 0.05).At day 11 after injury,the wound healing rate of group D was higher than that of group C,while it was lower than that of group C at other time points,but the difference was not significant(P > 0.05).(2)HE staining and Masson staining microscopic observation showed that the infiltration of inflammatory cells in the wounds of group B rats was delayed and lasted for a long time,the formation of blood vessels and collagen was blocked,and the new epidermis was weak and crawled slowly toward the center of the wounds.Compared with group A rats,the wound healing time was prolonged and the wound healing rate was decreased.However,as time went by,fibroblasts and neovascularization increased in the wound tissue of rats in groups C and D,the density of new collagen increased,and the distribution was uniform and regular,the epithelial crawling speed increased,and the new epidermis thickened.The above histomorphological changes were especially significant in rats in group E.(3)immunohistochemical staining showed that the expressions of TGF-1 and FGF-2 in group A and E were significantly higher than those in other groups,with the lowest expression in group B(P < 0.05)at day 3,7,11,15,21 after injury,and the highest expression in each group at day 15.At day 3 after injury,the expressions of TGF-1 in group A was higher than that in group E,while it was lower than that in group E at other time points,but the difference was not statistically significant(P > 0.05).At day 3 after injury,the expressions of TGF-1 in group C was higher than that in group D,while it was lower than that in group D at other time points,but the difference between the two groups was not significant(P > 0.05).At day 7 and day 11 after injury,the expressions of FGF-2 in group E was higher than that in group A,while it was lower than that in group A at other time points,but the difference was not statistically significant(P > 0.05).At day 11 and day 15 after injury,the expressions of FGF-2 in group D was higher than that in group C,while it was lower than that in group C at other time points,but the difference between the two groups was not significant(P > 0.05).At day 3 after injury,there was no statistically significant difference in CD34 expression between groups A,C,D and E(P > 0.05).At other time points,CD34 expression in groups A and E was higher than that in other groups,with the lowest expression in group B(P < 0.05),and the highest expression in each group at day 21.At day 3 after injury,the expressions of CD34 in group A was higher than that in group E,while it was lower than that in group E at other time points,but the difference was not statistically significant(P > 0.05).At day 11 and day 21 after injury,the expressions of CD34 in group D was higher than that in group C,while it was lower than that in group C at other time points,but the difference between the two groups was not significant(P > 0.05).Conclusion Topical application of rhGM-CSF combine with insulin can promote angiogenesis,collagen deposition,epithelialization,and significantly accelerate wound healing with II degree deep scald and improve the quality of wound healing.The mechanism may be associated with upregulation of TGF-beta1,FGF-2 and CD34 expression. |
