| ObjectiveFor the treatment of refractory diabetic wounds,the current treatment methods are various,and the commonly used methods include debridement,local wound medication and new dressings.Both insulin and bFGF have been used in the treatment of refractory wounds,and achieved good efficacy.The purpose of this study was to investigate the effect of the combined use of the two drugs on wound healing and whether the combined use of the two drugs can better promote wound healing.Methods1.150 mice were randomly selected for adaptive feeding for 7 days,followed by fasting and water for 24 hours.STZ solution diluted with 2% citric acid buffer was intraperitoneally injected at the calculated dose.With fasting blood glucose greater than 7.8mmol/L or random blood glucose greater than 16.7mmol/L combined with obvious polydipsia,polyphagia and polyuria symptoms as the modeling criteria.2.Mice were anesthetized with 10% chloral hydrate(20mg/Kg)after 1 week.Corneal trephine(1.0cm in diameter)was used to cut deep fascia in the middle of the spine.The full-layer skin of the wound was taken one day after the preparation of the wound,and HE staining sections confirmed the dermal-epidermal adhesion,the presence of microvesicles,collagen bundles and skin structures,and the infiltration of granulocytes.3.150 mice are divided into 5 groups according to random number table,blank control group(group A),respectively,recombinant basic fibroblast growth factor group(group B),insulin group(group C),human recombinant basic fibroblast growth factor + insulin shi fu group(group D),human recombinant basic fibroblast growth factor + insulin injection group(group E).4.The general condition of the wound was observed and photographed on the 2nd,4th,6th,8th and 12 th days after the intervention.Image Pro Plus6.0 Image analysis software was used to calculate the wound healing rate according to the following formula: wound healing rate =(initial wound area-unhealed wound area)/initial wound area x 100%.The histological changes were observed by HE staining.5.The OD value of bFGF in tissue at 2,4,6 and 12 days after insulin administration was determined by elisa6.Determination of glucose oxidase concentration and blood glucose value in the tissue: the glucose oxidase method was used to determine the content of GOD in the tissue,and the blood glucose concentration of the mice was detected with the blood glucose meter.7.All the data were statistically analyzed using SPSS17.0 software,and the experimental data were expressed as S.If the variance was homogenous,univariate analysis of variance wasused for comparison between groups,and SNK test was used for comparison between two groups.In order to determine whether there was interaction between two groups of drugs,factorial design analysis of variance was used for test.If the variance is not equal,the rank sum test is used.P <0.05 was considered to be statistically significant and different between the groups.Result1.On the second day after the establishment of the wound model,mice in each group were observed to have obvious edema in the soft tissue around the wound margin accompanied by exudation of pale yellow rarefied liquid,with the most obvious wound in group A,followed by group C and B,and the least severe wound in group D and E.On the 6th day,it was observed that the wounds of group D and group E were almost covered by pink granulation tissue,and the area of the wound was significantly reduced,while the amount of granulation tissue of group C and group B was less than that of group D and group E,and the extent of the wound reduction and the growth of the granulation were poor in group A.From day 8 to day 12,slight wound reduction was observed in mice in group A,while the wound reduction was significant in mice in group B/C/D/E compared with the initial wound.The most significant wound reduction was observed in group D,which reached about 80%.2.The wound healing rate of group A was(8.28 1.06)% on day 4,(28.62 1.41)% on day 6,(47.22 2.90)% on day 8,and(54.94 2.49)% on day 12.The wound healing rate of group B was(13.50 1.82)% on day 4,(31.56 1.61)% on day 6,(51.00 2.68)% on day 8,and(66.86 2.10)% on day 12.The wound healing rate of group C was(16.65 1.43)% on day 4,(33.52 2.57)% on day 6,(56.20 3.65)% on day 8,and(68.80 2.81)% on day 12.The wound healing rate of group D was(20.92 1.43)% on day 4,(45.40 5.90)% on day 6,(73.82 3.41)% on day 8,and(87.36 1.78)% on day 12.The wound healing rate of group E was(17.94 1.78)% on day 4,(37.84 2.75)% on day 6,(63.10 3.58)% on day 8,and(78.48 1.53)% on day 12.3.On the second day after injury,the epidermal structure of the wound surface of mice in the A/B/C group was destroyed,collagen fiber edema and degeneration,and A small amount of inflammatory cells infiltrated.In the D/E group,there were a large number of inflammatory cells,and a small number of inflammatory cells infiltrated into the deep dermis.Obvious new granulation tissue was observed on the wound surface of mice in each group.On the 4th day after the intervention,the number of infiltration of inflammatory cells on the wound surface was significantly increased in each group under the microscope,and significant fresh granulation tissue was observed in group D and group E,and the number of fibroblasts and new capillaries in group D was significantly higher than that in group E.However,only A small amount of new granulation tissue was found in group A,and only A small amount of granulation tissue and new capillaries were found in group B/C.The number of inflammatory cells in group D/E was extremely small,especially in group D,at 6-8 days after the intervention.In the group A/B,there were still more inflammatory cells in the field of view,but more fibroblasts and A small amount of collagen fibers appeared than 4 days ago.There were more granulation tissue and fibroblasts in group C.On the 12 th day,the wound surface of group D was basically epithelialized,with morphological characteristics of laminated epidermal cells and relatively homogenous arrangement of new collagen.Most of the wounds in group E were epithelialized,with a small number of inflammatory cells and a large amount of new collagen.The degree of epithelialization in group C was worse than that in group D/E,and there was still a small amount of new collagen.Similar to group B,group A has not yet formed epithelial tissue,and there are still A largenumber of inflammatory nuclear granulation tissues in the dermis,among which group A is more severe than group B.4.The OD value of bFGF in the tissues of mice was determined by enzyme-linked immunosorbent assay,and there was no statistically significant difference between the two groups(P > 0.05).On day 6,there was no significant difference in the OD value of bFGF in tissues between the groups by rank-sum test(homogeneity of variance test means variance is not homogeneous,use rank-sum test)(P > 0.05),but it was observed that the OD value of bFGF in tissues of the groups on day 6 was higher than that on day 6.After one-way anova test(homogeneity of variance test means homogeneity of variance),there was no statistically significant difference in the OD value of bFGF in tissues between the groups on day 12(P > 0.05),but it was observed that the OD value of bFGF in tissues of the groups on day 12 was slightly lower than that on day 6.5.By enzyme-linked immunosorbent assay was developed for the determination of the mice in the organization the OD value of insulin,insulin dosing day 2 mice tissues group E highest OD value(P < 0.05),and 4,6,8,12 tissue insulin OD values were higher than other groups(P < 0.05),the whole change trend rising slowly,4 days to spike,down slowly,after 12 days appeared slightly elevated.Group C and group D 2 days no statistical difference between tissue insulin OD value,but are lower than that of group E(P < 0.05),4 days group D insulin OD value of the lowest in the organization,group C slightly tall,the highest group E,statistically significant differences between groups(P < 0.05),the sixth day the tissue insulin OD value group D was higher than group C but lower than that of group E(P < 0.05),8 days there was no statistically significant difference between group C and group D(P > 0.05),but lower than that of group E(P < 0.05),minimum 12 days D group,group C higher than that of group D,the highest group E,The difference between the groups was statistically significant(P < 0.05).There was no significant change of insulin OD value in group C and group D.6.after injury 2,4,6,8,12 days,measuring blood sugar levels,groups of mice after statistical analysis found that: each time point of the mice blood sugar levels are much higher than normal,and conforms to the standard ChengMo Ⅰ diabetes mice.The difference between the groups showed that the blood glucose of group E was the lowest at day 2,4,6 and 12(P < 0.05),and there was no significant difference between group C and group D at day 8(P > 0.05),and the trend of the whole blood glucose was not obvious.The blood glucose of group A and group B was the highest,which was different from that of group C/D/E(P < 0.05),while there was no significant difference between the two groups(P > 0.05).The blood glucose of group C and group D was slightly lower than that of group A/B(P < 0.05),but higher than that of group E(P < 0.05),and there was no significant difference between the two groups(P > 0.05).Glucose oxidase protein concentration in tissues of each experimental group was measured by oxidase method to indirectly evaluate the glucose content in tissues.According to the statistical analysis,the glucose oxidase protein concentration in group E was the lowest at all sampling points(P < 0.05).Glucose oxidase protein concentration was the highest in group A/B,which was different from that in group C/D/E(P < 0.05),while there was no significant difference between groups(P > 0.05).The glucose oxidase protein concentration in group C and group D was lower than that in group A/B(P < 0.05),but higher than that in group E(P < 0.05),and there were significant differences between the two groups(P < 0.05).Conclusion1.The wounds in mice with diabetes typeⅠtopically human recombinant basic fibroblast growth factor shi fu joint insulin can obviously promote wound healing and the effect is better than the other group.2.External recombinant human basic fibroblast growth factor can effectively promote the proliferation and migration of epithelial cells,and accelerate the wound healing.3.Local wet compress insulin can reduce the glucose content in local tissue,reduce the adverse effect of high sugar state on wound surface,and promote wound healing. |
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