The Empirical Study Of The Mechanism Of Recombinant Human Epidermal Growth Factor For External Use Early Affecting Wound Healing In Diabetes Mellitus Combined With Burn

Background In our country, the morbidity of diabetes mellitus increases unceasingly with society's population aging, and the occurrence of diabetes mellitus have the trend to become younger in average with the raising of the living standard with people and the adjustment of diet structure. According to the statistics, the adult morbidity of diabetes mellitus will rise to 3.2% in 1996 from less than 1% in 1979, and estimates that will reach 4.2% in 2015. It can be seen that the incidence rate of the combined with the burn will also display the rising trend gradually; And the diabetes mellitus itself being long-term will have a lot of complication, especially impaired wound healing; The hepar and vicinity organization all exist insulin resistance phenomenon after burn, which will restrain insulin secretion, strengthen glyconeogenesis, raising glucogenesis, then make these patient blood sugar increasing further and aggravates state of illness; With the function of every visceral organ decreased gradually and sugar tolerance cut down, the probability complicated stress diabetes after severe burn will also increase obviously in the aged people.Objective Aiming directly at positive of epidermal growth factor(EGF) to wound healing, epidermal growth factor receptor(EGFR) may adjust and mediate several aspects during the wound healing; By detecting the change of the expression of EGFR and their messenger ribonucleic acid(mRNA) and influence to other endogenous growth factor, angiopoiesis and apoptosis with the EGF for external application early and the injection insulin to control blood sugar during the stage of wound healing in diabetes mellitus combined with burn. To explore the possible mechanisms of EGF for external use early promoting wound healing and the ones of impaired wound healing.Methods Part I: Choose 120 Wistar rats and divide them into 4 groups randomly (group A , B , C , D). Diabetes was induced in the former 3 groups by a single intraperitoneal injection of freshly prepared solution of streptozotocin (STZ) dissolved in 0.1% cold citrate buffer (pH 4.5) at a concentration of 200 mg/kg body weight. Rats in the group D were injected with buffer alone. After 3 d, blood glucose levels (from tail vein) of rats in the former 3 groups were measured by ACCU-CHEK Active and check the model of diabetes. 8 weeks after establishing the models,we make the the model of deep partial thickness scalding at the same time, then check the condition of destroying skin and the secreting insulin by HE staining and immunohistochemistry staining to verificate the model further.Then we operated in different way. Then we determine the healing rate of the wound in different days; and fetche the full-thick skin tissue respectively, and make use of two kinds of methods of immunohistochemistry and Western blot to detect the expression and location of EGFR, meanwhile make use of mRNA hybridization in situ to detect the expression of EGFR mRNA. Part II: The former treatments are equal to ones of Part I. Others,we make use of immunohistochemistry to detect the expression and location of EGF, bFGF and utilize mRNA hybridization in situ to detect the expression of EGF mRNA and FGF mRNA; And we also make use of the method of immunohistochemistry to check the expression of CD34 and reflect the microvessel density (MVD);Finally, we make use of the method of TUNEL to check the apoptosis rate and their distribution in different phases.Then analysis the correlation respectively between EGFR and EGF,bFGF.Results Part I :One week after establishing the diabetic models, there were some typical symptoms of diabetes mellitus observed in diabetic rats. Meanwhile ,blood sugar in diabetic rats were obviously higher. Further,through detecting with pathological methods,we found a great quantity pancreatic islet destroyed, the secretion of insulin weakened obviously. And the deep partial thickness scald of rats also were testified by pathological methods. The wound healing rate of former 3 group A,B,C compared with normal group D, the rate of group A nearly reached normal level(P>0.05),ones of group B,C were significantly lower than group D(P<0.01);Immunohistochemistry and Western blot used to detect the levels of EGFR in the group B and C the expression of EGFR were delayed obviously;In situ hybridization for EGFR mRNA expression of wound skin tissue, showed EGFR mRNA expression in group A grew fast;then in group B and C they grew slowly;Part II: Immunohistochemical detection of EGF,bFGF expression, their expressions showed the positive expression in group A and D raised quickly and in group B and C they grew slowly and reach low level;In situ hybridization for EGF mRNA and bFGF mRNA expression of wound skin tissue showed coherent changes;Immunohistochemical detection of CD34 for reflecting the microvessel density (MVD)of the wound, the positive expression in group A showed a faster raising and in group B an C, the expression grew slowly.The in Situ Cell Death detection is used to detect and quantify apoptotic cell death at single cell level in skin wound tissues in TUNEL.With the wound healing going on, the apoptosis rate in group A was higher early,while the rate in group B and C descended slowly and were higher than that of group D in the different phases.Conclusions1.External application of rhEGF when controlling blood sugar can accelerateobviously the wound healing in diabetes mellitus combined with burn;2.After controlling blood sugar, external application of rhEGF and lastingstimulation can boost obviously the expression of other growth factors andmRNA;3. The variation of EGFR can really affect the endogenous growth factors andtheir mRNA,then coordinate them for promoting the wound healing;4.After controlling blood sugar, external application of rhEGF and lastingstimulation can accelerate angiopoiesis obviously, formation of granulationtissue, blocking wound, and descending apoptosis, in order to coordinatelypromote the wound healing;5. Impaired wound healing in diabetes mellitus combined with burn is possibledue to the lack of growth factor and the sluggish of growth factor receptorexpressing, lasting high blood sugar uncontrolled, capillary dyspoiesis,enhanced apoptosis and the imbalance between cell proliferation and apoptosis.