Title SPECIFIČNOSTI IMUNOSNOG ODGOVORA U PROCESU CIJELJENJA RANA U MODELU DIJABETESA U CD26 DEFICIJENTNIM MIŠEVIMA
Title (english) SPECIFICITIES OF THE IMMUNE RESPONSE DURING THE PROCESS OF WOUND HEALING IN A DIABETES MODEL IN CD26 DEFICIENT MICE
Author Alaa Sharbini
Mentor Lara Batičić Pučar (mentor)
Committee member Dijana Detel (predsjednik povjerenstva)
Committee member Ester Pernjak-Pugel (član povjerenstva)
Committee member Lara Batičić Pučar (član povjerenstva)
Granter University of Rijeka Faculty of Medicine (Department of Chemistry and Biochemistry) Rijeka
Defense date and country 2018-07-27, Croatia
Scientific / art field, discipline and subdiscipline BIOMEDICINE AND HEALTHCARE Basic Medical Sciences
Abstract Uvod: Dipeptidil-peptidaza IV (DPP IV/CD26) multifunkcionalan je protein s značajnom proteolitičkom i kostimulacijskom ulogom čime utječe i na proliferaciju, angiogenezu, adheziju, migraciju i apoptozu stanica u procesu cijeljenja rana. Modulacijom biološke aktivnosti inkretina sudjeluje i u regulaciji koncentracije glukoze u krvi, međutim, patofiziološki procesi cijeljenja rana u dijabetesu nisu dovoljno razjašnjeni. Pretpostavka ovog istraživanja je da DPP IV/CD26 ima važnu ulogu u modulaciji imunosnog odgovora u procesu cijeljenja rana u hiperglikemiji.
Cilj istraživanja bio je ispitati utječe li nedostatak DPP IV/CD26 na makroskopske i/ili mikroskopske promjene tijekom procesa cijeljenja rana kože u dijabetesu, istražiti izražaj makrofaga i limfocita T kod CD26 deficijentnih te divljeg tipa životinja s induciranim dijabetesom te ispitati promjene u aktivnosti serumske DPP IV/CD26 tijekom uspostave eksperimentalne hiperglikemije i tijekom procesa cijeljenja rana kože u C57BL/6 životinjama.
Materijal i metode: CD26 deficijentnim (CD26-/-) te divljem tipu miševa (C57BL/6) induciran je model dijabetesa intraperitonealnom aplikacijom otopine streptozotocina u citratnom puferu u dozi od 50 mg/kg tijekom pet dana. Miševima s potvrđenim dijabetesom je potom na interskapularnom dijelu leđa učinjeno šest rana promjera 5 mm te su pojedine skupine pokusnih životinja žrtvovane drugog, četvrtog, sedmog, desetog te petnaestog dana. Primjenom različitih metoda analize makroskopskih i mikroskopskih uzoraka (patohistološkim, imunohistokemijskim, histomorfometrijskim i spektrofotometrijskim metodama) pratio se stupanj regeneracije pojedinih slojeva kože, stupanj proliferacije stanica bazalnog sloja epidermisa i fibroblastadermisa, izražaj limfocita T i makrofaga u vezivu koriuma oba soja ispitivanih životinja, te enzimska aktivnost serumske DPP IV/CD26 kod CD57BL/6soja tijekom procesa cijeljenja rana.
Rezultati dobiveni ovim istraživanjem ukazuju na relativno uspješniji proces cijeljenja rana kože u uvjetima nedostatka DPP IV/CD26 i razvijenog dijabetesa. Stupanj regeneracije koriuma kod CD26-/- miševa statistički značajno je veći (p<0,05) u odnosu na C57BL/6 miševe kao i broj Ki67 pozitivnih stanica, što upućuje na pojačanu proliferaciju stanica veziva uz obnovu ekstracelularnog matriksa u uvjetima nedostatka CD26. Izražaj limfocita T statistički je značajno veći (p<0,05) kod divljeg tipa životinja što ukazuje da je upalna faza manje izražena kod CD26-/- životinja. Porast izražaja makrofaga bio je brži i intenzivniji u uvjetima nedostatka CD26. Aktivnost serumske DPP IV/CD26 kodC57BL/6 miševa u uvjetima hiperglikemije je statistički značajno veća (p<0,05) u usporedbi s fiziološkim uvjetima dok je tijekom procesa cijeljenja rana značajno snižena drugog i četvrtog dana od indukcije rana.
Zaključak: Dobiveni rezultati potvrđuju pretpostavku kako molekula DPP IV/CD26 utječe na intenzitet i dinamiku cijeljenja rana kao i specifičnost imunosnog odgovora u uvjetima eksperimentalne hiperglikemije. Pokazano je da je kod CD26-/- miševa upalni odgovor manje izražen nego kod divljeg tipa miševa. Proces regeneracije i reparacije tkiva u uvjetima hiperglikemije uspješniji je kod nedostatka DPP IV/CD26 što potvrđuje važnost inhibitora ove molekule u terapijske svrhe kod oboljelih od dijabetesa.
Abstract (english) Introduction: Dipeptidyl peptidase IV (DPP IV/CD26) is a multifunctional protein with a significant proteolytic and costimulatory role, thus affecting the process of proliferation, angiogenesis, adhesion, migration and apoptosis of cells in the wound healing process. It is also involved in the regulation of blood glucose concentrations by modulating the biological activity of incretins. However, the pathophysiological processes of wound healing in diabetes are not sufficiently clarified. The hypothesis of this study is that DPP IV/CD26 plays an important role in modulating the immune response in the wound healing process in conditions of hyperglycemia.
The aim of this study was to determine whether DPP IV/CD26 deficiency affects macroscopic and/or microscopic changes during cutaneous wound healing process in diabetes. We aimed to determine the expression of macrophages and lymphocytes T in CD26 deficient and wild-type animals with induced diabetes and to investigate changes in serum DPP IV/CD26 activity during the development of experimental hyperglycemia and during the wound healing process in C57BL/6 animals.
Materials and Methods: A model of diabetes was induced in CD26 deficient (CD26-/-) and wild type (C57BL/6) mice by intraperitoneal administration of a streptozotocin solution in citrate buffer at a dose of 50 mg/kg for five days. After the induction of diabetes, six experimental wounds (5 mm in diameter) were induced on the interscapular dorsal part of animals. Groups of experimental animals were sacrificed the second, fourth, seventh, tenth and fifteenth day of wound healing. Different methods of analysis were used for macroscopic and microscopic examinations (pathohistological, imunohistochemical, histomorphometrical and spectrophotometrical methods). The degree of regeneration of different skin layers, the degree of proliferation of the basal layer of epidermis and fibroblasts of the dermis, and the expression of lymphocytes T and macrophages in wound tissues of both mice strains were determined. Likewise, serum DPP IV/CD26 activity during the wound healing process in C57BL/6 mice was analyzed.
The results of this study indicate a relatively more successful skin wound healing process under conditions of DPP IV/CD26 deficiency in diabetes. The rate of tissue regeneration in CD26-/- mice was statistically significantly higher (p<0.05) than in C57BL/6 mice as well as the number of Ki67 positive cells, indicating an increase drate of cell proliferation and regeneration of extracellular matrix in conditions of CD26 deficiency. The number of lymphocytes T is statistically significantly higher (p<0.05) in wild type mice indicating that the inflammatory phase is less pronounced in CD26-/-animals. The increase in macrophage expression was faster and more intense in the absence of DPP IV/CD26. The activity of serum DPP IV / CD26 in C57BL/6 diabetic mice was statistically significantly higher (p<0.05) compared to physiological conditions while it was significantly decreased the second and fourthday of wound healing.
Conclusion: Obtained results confirm the hypothesis that DPP IV/CD26 influences the intensity and dynamics of wound healing as well as the specificity of the immune response in experimental hyperglycemic conditions. It has been shown that CD26-/-mice show a less pronounced inflammation response than wild type mice. The process of tissue regeneration and reparation in hyperglycemia is more successful in conditions of DPP IV/CD26 deficiency, which confirms the importance of inhibitors of this molecule for therapeutic purposes in patients with diabetes.
Keywords
dijabetes
dipeptidil-peptidaza IV (DPP IV/CD26)
cijeljenje rana
limfociti T
makrofazi
proliferacija stanica
Keywords (english)
Diabetes
Dipeptidyl peptidase IV (DPP IV/CD26)
Cell proliferation
Lymphocytes T
Macrophages
Wound healing
Language croatian
URN:NBN urn:nbn:hr:184:927934
Study programme Title: Study of Sanitary Engineering Study programme type: university Study level: graduate Academic / professional title: magistar/magistra sanitarnog inženjerstva (magistar/magistra sanitarnog inženjerstva)
Type of resource Text
File origin Born digital
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Created on 2018-12-06 17:10:48