The Role And Mechanism Of CD26 In Dermal Fibroblasts And Cutaneous Wound Repair

Background:Skin wounds caused by various acute and chronic injuries,such as burns,mechanical injuries,surgical operations,chronic ulcers are common clinically and it imposes a heavy burden on families of patients and socioeconomic development.The complicated process of skin wound repair in which dermal fibroblasts play an important role is regulated by many factors,such as keratinocytes,fibroblasts,vascular endothelial cells,inflammatory cells,extracellular matrix,cytokines and so on.These factors are highly coordinated and mutually regulated.Dermal fibroblasts are activated by skin trauma.Their subsequent biological behaviors,such as proliferation,migration,differentiation,synthesis of extracellular matrix,secreting a variety of cytokines are the basis of wound repair.Therefore,any endogenous or exogenous factors that alter the biological properties and functions of dermal fibroblasts may affect cutaneous wound repair process.Studies have confirmed that dermal fibroblasts are heterogeneous.There are at least two groups of fibroblasts with unique phenotypes and functions in the skin.CD26 can not only be a new marker to differentiate fibroblast subgroups,but also play an important part in regulating the biological behavior of dermal fibroblasts and affecting cutaneous wound repair process.Objective:To elucidate the effect of CD26 on the biological behavior of dermal fibroblasts and the specific molecular mechanism involved.To further clarify the role of CD26 in cutaneous wound repair and dermal regeneration.To provide a new clue for improving and perfecting the molecular mechanism of wound repair and lay a theoretical foundation for exploring new drug targets and strategies to accelerate wound repair.Methods:We constructed primary cultured human dermal fibroblast models with CD26 gene low-expressed and over-expressed by means of recombinant adenovirus technique.The infection efficiency of adenovirus was detected by qRT-PCR and Western blot.Cell function test and flow cytometry were used to assess the effects of CD26 gene on the proliferative capacity,clonogenicity,migration,adhesion,synthesis of extracellular matrix,apoptosis and cell cycle distribution of dermal fibroblasts.Agilent gene chip was also used to predict and verify the key signaling molecules and the corresponding signaling pathways via differential gene screening,GO analysis,pathway analysis and cluster analysis of the CD26 low-expressed fibroblasts.We randomly divided the healthy,male,4-5 week old,SPF grade BALB/C nude mice into experimental group and control group.The anti-shrinkage full-thickness skindefects were made on the back of nude mice and 5×108 plaque-forming unit(PFU)of purified adenovirus was injected evenly at 8 points around the wound after mixing with 150?l normal saline.20?l was injected at each point and the rest 40?l was sprayed on the wounds.On the 0,3,7,11,15d after surgery,the wounds were photographed and two mice were sacrificed in each group.The whole wound and the adjacent normal skin were cut for histopathology,immunohistochemistry,immunofluorescence and molecular biological detection to evaluate the wound repair process and dermal regeneration.Results:1.We successfully constructed primary cultured human dermal fibroblast models with CD26 gene low-expressed and over-expressed by means of recombinant adenovirus technique.2.The capabilities of proliferation,clone formation,migration,adhesion,synthesis and secretion of extracellular matrix were weakened in CD26 low-expressed fibroblasts.These abilities of fibroblasts were enhanced after CD26 overexpression.3.Low expression of CD26 could induce apoptosis in fibroblasts and over expression of CD26 could inhibit the apoptosis process,especially early apoptosis.4.Low expression of CD26 could induce G0/G1 cell cycle arrest in fibroblasts.While the proportion of G0/G1 stage decreased and S+G2/M stage increased in CD26 overexpressed fibroblasts,which means more cells going through DNA replication and mitosis.5.CD26 participates in regulating the cell cycle distribution and proliferation of dermal fibroblasts through the PI3K/AKT mediated p21/p27/Cyclin D1 signal transduction pathway.CD26 regulates the migration ability of dermal fibroblasts through the PI3K/AKT mediated mTOR/p70S6K/4EBP1/MMP-9 signal transduction pathway.CD26 regulates the apoptosis process of dermal fibroblasts through the PI3K/AKT mediated Bad/Bcl-2/Caspase-3/PARP signal transduction pathway.6.The intradermal injected adenovirus could effectively infect nude mice wound tissues and significantly interfere or over-express CD26 expression level in the surrounding tissues of wounds.7.The time of cutaneous wound healing,the process of epithelialization and the extracellular matrix deposition were all obviously accelerated in the CD26 over-expressed nude mice.Conclusions:1.CD26 can regulate various biological behaviors and functions of dermal fibroblasts,which may further affect the process and outcome of cutaneous wound repair.2.PI3K/AKT mediated downstream signal transduction pathway may be a potential mechanism for CD26.The proliferation and cell cycle of fibroblasts are regulated by p21/p27/Cyclin D1 signal transduction.The migration of fibroblasts is influenced by mTOR/p70S6K/4EBP1/MMP-9 signal transduction.The apoptosis of fibroblasts is mediated by Bad/Bcl-2/Caspase-3/PARP signal transduction.