| BackgroundWound healing is a highly dynamic process which involves three overlapping stages includinginflammation,proliferation,and remodeling period.The inflammation period is the earliest and the most crucial procedure which determines whether the healing process can smoothly switch to the subsequent proliferation and remodeling phases,as well as ultimate healing of the wounds.As we all known,variety of immune cells such as neutrophils,macrophages,lymphocytes and mast cells etc are involved in this procedure.Although the important role of neutrophils and macrophages has been largely defined,the particular function of lymphocytes during wound-repair process was less promulgated.A growing body of related researches has shown that gamma-delta T cells,which is a subtype of T lymphocytes,locates in skin and plays an important role in maintaining the homeostasis of the skin.However,its'specific function and the underlying mechanism in wound repair are still not so clear.Several studies have revealed that gamma-delta T cells are associated with changes in such skin inflammatory or immune disorders as psoriasis,systemic lupus erythematosus and so on,which suggested that gamma-delta T cells may be involved in the process of wound healing,and play an important role in the inflammation phase.In addition,mice with gamma-delta T cell-deficient exhibit symptoms of smoother skin,keratinocytes decrease,and the thinning of skin layer.Meanwhile,the obese mice showed diminished ability of gamma-delta T cells to regulate skin homeostasis and similar structural changes in the skin of mice with gamma-delta T cell-deficient.Therefore,we speculated that epithelial layer thinning in diabetic mice or patients is related with quantity discrepancy and/or functional defect of gamma-delta T cells.It is also suggested that the abnormal gamma-delta T cells in the skin may be one of the main factors that affect the chronic wound healing of diabetes mellitus.In this study,based on the presence of gamma-delta T cells in mice and human skin,we intend to define the dynamic changes of gamma-delta T cells in wound healing by establishing a deep second-degree burn model in dorsal skin of mice,and to further clarify the role of gamma-delta T cells in the formation of refractory diabetic wounds.ObjectiveTo observe the dynamics variation process of gamma-delta T cells in wound healing.To explore the functional role of gamma-delta T cells in the refractory diabetic wound formation.MethodPart?Observe the expression of gamma-delta T cells in the epithelial tissue at the dorsal skin of mice by immunohistochemical techniques.Study the location and the dynamic motion in the epithelial tissue through the Laser Scan Microscope(LSM)at CXCR6-GFP transgenic mice(epithelial gamma-delta T cells express GFP)in vivo.In addition,observe the expression of gamma-delta T cells in the normal epithelial tissue of patients by immunohistochemical techniques.Part?Establish the 3-degree burns model at the dorsal skin in mice,cut off the wound tissueandconfirmthedepththroughtheparaffinhematoxylin-eosin staining techniques.Observe the healing procedure and the dynamic variation of gamma-delta T cells in the wound by immunohistochemical techniques.Meanwhile,detect the expression of the cytokines which were secreted by gamma-delta T cells(including IGF-1?FGF-7?FGF-10)and the gene(igf-1?fgf-7?fgf-10?jaml)in the healing procedure by ELISA and Real-time PCR technology.Besides,observe the expression of gamma-delta T cells in the injury epithelial tissue of patients by immunohistochemical techniques.Part? Establish the 3-degree burns model at the dorsal skin in diabetic mice,observe the healing procedure and the dynamic variation of gamma-delta T cells in the wound like in Part?.Detect the expression of the cytokines at gene and protein level.In addition,observe the expression of gamma-delta T cells in the refractory epithelial tissue of diabetic patients and in the injury epithelial tissue of non-diabetic patients by immunohistochemical techniques.ResultsPart?There are great deals of gamma-delta T cells located at mice skin,majority in epidermis(approximately 33?m subcutaneous)that are dendritic morphology and minority in dermis(approximately 58?m subcutaneous)that are not dendritic morphology just like alpha-beta T cells in circulation system.The dynamic observation show that the gamma-delta T cells move rapidly(at about 4?m/min speed),in the dermal layer and the gamma-delta T cells remain stationary in the epidermis.The location of the gamma-delta T cells in the epithelial tissue is similar in human beings to in murine.Although the expression of gamma-delta T cells in the dermal layer in human beings increased,the majority of??TCR positive lymphocytes located at epidermis.Part?The 3-degree burns mice model are confirmed through morphology observation and the paraffin hematoxylin-eosin staining techniques.The immunohistochemical detects that the expression of the??TCR positive lymphocytes during wounds healing.The results shown that??TCR positive cells decreased on 1d post-wounding,and began to increase on 3d.The expressions of??TCR raised up to highest levels on 14d,and then decreased on 10d,the numbers of positive cells reduced significantly on 21d post-wounding.??T cells related cytokines(FGF-7,FGF-10,IGF-1 and JAML)were detected at proteins and genes levels through ELISA and Real-time PCR,respectively.Consistent to results of IHC,the expressions of both proteins and genes were increased on 3d post-wounding,and up to the highest levels on 5d or 7d,and then decreased gradually,the levels rescued to close to the normal on 28d.On the other hand,the acute wounds from patients shown more??TCR positive cells than normal skin tissues.Part? The wound healing time post burn was significantly longer in the db~+/db~+mice model than in the db/m mice model(37.2±4.11 VS 28.2±3.75).The results of observing the paraffin through immunohistochemical staining techniques also show the dynamic changes that in the trend of the gamma-delta TCR positive lymphocytes variations in wound healing process after burns in the db~+/db~+mice model compared with which in the db/m mice model,??T lymphocytes became less 1day post burn,but began to increase 3day later,and the quantity became more and more 7day?10day?14day post burns gradually.The quantities of??T cells maintain in a high level,and never became less 21day post burns.Although,the total quantity of the variation trend of??T lymphocytes in the db~+/db~+mice model is much less than in the db/m mice model.In addition,detect the secretion of some cytokines and the expression of the gene by the method of ELISA and RT-PCR techniques,the expression of some cytokines increased post burns,and peaked 7day post burns,decline 10day post burns,the level of the cytokines expressing became near to the normal levels 28day post burns in the db/m mice model,the same as the results of the expressing of the genes.But in the db~+/db~+mice model,the secretion of some cytokines and expression of genes increased post burns,and peaked at 14day post burns gradually,and the expressing still maintain a high level at 28day post burns.Observe the??T cells in the patients'tissue,the results show that the numbers of??T cells in wound tissues is more than that in normal tissues,moreover,the numbers of the diabetic patients is less than of the non-diabetic patients in wound tissues.Conclusion1.Gamma-delta T cells located both in the epidermis and dermis,but the location are very different between in the murine and in the human beings;2.Gamma-delta T cells are involved in the process of wound healing;3.Gamma-delta T cells are related with the process of the refractory diabetic wounds. |
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