【作者】 徐鹏；

【导师】 姚敏；

【作者基本信息】 上海交通大学 ， 外科学（烧伤整形）， 2016， 博士

【摘要】 研究背景创伤愈合过程是一个完整、动态的过程,基本分为三个互相重叠的阶段:炎症反应、组织细胞增殖和重塑期。炎症反应期是创面修复最早发生并且最重要的阶段,决定着愈合过程能否顺利进入增殖和重塑期,并完成正常的愈合。目前已知多种免疫细胞参与了该阶段的修复,如中性粒细胞、巨噬细胞、淋巴细胞、肥大细胞等。中性粒细胞和巨噬细胞的重要作用已基本明确,然而淋巴细胞在创面修复中的作用研究较少。尽管目前相关研究已表明淋巴细胞的一个亚型γδT淋巴细胞在皮肤中存在定植,并在维持皮肤的稳态中发挥着重要作用,但其在创面修复中具体的作用及其机制仍未明确。多个研究表明γδT细胞一些皮肤炎性疾病或者免疫性疾病的皮肤改变相关,如银屑病、系统性红斑狼疮等。这提示我们γδT细胞可能参与了创面愈合过程,并在炎性反应中发挥重要的角色。另一方面,γδT细胞缺陷的小鼠皮肤表现出皮肤更加平坦,角蛋白细胞减少,皮肤表皮层变薄等改变,而肥胖的小鼠皮肤γδT细胞调节皮肤稳态功能减弱,并表现出与γδT细胞缺陷的小鼠皮肤相似的结构改变。因此我们推测糖尿病小鼠和患者的上皮层变薄是否与γδT细胞的数量或功能缺陷相关,这也进一步提示我们皮肤中γδT细胞的异常可能是糖尿病慢性创面的影响因素之一。本研究拟在验证γδT细胞在小鼠及人皮肤中存在定植的基础上,通过建立小鼠背部皮肤三度烧伤模型探讨γδT细胞在创面愈合中的动态变化,并进一步明确γδT细胞在糖尿病难愈创面形成中的作用。目的观察γδT细胞在创面愈合中的动态变化,探讨γδT细胞在糖尿病难愈创面形成中的作用。方法第一部分:选取小鼠背部皮肤,通过免疫组化技术验证γδT淋巴细胞在皮肤组织中的表达,应用激光扫描共聚焦显微镜(Laser scan microscope,LSM)在体动态观察CXCR6-GFP转基因小鼠(皮肤的γδT细胞表达GFP)皮肤中γδT淋巴细胞在皮肤组织中的空间分布和随时间的动态变化情况。同时,收集患者的正常皮肤组织,通过免疫组化染色观察γδT淋巴细胞在人皮肤组织中的表达情况。第二部分:建立小鼠背部皮肤三度烫伤模型,并通过创面组织HE染色验证模型。观察创面的愈合过程,应用免疫组织化学技术检测γδT细胞在创面愈合过程中的动态变化。同时,应用ELISA和Real-time PCR技术分别在蛋白和基因水平检测γδT细胞相关的细胞因子(IGF-1、FGF-7、FGF-10)在创面愈合过程中的表达变化。另一方面,收集临床患者的皮肤组织标本,并通过免疫组织化学技术检测γδTCR在创面组织中的表达。第三部分:建立糖尿病小鼠背部皮肤三度烧伤创面模型,观察创面的愈合时间。应用免疫组织化学检测创面组织中γδTCR的表达,同时应用ELISA和Real-time PCR技术检测γδT细胞相关的细胞因子的蛋白(IGF-1、FGF-7、FGF-10)和基因(igf-1、fgf-7、fgf-10、jaml)表达,并观察以上指标在创面愈合过程中表达的变化。而且,收集临床糖尿病和非糖尿病患者的慢性创面组织,通过免疫组织化学染色观察了γδTCR在糖尿病患者慢性创面组织中的表达变化。结果第一部分:小鼠皮肤中有γδT细胞的定植,表皮层的γδT细胞居多,形态类似DC,有很多树突,可在皮肤表面约33μm观察到,少量位于真皮浅层的(约58μm处)γδT细胞则没有树突,形态类似循环中的αβT细胞。通过动态观察发现真皮层中的γδT细胞在无任何刺激因素下即以较快的速度移动(约4μm/min),而表皮层的γδT细胞则始终保持静止状态。γδT细胞在人的皮肤组织的分布与小鼠γδT细胞在小鼠皮肤中的表达相似,γδTCR阳性的淋巴细胞主要存在于人皮肤组织的表皮层。然而,尽管γδTCR在人真皮层表达比在小鼠真皮层的表达增多。第二部分:小鼠烧伤皮肤表现及HE染色显示成功建立三度烧伤模型。免疫组织化学方法检测创面组织中γδTCR阳性的淋巴细胞表达变化,结果显示正常皮肤中阳性表达的细胞量较少,烧伤后3dγδTCR阳性的细胞数量开始增加,5d,7d以及10d表达水平明显增高,烧伤后14d阳性细胞数达到高峰,以后表达水平开始下降,直到烧伤后21dγδTCR阳性的细胞数量明显减少。小鼠烧伤创面组织中FGF-7、FGF-10和IGF-1的含量均在烧伤后3d开始增高,烧伤后表达5d达到高峰,7d有所下降,在烧伤后10d蛋白含量较前进一步降低,28d时蛋白含量恢复至0d的水平或较0d稍高水平。Real-time PCR检测小鼠创面组织中fgf-7、fgf-10、igf-1、jaml等基因的表达,结果与免疫组织结果一致,基因的表达在烧伤后3d开始增高,5d,7d,10d维持在高表达水平,烧伤后14d较前表达降低,烧伤后28d基本恢复至正常组织表达水平。临床患者的急性皮肤创面组织中γδT淋巴细胞数量明显多于正常的皮肤组织。第三部分:db/db小鼠烧伤创面的愈合时间较db/m小鼠明显延长(37.2±4.11 d vs 28.2±3.75 d)。免疫组织化学染色结果显示,在db/db小鼠烧伤创面的愈合过程中,γδTCR阳性的淋巴细胞同样表现为动态的改变,但与对照组db/m小鼠变化趋势不同,烧伤后1dγδT淋巴细胞较少,3d开始逐渐增多,烧伤后7d,10d,14dγδTCR阳性的淋巴细胞数目明显增多,并维持高表达水平,烧伤后21d仍无明显减少。并且,γδT淋巴细胞在db/db小鼠烧伤创面愈合3d,7d,10d,14d的表达均比在db/m小鼠减少。ELISA和Real-time PCR检测γδT淋巴细胞分泌的一些细胞因子的蛋白及基因的表达,结果均显示db/m小鼠烧伤后各因子表达在5d,7d达到高峰,10d开始下降,烧伤后28d表达明显下降至接近正常水平,而db/db小鼠烧伤后相关蛋白或基因的表达逐渐上升,10d,14d达到高峰,28d仍维持高水平表达。对临床患者创面组织中的γδTCR进行免疫组化染色,结果显示无论糖尿病和非糖尿病患者中,创面组织中γδTCR阳性的细胞数均高于其在正常组织中的表达,而在创面组织中的γδTCR阳性细胞数,糖尿病患者明显低于非糖尿病患者。结论1.γδT淋巴细胞在皮肤组织的表皮层和真皮层均有定植,但在人和小鼠皮肤中的分布不完全相同;2.γδT细胞参与了创面的愈合过程;3.γδT细胞与糖尿病难愈创面的形成相关。更多还原

【Abstract】 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.MethodPartⅠObserve 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.PartⅡEstablish 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.PartⅢ 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 PartⅡ.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.ResultsPartⅠThere are great deals of gamma-delta T cells located at mice skin,majority in epidermis(approximately 33μm subcutaneous)that are dendritic morphology and minority in dermis(approximately 58μm 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 4μm/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 ofγδTCR positive lymphocytes located at epidermis.PartⅡThe 3-degree burns mice model are confirmed through morphology observation and the paraffin hematoxylin-eosin staining techniques.The immunohistochemical detects that the expression of theγδTCR positive lymphocytes during wounds healing.The results shown thatγδTCR positive cells decreased on 1d post-wounding,and began to increase on 3d.The expressions ofγδTCR raised up to highest levels on 14d,and then decreased on 10d,the numbers of positive cells reduced significantly on 21d post-wounding.γδT 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 moreγδTCR positive cells than normal skin tissues.PartⅢ 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,γδT 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 ofγδT cells maintain in a high level,and never became less 21day post burns.Although,the total quantity of the variation trend ofγδT 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 theγδT cells in the patients’tissue,the results show that the numbers ofγδT 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.更多还原