【作者】 王涛；

【导师】 官大威；

【作者基本信息】 中国医科大学 ， 法医学， 2010， 硕士

【摘要】 目的皮肤损伤愈合是一个复杂的过程,包括血液成分凝集、炎症反应、肉芽组织形成、新生上皮覆盖、结缔组织收缩和纤维化等过程。迷走神经递质乙酰胆碱可被非神经细胞(如角质形成细胞、内皮细胞、黑色素细胞等)合成并通过自分泌、旁分泌及内分泌等方式参与皮肤的许多基本功能,如增殖、分化、粘附和迁移,微循环,血管发生及许多免疫反应。实验表明,参与皮肤损伤愈合的中性粒细胞、单核巨噬细胞、成纤维细胞、角质形成细胞及内皮细胞等均发现非神经胆碱能系统或其组成成分。M3R (M3 subtypes of muscarinic receptor)是毒蕈碱乙酰胆碱受体的一个亚型,以往的研究表明M3R参与炎症反应和组织器官纤维化等。本研究应用免疫组织化学和Western blot技术,检测小鼠皮肤切创后损伤区及周边区各时间段M3R的表达情况,探讨M3R在皮肤损伤愈合中的作用及其在损伤时间推断上应用的可能性。实验材料与方法健康成年清洁级昆明小鼠50只,雌雄不限,体重35-40g,随机分为10组。其中9组为皮肤切创组,另一组为正常对照组,每组均为5只小鼠。乙醚气体吸入麻醉,背部剪毛处理后,常规手术消毒,在背部中央做一纵行长1.5cm皮肤全层切口,深达筋膜。分别于伤后Oh、6h、12h、1d、3d、5d、7d, 10d和14d将小鼠麻醉后脱颈椎处死,取伤口处1.5cm×1.0cm皮肤组织；正常对照组小鼠麻醉后背部剪毛,不做切创,观察14d,脱颈椎处死,取相同部位同等大小皮肤。以备HE染色、免疫组织化学染色和Western blot检测应用。应用免疫组织化学和Western blot技术检测50只小鼠皮肤切创后各时间段M3R变化情况。在显微镜×400倍下,于损伤区及损伤周边区随机选取10个视野,计细胞总数、阳性细胞数及阳性细胞率。应用Scion image软件检测各时间段的平均光密度值,测得数据用均数±标准差(x±s)表示。用SPSS13.0 for Windows进行单因素方差分析,以P<0.05为差异有统计学意义。结果正常对照组小鼠皮肤的表皮、毛囊、皮脂腺及皮肌层中均表达M3R。皮肤切创伤后6h,可见少数多形核粒细胞有M3R表达；伤后12-24h,大量的多形核粒细胞和部分单个核细胞M3R呈阳性；伤后1-3d随着时间的延长,M3R阳性细胞主要以单个核细胞为主；伤后5-14d, M3R主要表达在成纤维细胞中。伤后0～6h,M3R阳性细胞率较低,随着伤后时间的延长,M3R阳性细胞率逐渐增加,12h达到高峰,伤后1-5d一直呈高表达状态,伤后7d M3R阳性细胞率再次达到高峰,随后逐渐下降。各时间组与相邻上组的细胞总数及阳性细胞率差异均有统计学意义(P<0.05)。Western blot结果显示正常对照皮肤有M3R阳性条带,皮肤切创伤后各时间段组M3R表达强度有一定规律。伤后0-12h,阳性条带逐渐增强,1d,稍有下降,3d、5d,条带变化不明显,7d, M3R阳性条带再次达到高峰,伤后10d,M3R减弱,到伤后14d, M3R阳性条带基本与正常对照组条带情况一致。除Oh与正常对照组之间平均光密度值差异无统计学意义外(P<0.05),其余各组与其相邻上组之间平均光密度值差异均具有统计学意义(P<0.05)。结论1、正常对照组小鼠皮肤的表皮、毛囊、皮脂腺及皮肌层中均表达M3R。2、小鼠皮肤切创愈合过程中多形核粒细胞、单个核细胞及成纤维细胞均表达M3R。3、小鼠皮肤切创愈合过程中M3R表达的规律性变化可用于皮肤损伤时间的推断。更多还原

【Abstract】 ObjectiveSkin incised wound healing is a complex process, including clotting of blood components, inflammation, granulation tissue formation, re-epithelialization, connective tissue retraction and fibroplasia. The parasympathetic neurotransmitter acetylcholine is synthesised and secreted by non-neuronal cells (keratinocyte, endothelial cell, melanocyte et al) in an autocrine, paracrine and endocrine fashion which influenced a plethora of cutaneous basic functions, such as cell proliferation, differentiation, adhesion and migration, microcirculation, angiogenesis, as well as a great deal of immune responses. Experiments demonstrate the non-neuronal cholinergic system and its components expresse in neutrophil, mononuclear macrophage, fibroblast, keratinocyte and endothelial cell which participate in skin incised wound healing. Experiments show M3 subtypes of muscarinic receptor take part ininflammatory reactions and tissue and organ fibrous degeneration, In the present study, the level of M3R in incised skin wound was detected by immunohistochemical technique and Western blot. The investigation is aimed at exploring the possible role of M3R in skin wound healing and its applicability to the wound age estimation.Materials and MethodsA total of 50 adult, healthy mice of either sex, each weighting 35-40g, were divided into ten groups randomly, one control group and nine skin incision groups. A 1.5cm-long full-thickness incision deep to the fascia was made with a scalpel in the skin layer on the central dorsum of each mouse under sterile technique after the mouse was anesthetized by inhalating diethylether. After injury, each mouse was housed individually and fed with sterilized food and distilled water to prevent from infection. The 1.5cm×1.0cm specimens were taken from the wounded site after the mice were executed by cervical dislocation at Oh,6h,12h, 1d,3d,5d,7d, 10d and 14d post-injury to prepare for the subsequent procedures. The level of M3R in mouse skin incised wound was detected by immunohistochemical staining and Western blot, and the non-incised mouse skin was used as control. The number and ratio of M3R-positive cells were counted and analyzed by microscope. The band average optical value was analyzed by software of Scion image. The data were analyzed comparatively by the software of SPSS 13.0 for Windows.ResultsImmunoreactivity of M3R was detected in epidermis, hair follicle, sebiferous gland and dermomuscular layer in normal mouse skin. Expression of M3R was detectable in polymorphonuclear cells (PMNs) in the wound specimens aged 6h. In the wound specimens aged from 12h to 24h after injury, M3R-positive immunoreactivity was identified in a large number of infiltrating PMNs and part of mononuclear cells (MNCs). Afterwards from Id to 3d post-wounding, the M3R-positive cells were mainly found of MNCs and spindle-shaped fibroblastic cells (FBCs) and mainly in FBCs from 5d to 14d post-injury. Morphometrically, the ratios of the number of M3R-stained PMNs, MNCs and FBCs to total number of the cells in the wounds were evaluated and calculated. The ratio of the M3R-positive cells was low in the wounded specimens aged from Oh to 6h, and elevated in the wound specimens aged 12h and kept a highly level from Id to 5d post-injury, with a peak at 12h. Thereafter, the ratios decreased from 7d to 14d and minimized in the specimens aged 14d, with the second peak at the 7d. There were significant differences in ratios of M3R-positive cells between the neighboring time groups (P<0.05) except that between Oh post-injury and normal group.Conclusions 1. M3R is dectected in epidermis, hair follicle, sebaceous gland and dermomuscular layer of normal mouse skin.2. M3R is dectected in PMNs, MNCs and FBCs during the skin incised wound healing in mice.3. The time-dependent expression of M3R may be used as a marker for the wound age determination of the incised skin.更多还原