表柔比星复合皮肤缺损建立大鼠氧化应激难愈性创面模型的研究

【作者】 王顺梅；

【导师】 郝钰；

【作者基本信息】 北京中医药大学 ， 中医学， 2012， 硕士

【摘要】 研究背景慢性难愈合创面(俗称溃疡)是由多种创伤和疾病所致,不能通过正常及时有序的修复过程达到解剖和功能上的完整状态的创面。临床上多指经1个月以上治疗未能愈合、也无愈合倾向的创面。主要包括创伤性溃疡、下肢静脉性溃疡、压迫性溃疡以及糖尿病溃疡等。它发生在体表,具有病因复杂、病程长、治疗难度大、并发症多、愈后极易复发以及少数尚有癌变可能等特点,对患者的生活和工作质量造成了极大的危害。目前西医对于慢性难愈性创面的一般处理方法为早期清创,控制感染,应用适当的创面敷料,处理手段单一。而一些特殊治疗方法如外源性生长因子应用、组织工程皮肤等临床应用不广泛,故西医在促进慢性创面愈合方面总体疗效不理想。中医采用分期辨证论治,全身整体内治结合局部外治,在促进慢性创面愈合确有疗效。目前大量研究集中在探讨中药在慢性创面愈合过程中的作用机制。研究创伤愈合机理与评价创伤修复药物的基础是建立符合临床发病过程和建立符合影响创面愈合主要因素的动物模型。慢性创伤修复模型应体现慢性难愈性创面发病机制的复杂性、多因素性及慢性创面本身存在感染与慢性炎症反应等特点。在研究复制动物模型时应抓住影响模型复制的一个或几个主要因素加以研究。研究表明氧化应激反应是导致创伤修复延迟的一个重要机制。氧化应激是氧化和抗氧化间的稳态失衡,自由基产生增多,和(或)机体或组织抗氧化能力下降的一种机体状态。创面微环境中的许多细胞都能不同程度地产生活性氧等自由基,适量的自由基对创面的愈合能起到有利的作用,过量的自由基将导致组织损害,不利于创面愈合。进一步研究氧化应激对创面愈合的影响机制,将为治疗慢性难愈合创面提供更广阔的思路和途径。目的通过采用皮肤缺损-表柔比星干预复合因素叠加法建立大鼠氧化应激难愈性创面模型。方法将大鼠分为正常组(无皮肤缺损)、单纯组(单纯皮肤缺损)、模型组,单纯组和模型组大鼠用特制打孔器在背部打一个直径为1.5cm的圆孔,形成大鼠皮肤全层缺损开放性创面,模型组在此基础上沿创面边缘皮内注射表柔比星(2mg/ml)0.15ml。观察大鼠创面一般情况,于第3、6、11、20天观察记录创面面积并计算创面愈合率。于第3、7、14、22天取材。将1/2创面组织于10%甲醛固定,石蜡包埋,切片,HE、Masson染色,观察创面组织学变化。1/2创面组织制成组织匀浆,并于大鼠腹主动脉取血,生化法检测各组大鼠皮肤匀浆和血清的SOD活力、MDA、H2O2水平。进行统计学分析,各组间进行比较。结果模型组和单纯皮肤缺损组比较,创面愈合时间延长,单纯组创面到第20天已基本愈合,而模型组创面到第28-30天才基本愈合。在相同时间内的模型组创面愈合率明显低于单纯组(P<0.01)。通过创面组织学观察,单纯组第3天创面可见许多炎性细胞,第7天可见成纤维细胞分布,真皮层可见新生毛细血管,第14天创面可见大量成纤维细胞,新生胶原数量多,到第22天可见新的角质层形成,新生胶原排列整齐。与单纯组相比,模型组创面从第3天到第22天炎性细胞持续浸润,成纤维细胞数量少,新生胶原数量少,排列紊乱。测定各组大鼠皮肤匀浆和血清的SOD、MDA、H2O2水平,在第3、7天,模型组和单纯组与正常组相比,SOD活力明显下降(P<0.01), MDA、H2O2水平显著升高(P<0.01)。模型组与单纯组比较,SOD活力更低,MDA、H2O2水平更高,均有统计学差异(P<0.05)。到第14天,单纯组SOD活力和MDA、H2O2水平都与正常组无明显差异(P>0.05),而模型组仍有显著差别(P<0.01),SOD活力低于正常组,MDA、H2O2水平高于正常组。结论采用皮肤缺损叠加表柔比星干预复合因素造模方法可成功复制慢性氧化损伤性创面模型。该模型创面愈合时间明显延长,愈合率下降,创面处于氧化损伤状态。可以应用其研究药物对创面的治疗作用和对创面氧化应激状态的影响。更多还原

【Abstract】 BackgroundChronic wounds (commonly known as ulcers) are caused by a variety of wounds and disease, can not through the normal timely and orderly repair process to complete anatomical and functional healing. In clinical, it refers to the wounds that has no healing or healing tendency by the treatment more than a month, including traumatic ulcers, lower extremity venous ulcers, pressure ulcers and diabetic ulcers. It occurs in the surface, has a complex etiology, duration, difficulty in treatment, more complications, vulnerability to relapse and cancerous possibility, it causes great harm on the quality of patients’ lives and work. Currently, the general treatment of western medicine for chronic refractory wounds is early debridement, infection control, application of appropriate wound dressings. It is a single processing means. Some special treatment, such as the clinical application of exogenous growth factors, tissue engineering skin is not extensively used in clinical, the overall effect in promoting chronic wound healing are not ideal. The method of TCM stage diagnosis and treatment, the body overall governance combined with local external treatment does have efficacy in promoting chronic wound healing. Large number of studies are expanded to explore mechanism of traditional Chinese medicine action in chronic wound healing process. The basis of the research of wound healing mechanism and evaluation of wound healing drugs is to establish animal models in line with the clinical disease process and the main factors of impacting wound healing. Chronic wound healing model should reflect the complexity of the pathogenesis of chronic dermal wounds, multiple factors, chronic wound infection and chronic inflammation. In the study for replication of animal model should seize one or several of the main factors affecting the replication of the model.The study showed that oxidative stress is an important mechanism for the delay wound healing. Oxidative stress is a state of decline in a body from oxidation and anti-oxidation homeostasis. It is caused by that free radicals increases, and (or) the body or tissue antioxidant capacity decreases. Many cells can generate oxygen free radicals in varying degrees in the wound microenvironment, the amount of free radicals can play a beneficial role in wound healing, excessive free radicals leading to tissue damage is not conducive to wound healing. Further study the mechanism of oxidative stress on wound healing, for the treatment of chronic wounds to provide a broader ideas and approaches.Objective Through the use of skin defects and Epirubicin intervention composited, a chronic wound model in oxidative stress state has been established.MethodsThe rats were divided into normal group (no skin defects), the simple group (skin defects), model group, simple group and model group rats were punched a diameter of1.5cm hole on the back playing with a special hole punch, forming a open wound of full-thickness skin defect. On this basis, model group rats were injected epirubicin (2mg/ml) of0.15ml in the wound edge skin. The changes were observed in rat wounds in general, calculated wound area and wound healing rate on the day3,6,11,20, drawn on the day3,7,14,22.1/2in wound tissue was performed with10%formalin-fixed, paraffin-embedded, sliced, with HE, Masson staining to observe the wound histological changes.1/2wound tissue is made to homogenates. Blooding in the abdominal aorta in rats to make serum. Biochemical methods used to measure the level of SOD, MDA, H2O2of skin homogenate and serum. For statistical analysis, comparison between the two groups.ResultModel group and the skin defect group, the wound healing time, to20days simple group wound has almost healed wounds of the model group to the first28-30days of basic healing. At the same time, the wound healing rate of model group was significantly lower than the skin defect group (P<0.01). Observation of the simple group wound tissue slice shows many inflammatory cells in3rd day, visible new capillaries in dermis and fibroblast distribution in7th day, a large number of fibroblasts and new collagen in wound tissue in14th day, the new cuticle formation and new collagen arranged in neat rows in22nd day. Compared with the simple group, model group wound sustained infiltration of inflammatory cells from3rd day to22nd day, as a small number of fibroblasts, a small number of new collagen, arranged in disorder. Determination of each group of rat skin homogenate and serum SOD, MDA,, H2O2, level, in the third and seventh day, the model group and the skin defect group compared with the normal group, SOD activity decreased significantly (P<0.01), MDA, the level of H2O2significantly increased (P<0.01). Model group shows the lower the activity of SOD, MDA, H2O2, a higher level, with significant differences (P<0.05) compared with skin defect group.14th day, the simple group SOD activity and MDA level of H2O2was compared with the normal group without significant difference (P>0.05), model group is still a significant difference (P<0.01), the activity of SOD is lower than the normal group of MDA, H2O2, higher than the normal group.ConclusionThe superposition method using the complex factors of skin defects interfere with epirubicin to establish a rat dermal wound model used in the experiment, the method can be successfully copied oxidative stress refractory wound model in rats. The model is applied to the study of Chinese medicine to promote wound healing mechanism through the oxidative damage.更多还原