曲尼司特对小鼠烧伤创面愈合的影响及给药时间的研究

发布时间：2018-06-05 09:38

本文选题：曲尼司特 + 瘢痕　；参考：《南方医科大学》2017年硕士论文

【摘要】：1背景增生性瘢痕是伤口异常愈合所导致,是烧伤和外伤后常见的并发症,常伴有疼痛、瘙痒以及关节活动受限。烧伤后瘢痕增生常导致患者性格孤僻、社交恐惧、甚至自尊受损,严重影响其日常生活质量。以往的研究证实,瘢痕增生是由于生物体内细胞因子分泌紊乱,成纤维细胞异常增生,Ⅰ型和Ⅲ型胶原不成比例的合成与降解造成的纤维化疾病。胎儿无瘢痕愈合实验验证,炎症反应是瘢痕形成的主要原因,且炎症反应程度的高低与肥大细胞(mastcells,MC)的含量呈正相关,当组织受到损伤时,MC立即被激活并发生局部聚集,参与机体的各种损伤修复、免疫等反应。因此降低MC含量或抑制其活性能调节伤口愈合过程中胶原的合成及Ⅰ、Ⅲ型胶原比例,从而有效抑制瘢痕的增生。曲尼司特(Tranilast,TNL)作为一种过敏介质阻释剂,是不同于H1、H2受体竞争类抗组胺药的新型抗变态反应药物,能有效预防和治疗一些纤维化疾病,其中包括增生性瘢痕,因此也被认为是一种新型的抗瘢痕治疗药物,这在一定程度上已被我们前期的研究所证实。但关于TNL最佳用药时间及TNL外用药作用研究甚少,因此本研究共设计两个实验,均以深Ⅱ度烧伤小鼠创面作为观测对象,实验一通过不同时间点开始给予口服TNL干预后,定期取创面的新生组织行病理学观察,以发现TNL抑制瘢痕增生的作用是否与药物开始干预的时间有关并找到最佳干预时间点;实验二用TNL滴眼液外滴创面,观察TNL外用能否同样有抑制瘢痕增生的作用,也为TNL临床用于治疗瘢痕类疾病提供一定的理论基础。2方法2.1实验动物选取、分组及烧伤模型建立实验一 7～8周龄清洁级雄性昆明小鼠66只,于小鼠背部制备深Ⅱ度烧伤创面模型,然后随机分为对照组(n=18)、早期干预组(n=18)、中期干预组(n=18)和晚期干预组(n=12);实验二 7～8周龄清洁级雄性昆明小鼠18只,于小鼠背部制备深Ⅱ度烧伤创面模型,然后随机分为空白对照组(n=6)、对照组(n=6)、实验组(n=6)。2.2给药方法实验一早期干预组、中期干预组、晚期干预组分别于烧伤创面形成后当天、7d、14d开始给予TNL200mg/(kg·d)灌胃,对照组于烧伤后当天开始每天给予等体积生理盐水灌胃;实验二空白对照组不做处理,对照组及实验组分别于烧伤创面形成后当天开始给予无菌生理盐水和TNL滴眼液(1ml注射器2-4滴,以覆盖创面为准)外滴创面。2.3取材定期观察创面愈合情况,实验一各组分别于创伤形成后14、28、42 d(晚期干预组于28、42d)随机选取6只小鼠取创面新生组织,实验二各组于烧伤创面形成后42天统一处死。2.4标本固定所取标本一式三份,一份甲醛固定,一份戊二醛固定,一份PBS缓冲液固定后置-80℃冰箱保存。2.5检测方法2.5.1石蜡包埋2.5.2切片2.5.3 HE 染色2.5.4甲苯胺蓝染色2.5.5 Masson 染色2.5.6抗体竞争法2.5.7 ELISA 法2.5.8透射电镜技术。3结果各组小鼠创面愈合时间比较差异无统计学意义(F=1.105,P=0.371)。口服TNL和外用TNL滴眼液均能使瘢痕组织中肥大细胞数量、总胶原含量、Ⅰ/Ⅱ型胶原比值、TGF-β1含量及组胺含量显著降低;且实验一中早期干预组各时间点肥大细胞数量、总胶原含量、Ⅰ/Ⅲ型胶原比值、TGF-β1含量及组胺含量均显著低于其它组(P0.05),与对照组相比,早、中、晚期干预组成纤维细胞的超微结构均发生明显改变。4结论TNL对小鼠烧伤创面愈合无明显影响;但口服TNL或外用TNL滴眼液干预后能明显降低烧伤后创面新生组织中MC的数量,组胺和TGF-β1的含量,抑制成纤维细胞胶原合成的能力及调节Ⅰ、Ⅲ型胶原合成的比例,从而抑制瘢痕的形成,且烧伤后即刻进行曲尼司特干预对瘢痕形成的抑制作用最为明显。
[Abstract]:1 background hypertrophic scar is caused by abnormal healing of the wound. It is a common complication after burn and trauma, often accompanied by pain, itching and limited joint activity. The hypertrophic scar after burn often causes the patient's character isolation, social fear and even the loss of self-esteem, which seriously affect the quality of daily life. Previous studies have confirmed that scar hyperplasia is due to Cytokine secretion disorders, fibroblast dysplasia, type I and type III collagen disproportionate synthesis and degradation of fibrotic diseases. Fetal scar free healing experiments verify that the inflammatory reaction is the main cause of scar formation, and the high degree of inflammatory reaction is positively related to the content of mastcells, MC. When the tissue is damaged, MC is immediately activated and locally aggregated to participate in various injury repair and immune responses. Therefore, reducing the content of MC or inhibiting its activity can regulate the synthesis of collagen and the proportion of type I and type III collagen during the wound healing process, thus effectively inhibiting the proliferation of the scar marks. Tranilast (TNL) is used as a kind of hypertrophy. Sensitive medium resistance release agent, a new antiallergic drug that is different from H1, H2 receptor antihistamine, can effectively prevent and treat some fibrotic diseases, including hypertrophic scars, so it is also considered as a new type of anti scar treatment drug, which has been confirmed by our previous research in a certain range. But on the most TNL There are few studies on the time of good drug use and the effect of TNL external use. Therefore, two experiments were designed in this study. All the wounds of deep second degree burn mice were taken as the observation objects. The experiment one was given the prognosis of oral TNL by oral administration at different time points. The pathological observation of the newborn tissues of the wound was taken regularly to find out whether the effect of TNL on the inhibition of scar hyperplasia was related to the drug. The time of the intervention was related and the best time for intervention was found. Experiment two with TNL eye drops was used to observe whether the external use of TNL could also inhibit scar hyperplasia. It also provided a theoretical basis for the clinical application of TNL in the treatment of cicatricial diseases by.2 method 2.1 experimental animal selection, group and burn model establishment experiment 7~8. 66 week old male Kunming mice were prepared for deep second degree burn wound model on the back of mice, and then randomly divided into control group (n=18), early intervention group (n=18), medium-term intervention group (n=18) and late intervention group (n=12), and 18 mice of two 7~8 weeks old male clean grade mice were tested on the back to prepare deep second degree burn wound model in the back of mice, and then the model of deep second degree burn wound was made in the back of mice. Then, the model of deep second degree burn wound was made in the back of mice. Randomly divided into the blank control group (n=6), the control group (n=6), the experimental group (n=6).2.2 administration method experiment one early intervention group, the middle intervention group, the late intervention group on the day after the burn wound formation, 7d, 14d began to give TNL200mg/ (kg. D) to the stomach, the control group began to give the same volume of normal saline day after the day after the burn; the experiment was two empty. The white control group did not do the treatment. The control group and the experimental group began to observe the wound healing by using the aseptic saline and TNL eyedrops (2-4 drops of 1ml syringe and covering the wound surface) on the same day after the formation of the burn wound. The experimental groups were respectively 14,28,42 d after the formation of the trauma (late intervention group in 28,42d). 6 mice were randomly selected to take the wound tissue of the wound. In the experiment two, 42 days after the formation of the burn wound, three parts of the.2.4 specimens were executed. One portion of formaldehyde was fixed, a glutaraldehyde was fixed, a PBS buffer was fixed after -80 centigrade and.2.5 was stored in the freezer, 2.5.1 paraffin embedded 2.5.2 section 2.5.3 HE and 2.5.4 a HE 2.5.5 Masson staining with aniline blue staining 2.5.6 antibody competition method 2.5.7 ELISA 2.5.8 transmission electron microscope technique.3 results there was no significant difference in the wound healing time of each group (F=1.105, P=0.371). The number of mast cells, the total collagen content in the scar tissue, the ratio of type I / II collagen, TGF- beta 1 and the ratio of type I / II collagen in the scar tissue were not statistically significant (F=1.105, P=0.371). Content and histamine content decreased significantly, and the number of mast cells, total collagen content, type I / III collagen ratio, TGF- beta 1 content and histamine content in the early intervention group were significantly lower than those of the other groups (P0.05). Compared with the control group, the ultrastructure of the early, middle and late intervened fiber cells significantly changed the.4 conclusion TNL There was no obvious effect on the healing of burn wounds in mice, but the number of MC, the content of histamine and TGF- beta 1, the ability to inhibit the synthesis of collagen in fibroblasts and the regulation of the synthesis of type I and type III collagen were significantly reduced after the oral TNL or external TNL eye drops. The inhibition of cicrst intervention on scar formation was most obvious.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R644

【参考文献】