肝素在动物实验外伤性鼓膜穿孔愈合中的作用

发布时间：2018-12-12 04:08

【摘要】： 研究背景：外伤性鼓膜穿孔是临床常见的疾病,常因直接或间接外力作用所致,虽大部分可自行愈合,但一旦感染,将成为永久性穿孔。鼓膜贴补法简单,安全,廉价,近年来贴补药物的选择一直是研究的热点。表皮生长因子(EGF)作为贴补药物治疗鼓膜穿孔的作用已被证实。在动物实验中,国内已有学者通过肉眼观察应用肝素作为贴补药物,可促进外伤性鼓膜穿孔的愈合。鼓膜穿孔愈合过程需要多种体内细胞因子的参与,如表皮生长因子(EGF)、转化生长因子-α(TGF-α)、碱性成纤维细胞生长因子(bFGF)、血小板衍生生长因子(PDGF)、角质形成细胞生长因子(KGF)等。研究发现在创伤愈合过程中上述生长因子都以与肝素结合的形式存在,不仅可提高生长因子生物学效应,而且可以保护其免于被各种理化因素灭活。肝素有利于创伤修复已被国内外学者所证实。肝素在鼓膜穿孔中对转化生长因子-α(TGF-α)、碱性成纤维细胞生长因子(bFGF)的影响至今尚未见报道。探讨在外伤性鼓膜穿孔愈合过程中肝素对生长因子TGF-α、bFGF的影响进而促进鼓膜穿孔的愈合是本研究的重点。研究目的：探讨局部应用肝素在外伤性鼓膜穿孔愈合中的作用。研究方法：①建立大鼠外伤性鼓膜穿孔模型：Wistar大鼠50只,所有动物先用戊巴比妥钠腹腔麻醉(0.3ml／100g),在电耳镜下用灼热针尖在大鼠双侧鼓膜后下象限制成约2mm×2mm紧张部的圆形穿孔。术后用氨苄青霉素(150mg／kg)肌肉注射预防感染,每天一次,连续5天。②实验方法：50只大鼠随机分为两组,第一组25只左耳外耳道置无菌干棉球；右耳肝素明胶海绵贴补。第二组25只左耳EGF明胶海绵贴补；右耳肝素明胶海绵贴补。分别于术后1天、3天、5天、7天和10天时,每组处死5只大鼠,解剖颞骨,剥离听泡外侧壁,取出完整鼓膜。对取得的鼓膜标本用10%甲醛固定,石蜡包埋,切片,常规HE染色,采用免疫组化两步法检测。免疫组化结果用配对样本(同体比较)的t检验,以P0.05为差异具有统计学意义。研究结果： 1.光镜下肝素组三天时上皮细胞增厚,细胞由扁平细胞变成多边形,细胞间距增加,核质比增大,上皮层与纤维层分离,上皮增生明显。纤维层也增厚,达5-6层,内层柱状上皮也增生。五天时鼓膜已基本愈合,且结构与正常鼓膜接近。 2.免疫组化图像分析,肝素组与空白组相比TGF-α、bFGF表达值差异有统计学意义,p0.05。肝素组与EGF组之间TGF-α、bFGF表达值差异无统计学意义,P0.05。研究结论： 1.肝素可提高TGF-α、bFGF在鼓膜穿孔愈合中的表达,并延长其表达时间。 2.局部应用肝素可缩短穿孔鼓膜的愈合时间,促进穿孔愈合。 3.肝素贴补法治疗鼓膜穿孔,愈合更接近正常鼓膜组织结构。
[Abstract]:Background: traumatic tympanic membrane perforation is a common clinical disease, often caused by direct or indirect external force. Tympanic membrane patch is simple, safe and cheap. The effect of epidermal growth factor (EGF) on tympanic membrane perforation has been confirmed. In animal experiments, heparin is used as a patching drug to promote the healing of traumatic tympanic membrane perforation. The healing process of tympanic membrane perforation requires a variety of cytokines, such as epidermal growth factor (EGF), transforming growth factor- 伪 (TGF- 伪), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), Keratinocyte growth factor (KGF). It was found that these growth factors existed in the form of binding with heparin during wound healing, which not only enhanced the biological effects of growth factors, but also protected them from being inactivated by various physical and chemical factors. Heparin is beneficial to wound repair and has been confirmed by domestic and foreign scholars. The effect of heparin on transforming growth factor- 伪 (TGF- 伪) and basic fibroblast growth factor (bFGF) during tympanic membrane perforation has not been reported. To investigate the effect of heparin on growth factor TGF- 伪, bFGF during the healing of traumatic tympanic membrane perforation, and to promote the healing of tympanic membrane perforation is the focus of this study. Objective: to investigate the effect of local application of heparin on the healing of traumatic tympanic membrane perforation. Methods: 1 the rat model of traumatic tympanic membrane perforation was established: 50 Wistar rats were treated with pentobarbital sodium intraperitoneal anesthesia (0.3ml/100g). A circular perforation of the 2mm 脳 2mm tension region was limited to the posterior inferior image of the bilateral tympanic membrane with a scorching needle under the ear mirror. After operation, ampicillin (150mg/kg) was injected intramuscularly to prevent infection, once a day for 5 days. Methods: 50 rats were randomly divided into two groups: the first group (25 rats) had sterile dry cotton balls in the external auditory canal of the left ear, and the right ear heparin gelatin sponge patch. Group 2: EGF gelatin sponge patch in left ear and heparin gelatin sponge patch in right ear. At 1 day, 3 days, 5 days, 7 days and 10 days after operation, 5 rats in each group were killed, temporal bone was dissected, lateral wall of auditory vesicle was removed and intact tympanic membrane was removed. The tympanic membrane samples were fixed with 10% formaldehyde, embedded in paraffin, sectioned and stained with conventional HE, and detected by immunohistochemical two-step method. Immunohistochemical results of paired samples (compared with the t test), with P0.05 as the difference was statistically significant. Results: 1. In the heparin group, the epithelial cells became thicker, the cells changed from flat cells to polygons, the cell spacing increased, the ratio of nucleus to cytoplasm increased, the epithelial layer separated from the fibrous layer, and the epithelium proliferated obviously. The fibrous layer also thickened to 5-6 layers and the inner columnar epithelium also proliferated. At 5 days, the tympanic membrane was basically healed and its structure was close to that of the normal tympanic membrane. 2. Immunohistochemical analysis showed that the expression of TGF- 伪 and bFGF in heparin group was significantly different from that in blank group (p 0.05). There was no significant difference in the expression of TGF- 伪 and bFGF between heparin group and EGF group (P 0.05). Conclusion: 1. Heparin increased the expression of TGF- 伪 and bFGF in the healing of tympanic membrane perforation and prolonged the expression time. 2. Local application of heparin can shorten the healing time of perforated tympanic membrane and promote perforation healing. 3. Heparin patch therapy for tympanic membrane perforation, healing closer to the normal tympanic tissue structure.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R764

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