色甘酸钠影响大鼠增生性瘢痕形成的实验性研究

发布时间：2018-02-14 21:34

本文关键词： 增生性瘢痕色甘酸钠肥大细胞类胰蛋白酶　出处：《西南医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:本实验拟建立大鼠深度创面模型,使用肥大细胞膜稳定剂色甘酸钠干预创面愈合过程中肥大细胞的作用,通过观察愈合后瘢痕增生情况并检测肥大细胞数量、类胰蛋白酶等相关指标,探究以肥大细胞作为瘢痕预防和治疗靶点的可行性。方法:选取40只雄性SD大鼠,随机将其等分为A、B、C、D四组,每组10只,A、B、C组为实验组,其中A组为大剂量色甘酸钠组(40mg/kg),B组为中剂量色甘酸钠组(20mg/kg),C组为小剂量色甘酸钠组(10mg/kg),D组为阴性对照组。使用脱毛剂对所有实验对象进行背部脱毛后,麻醉条件下于背部脊柱两侧用手术刀形成两个对称的面积约15mm*10mm*2mm的切割伤深度创面。创面以2%稀释碘伏溶液消毒后予以暴露,每日观察大鼠背部创面情况(是否有渗血和异常渗液)。分别于术前30min;术后第24、第48、第72小时,对A、B、C组所有实验对象腹腔注射对应剂量色甘酸钠溶液,D组注射相等体积生理盐水。记录创面初始面积,定期观察创面面积大小,并在术后第3d取背部左侧创面组织,对组织进行固定脱水及常规HE染色,行甲苯胺蓝染色(肥大细胞计数)、免疫组化染色(类胰蛋白酶)等处理。记录全部实验对象创面愈合时间,组间取其均数进行比较。术后第42d处死所有实验对象,并收集背部右侧瘢痕组织,对其进行常规HE染色。肥大细胞计数:组织切片后使用甲苯胺蓝染色法,用Image-pro6.0图像分析软件系统,在双盲高倍镜下(×400)连续计数单位面积(个/mm2),每张随机挑选5个视野,计数其中肥大细胞的总数后,取其平均值作为该实验对象最终数据。并对脱颗粒和未脱颗粒进行定义。脱颗粒后肥大细胞胞膜不完整,边界模糊,且周围可见散在颗粒物质分布。而完整肥大细胞形态相对完整规则,与周边组织交界分明。类胰蛋白酶分析方法:免疫组化染色后,用image-pro6.0图像分析软件系统,双盲高倍镜下(×400)连续计数单位面积(个/mm2),同样每张切片随机选取5个视野,记录其中胰蛋白酶阳性肥大细胞数,并取其平均值作为最后结果。结果:1、创面初始面积:通过方差分析进行组间比较后再两两比较均可得p0.05,提示所有实验对象创面初始面积无统计学差异。2、创面愈合时间:通过方差分析进行组间比较后再两两比较均可得p0.05,提示所有实验对象创面愈合时间无统计学差异。3、瘢痕面积及硬度:术后第42天,见所有实验对象背部创面均出现瘢痕愈合,颜色呈淡红或苍白色,组间比较瘢痕面积大小关系提示:c组d组b组a组。d组瘢痕组织较硬;a、b组两组瘢痕质韧,组间无明显差异;而c组位于两者之间。4、he染色后镜下观察,术后第3天:a、b两组创面情况类似,可见炎性细胞、成纤维细胞散在分布,但新生毛细血管数量不多;d组镜下见细胞结构混乱,炎性细胞及成纤维细胞数量多,分布凌乱,且见丰富毛细血管。c组炎性细胞及成纤维细胞不及d组丰富,但远多于a、b两组。术后第42天:d组瘢痕组织内大量成纤维细胞、细胞外基质和毛细血管分布,部分呈环形或螺旋样分布,炎性细胞数量不多,纤维组织散在分布,无明显条理性;c组瘢痕组织颜色较d组浅,镜下可见成纤维细胞明显少于d组,排列尚规则,胶原纤维排列较d组整齐,细胞外基质不多,散在分布少量毛细血管;而a组、b组瘢痕组织增生不明显,镜下见成纤维细胞排列较紧密、有序,纤维组织排列整齐有序。5、肥大细胞计数:切片行甲苯胺蓝染色法后,在显微镜下进行计数,发现各组间肥大细胞总数差异无统计学意义,但a、b两组脱颗粒细胞数明显少于D组,C组位于两者之间,相应脱颗粒肥大细胞数/肥大细胞总数比例:D组C组A组≈B组。6、类胰蛋白酶计数:术后第3天,接受到刺激信号后的肥大细胞开始表达类胰蛋白酶,D组类胰蛋白酶阳性肥大细胞数量明显少于A组、B组,C组数量位于两者之间。类胰蛋白酶阳性肥大细胞数:B组A组C组D组。结论:1、色甘酸钠可以显著抑制肥大细胞脱颗粒,减轻早期炎症反应;但不影响局部组织中肥大细胞数量,且不影响创面愈合时间;2、色甘酸钠可以有效减少创面愈合后瘢痕的形成;3、通过对比使用不同剂量色甘酸钠,发现抑制大鼠肥大细胞脱颗粒的最佳有效剂量为20mg/kg;4、色甘酸钠影响肥大细胞类胰蛋白酶释放,可能是其影响成纤维细胞活化,并最终减少瘢痕形成的机制之一。
[Abstract]:Objective: This study intends to establish a rat model of deep wounds, mast cells using mast cell membrane stabilizer cromolyn sodium intervention in the process of wound healing effect, through the observation of the healing scar hyperplasia and detected the number of mast cells, tryptase and other related indicators, to explore the feasibility of mast cells as targets for prevention and treatment of scar. Methods: 40 male SD rats were randomly divided into the A, B, C, D four groups, 10 rats in each group, A, B, C group, A group for a large dose of sodium cromoglycate (40mg/kg) group, B group middle dose of cromolyn sodium group (20mg/kg), C group of small dose of cromolyn sodium group (10mg/kg), group D was negative. The use of hair removal agent back hair removal to all subjects after anesthesia conditions on the back on both sides of the spine with a scalpel to form two symmetrical area of about 15mm*10mm*2mm the cutting depth to wound wound. 2% dilute Release the iodophor solution after disinfection to be exposed, the daily observation of back wounds in rats (whether there is abnormal blood seepage and seepage). Respectively at 30min before operation; after twenty-fourth, forty-eighth, seventy-second hours of A, B, C group all subjects corresponding dose of intraperitoneal injection of sodium cromoglycate solution, group D injection equal volume of saline. The initial recording wound area, regular observation of wound size, and after the 3D take the left side of the back wound tissue were fixed, dehydration and conventional HE staining on tissue, toluidine blue staining (mast cell count), immunohistochemical staining (tryptase) treatment. The recording time of all experimental objects the wound healing, the number of groups were compared. Postoperative 42d were all subjects, and collect the scar right back, routine HE stain on it. Mast cell count: tissue sections using toluidine blue staining method, use Image-pro6.0 diagram Image analysis software system in double blind high magnification (* 400) continuous count per unit area (/mm2), each of 5 randomly selected fields, the counting of mast cells in total, the average value of the experimental data and the final object. Degranulation and degranulation were not defined off. The particles after mast cell membrane is not complete, fuzzy boundaries, and could be seen around the scattered particles. And the complete form of mast cells relatively complete rules, and the surrounding tissue of the border clear. Tryptase analysis method: immunohistochemical staining and image analysis software system with image-pro6.0, double blind high magnification (* 400) count per unit area (/mm2), the same in each section was randomly selected 5 horizons, which records of tryptase positive mast cell number, and take the average value as the final result. Results: 1, the initial wound area: through the analysis of variance were compared between the two groups after Then 22 can get P0.05, suggesting that all initial experiments showed no significant difference.2 the wound area, wound healing time: by the analysis of variance were compared between the two groups after 22 can get P0.05, suggesting that all subjects had no significant difference.3 wound healing time, scar area and hardness: forty-second days after operation, see all subjects back scar wounds were healed, the color was light red or pale, prompting groups scar area size relationship: C group D group B group a group.D group scar is hard; a, B two groups of scar quality and tough, no significant differences between two groups; group C in between.4 and he staining microscopically observed after third days after operation: A, B two groups were similar, inflammatory cells, fibroblasts scattered, but the number of new capillaries; D group microscopically, cell structure disorder, inflammatory cells and fibroblast cell number, distribution Messy, and abundant capillary.C group of inflammatory cells and fibroblasts in D group than the rich, but far more than a, B two groups. After forty-second days, the scar tissue in the D group, a large number of fibroblasts and extracellular matrix and capillary distribution, part of a circular or spiral like distribution, the number of inflammatory cells not much, fibrous tissue scattered, no obvious rational; scar tissue in group C than in group D color light microscope fibroblasts significantly less than group D, are still rules, collagen fibers were compared with the D group neatly, extracellular matrix is not much, scattered in small capillaries; and group A, B group of scar tissue hyperplasia was not obvious, microscopically fibroblasts arranged more closely and orderly, neat and orderly.5 fiber tissue, mast cell count: sections were stained with toluidine blue staining, were counted under the microscope, found no statistically significant difference between the groups, the total number of mast cells, but a, B two Group degranulation was less than the D group, C group is located in between the corresponding number of mast cells degranulation of mast cells / total ratio: D group C group A group is B group.6, tryptase count: third days after operation, to accept the signal after the start of stimulation of mast cell tryptase expression quantity. D group of tryptase positive mast cells was significantly less than that of A group, B group, C group is located between the two. The number of tryptase positive mast cell number: B group A group C group D group. Conclusion: 1, cromolyn sodium can significantly inhibit the degranulation of mast cells, reducing inflammatory reaction; but does not affect the the number of mast cells in the local tissues, and does not affect the wound healing time; 2, cromolyn sodium can effectively reduce the wound healing after scar formation; 3, cromolyn sodium with different doses by comparison, found that the best effective dose inhibits rat mast cell degranulation of 20mg/kg; 4, cromolyn sodium. The release of the ringing mast cell tryptase may be one of the mechanisms that affect the activation of fibroblasts and ultimately reduce the formation of cicatricial cells.

【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R622

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