犬声带瘢痕模型的建立及其细胞外基质中赖氨酰氧化酶、热休克蛋白70和透明质酸表达的研究
发布时间：2018-08-16 15:28
【摘要】：目的:建立犬声带瘢痕模型,分析损伤后不同时间段内声带愈合的形态学、组织病理学及细胞外基质(ECM)相关影响因子的变化。方法:5只实验犬随机分为对照组(1只)和实验组(4只),对照组不做特殊处理,实验组行支撑喉镜下CO_2激光双侧声带烧灼术。各只实验犬分别在5个不同时间(术前和术后6h、3周、8周和12周)于支撑喉镜下观察声带损伤的大体形态学变化后处死,取材。采用苏木精-伊红染色、免疫荧光等方法观察声带各层组织病理学变化和超微形态学变化。观测声带组织内赖氨酰氧化酶(LOX)、热休克蛋白70(HSP70)和透明质酸(HA)的累积光密度值(IOD)的变化规律。结果:1喉内镜示,损伤后3周声带创面轻度充血水肿及炎性渗出;术后8周创面充血水肿消失,局部挛缩凹陷,无粘连和肉芽形成;术后12周双侧声带表面光滑,局部挛缩,声带瘢痕形成,声嘶明显。2苏木精-伊红染色示,术后6h声带创面大量炎性细胞浸润、红细胞渗出,细胞充血水肿;术后3周声带创面成纤维细胞增生,新生血管形成,大量纤维组织无序排列;术后8周声带创面各层纤维组织增生紊乱,血管增粗;术后12周时,声带创面固有层增厚,大量胶原纤维增多,排列紊乱,呈团状或束状,纤维间较多不规则间隙;血管和腺体少见或消失。3免疫荧光示LOX、HSP70、HA均定位于细胞质和细胞核,术后不同时间段表达的阳性程度不同,大多在炎性细胞、血管内皮细胞和腺体内表达较强烈。4IOD值比较,在不同时间段LOX的IOD值之间均有差异(P0.05);HSP70在术前与术后12周的IOD值无明显异常,余各组间差异有统计学意义(P0.01);HA在术后3周与12周间无异常,余各组间差异有统计学意义(P0.05)。5LOX在术后6h表达减弱,术后3~12周呈上升趋势;HSP70在术后6h升至峰值,术后3~12周呈下降趋势;HA在术后6h表达减弱,术后8周升至峰值,术后8~12周下降。6透射电镜显示,声带术后3~8周固有层成纤维细胞极度活跃状态,细胞肿胀,细胞器丰富;术后12周时,声带固有层内仍功能活跃,纤维细胞呈网状排列紊乱,纤维间隙较大。结论:支撑喉镜下CO_2激光声带烧灼术建立犬声带瘢痕模型可靠,损伤后3个月声带瘢痕基本形成。LOX、HSP70、HA在声带瘢痕形成的早中晚期发挥着不同的作用,可以作为检测声带瘢痕形成较敏感的指标。
[Abstract]:Aim: to establish canine vocal cord scar model and analyze the changes of morphology histopathology and related factors of extracellular matrix (ECM) related to vocal cord healing in different time after injury. Methods five experimental dogs were randomly divided into two groups: control group (1 dog) and experimental group (4 dogs). The control group did not receive special treatment. The experimental group received CO_2 laser cauterization of bilateral vocal cords under laryngoscope. The experimental dogs were killed at 5 different times (3 weeks before and 6 hours after operation, 8 weeks and 12 weeks after operation) after observing the morphological changes of vocal cord injury under supporting laryngoscope. The histopathological and ultrastructural changes of vocal cord were observed by hematoxylin-eosin staining and immunofluorescence. The changes of cumulative optical density (IOD) of (LOX), heat shock protein 70 (HSP70) and hyaluronic acid (HA) in vocal cord tissues were observed. Results 1 laryngeal endoscopy showed slight hyperemia and edema and inflammatory exudation of vocal cord wound at 3 weeks after injury, hyperemia and edema disappeared at 8 weeks after injury, no adhesion and granulation formation in local contracture, smooth surface of bilateral vocal cord and local contracture at 12 weeks after operation. The vocal cord scar formation, hoarseness obvious .2 hematoxylin-eosin staining showed that the vocal cord wound surface was infiltrated by a large number of inflammatory cells, red blood cell exudation, hyperemia and edema at 6 hours after operation, fibroblast proliferation and angiogenesis of vocal cord wound at 3 weeks after operation. At 8 weeks after operation, the lamina propria of vocal cord wound was thickened, and a large number of collagenous fibers were arranged in disorder, in the shape of mass or bundles, the lamina propria of vocal cord wound was thickened and a large number of collagenous fibers were increased at 12 weeks after operation. There were many irregular interspaces between the fibers. 3. 3 immunofluorescence showed that Loxus HSP70HA was located in cytoplasm and nucleus, and the positive degree was different at different time after operation, most of which were in inflammatory cells. The expression of IOD in endothelial cells and glands was significantly higher than that in LOX at different time points (P0.05). The IOD values of HSP70 were not abnormal before and 12 weeks after operation. There was no significant difference in HA between 3 and 12 weeks after operation (P0.01). There was a significant difference between the other groups (P0.05) .5LOX expression decreased at 6 hours after operation, and the HSP70 increased to a peak at 6 hours after operation at 3 and 12 weeks after operation. The expression of HA decreased at 6 hours after operation and rose to the peak at 8 weeks after operation. A decrease of .6 transmission electron microscope showed that fibroblasts in lamina propria were extremely active, cells were swollen and organelle was abundant at 3 and 8 weeks after vocal cord operation. At 12 weeks after operation, the vocal cord lamina propria was still active, the fibrous cells were disorganized and the fiber gap was larger. Conclusion: the canine vocal cord scar model established by CO_2 laser cauterization under bracing laryngoscope is reliable. The cicatricial formation of vocal cord in 3 months after injury. Loxo HSP70HA plays different roles in the early, middle and late stage of vocal cord scar formation. It can be used as a sensitive index for the detection of vocal cord scar formation.
【作者单位】： 武汉大学附属同仁医院武汉市第三医院耳鼻咽喉科;
【基金】：武汉市卫计委中青年医学骨干培养计划工程[No:武卫生计生(2014)77号文件]
【分类号】：R-332;R767.4
本文编号：2186394
[Abstract]:Aim: to establish canine vocal cord scar model and analyze the changes of morphology histopathology and related factors of extracellular matrix (ECM) related to vocal cord healing in different time after injury. Methods five experimental dogs were randomly divided into two groups: control group (1 dog) and experimental group (4 dogs). The control group did not receive special treatment. The experimental group received CO_2 laser cauterization of bilateral vocal cords under laryngoscope. The experimental dogs were killed at 5 different times (3 weeks before and 6 hours after operation, 8 weeks and 12 weeks after operation) after observing the morphological changes of vocal cord injury under supporting laryngoscope. The histopathological and ultrastructural changes of vocal cord were observed by hematoxylin-eosin staining and immunofluorescence. The changes of cumulative optical density (IOD) of (LOX), heat shock protein 70 (HSP70) and hyaluronic acid (HA) in vocal cord tissues were observed. Results 1 laryngeal endoscopy showed slight hyperemia and edema and inflammatory exudation of vocal cord wound at 3 weeks after injury, hyperemia and edema disappeared at 8 weeks after injury, no adhesion and granulation formation in local contracture, smooth surface of bilateral vocal cord and local contracture at 12 weeks after operation. The vocal cord scar formation, hoarseness obvious .2 hematoxylin-eosin staining showed that the vocal cord wound surface was infiltrated by a large number of inflammatory cells, red blood cell exudation, hyperemia and edema at 6 hours after operation, fibroblast proliferation and angiogenesis of vocal cord wound at 3 weeks after operation. At 8 weeks after operation, the lamina propria of vocal cord wound was thickened, and a large number of collagenous fibers were arranged in disorder, in the shape of mass or bundles, the lamina propria of vocal cord wound was thickened and a large number of collagenous fibers were increased at 12 weeks after operation. There were many irregular interspaces between the fibers. 3. 3 immunofluorescence showed that Loxus HSP70HA was located in cytoplasm and nucleus, and the positive degree was different at different time after operation, most of which were in inflammatory cells. The expression of IOD in endothelial cells and glands was significantly higher than that in LOX at different time points (P0.05). The IOD values of HSP70 were not abnormal before and 12 weeks after operation. There was no significant difference in HA between 3 and 12 weeks after operation (P0.01). There was a significant difference between the other groups (P0.05) .5LOX expression decreased at 6 hours after operation, and the HSP70 increased to a peak at 6 hours after operation at 3 and 12 weeks after operation. The expression of HA decreased at 6 hours after operation and rose to the peak at 8 weeks after operation. A decrease of .6 transmission electron microscope showed that fibroblasts in lamina propria were extremely active, cells were swollen and organelle was abundant at 3 and 8 weeks after vocal cord operation. At 12 weeks after operation, the vocal cord lamina propria was still active, the fibrous cells were disorganized and the fiber gap was larger. Conclusion: the canine vocal cord scar model established by CO_2 laser cauterization under bracing laryngoscope is reliable. The cicatricial formation of vocal cord in 3 months after injury. Loxo HSP70HA plays different roles in the early, middle and late stage of vocal cord scar formation. It can be used as a sensitive index for the detection of vocal cord scar formation.
【作者单位】： 武汉大学附属同仁医院武汉市第三医院耳鼻咽喉科;
【基金】：武汉市卫计委中青年医学骨干培养计划工程[No:武卫生计生(2014)77号文件]
【分类号】：R-332;R767.4
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