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

Developing a canine vocal told scar model by CO_2 laser and studying the LOX,HSP70 and HA expression in its extra celluar matrix

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(P<0.05);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(P<0.01);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(P<0.05)。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。

Objective：Building a canine vocal fold scar model and analyzing the changes in morphology,histopathology and related factors of extra cellular matrix（ECM）of vocal cord healing at different time periods.Method：Five experimental dogs were randomly divided into the control group（one dog）and the experimental group（four dogs）.No special treatment was done on the control group,and the experimental group was given CO2 laser through laryngoscope with minimally invasive surgery on bilateral vocal cords.Observation of the morphological changes of injured vocal cords was made at five different time,pre-operation and 6h,3w,8wand 12wpost-operation.HE staining,immunofluorescence,were used respectively to observe the histopathological and ultrastructural changes of each layer of vocal cord.Observation were made on the changing pattern of lysyl oxidase（LOX）,heat shock proteins70（HSP70）,and the integrated optical density index（IOD）of Hyaluronic acid（HA）in vocal cord tissues.Result：1Laryngoscope shows there were mild congestion and edema and inflammatory exudation on the wound surface of vocal cord 3wpost-operation.On the wound surface of vocal cord 8wpost-operation,the congestion and edema disappear,the local contraction sink,and no adhesion and granulation form.12wpost-operation,the surface of the vocal cord was smooth,there were local contractures,vocal fold scar form,and hoarseness in the bilateral vocal cords was obvious.2HE staining shows 6hpost-operation there were a large number of inflammatory cell infiltration,red blood cell leakage,and cell congestion and edema on the wound surface of vocal cord.3wpost-operation,there were fibroblast proliferation,angiogenesis,and a large number of fibrous tissues disorderly arranged on the wound surface of vocal cord.8wpost-operation,each layer of fibrous tissues were in hyperplasia and the blood vessels was thicken after on the wound surface of vocal cord,12 wpost-operation,a large number of collagens increases and were in group or fasciculation disorder.There were many irregular gaps in fibers,and blood vessels and glands become rare or disappear.3The immunofluorescence showed LOX,HSP70 and HA were all localized in the cellular cytoplasm and nucleus.The expression levels were different at different postoperative time,and they were mainly relatively strong expressed in the inflammatory cells,vascular endothelial cells and the glands.4The comparison of IOD values：The IOD values of LOX were different at different time periods（P〈0.05）.There were no significant differences in IOD of HSP70 between pre-operation and 12 wpostoperation,but there are significant differences among other groups（P〈0.05）.There were no significant differences in IOD of HA between pre-operation and 12wpost-operation,but there were significant differences among other groups（P〈0.01）.5The expression of LOX decreased 6hpost-operation and increased 3-12wpost-operation.The expression of HSP70post-operation reached the peak 6hpost-operation and decreased 3-12 wpostoperation.The expression of HA decreased 6hpost-operation,increased to the peak 8wpost-operation,and decreased 8-12wpost-operation.6The transmission electron microscope showed 3wto 8wpost-operation,in the intrinsic layer of the vocal cord,the fibroblasts were extremely active,the cells swelled,and the organelles were abundant.12wpost-operation,there were a small number of fibroblasts in the active state,and larger gaps between the fibers and fewer and thinner elastic fibers.Conclusion：CO2laser ablation of vocal cords under the MicroPost-Laryngoscope could establish reliable animal model of vocal fold scar,and 12 wreticular the vocal fold scars basically form.LOX,HSP70,HA play different roles at early,middle,and late stages in the vocal fold scar formation,and they can be used as a more sensitive index for vocal fold scar formation.