目的 采用低温等离子消融术构建犬声带瘢痕模型,并初步筛选出与声带瘢痕形成密切相关的靶基因.方法 对4只中华田园犬行低温等离子消融术,损伤左侧声带至肌层,对侧声带不予处理.于术前、术后即刻、术后3周和术后12周对犬双侧声带大体形态进行观察,通过HE染色观察声带病理结构变化,透射电镜观察声带超微结构变化.采用高通量测序分析双侧声带基因表达的差异,筛选出显著差异表达的靶基因.结果 术后3周术侧声带组织充血肿胀,边缘不齐,可见红色肉芽组织形成;术后12周声带创面局部挛缩凹陷,瘢痕形成;HE染色见瘢痕声带鳞状上皮层明显增厚,纤维层增厚、排列紊乱,局部成团状或束状聚集,肌层亦可见散在的纤维束;透射电镜下见间质增厚、密度不均,细胞肿胀,细胞间界线不清,细胞核增生、线粒体增多,细胞处于活跃状态.高通量测序分析发现众多基因家族参与了声带的瘢痕修复过程,其中密切相关的基因家族有IL家族、趋化因子CCL和CXCL家族、MMPs家族及其抑制剂TIMPs家族、Wnt家族、HSP家族、MAPK家族和TGF-β家族等.结论 本研究成功构建了犬声带瘢痕模型,并初步筛选出了与声带瘢痕形成密切相关的靶基因,为声带瘢痕机制的探索提供了一定的参考价值.
Objective To construct a canine model of vocal cord scar by low-termperature plasma ablation and screen the target genes closely related to the formation of vocal cord scar.Methods Four Chinese rural canines were treated with plasma ablation under the support of laryngoscope and endoscope,and the left vocal cords were injured to the muscle layer.The contralateral sides were left untreated.The gross morphology of vocal cord was observed before operation,immediately after operation,3 weeks after operation and 12 weeks after operation.The pathologi-cal structure of vocal cords was observed by HE stainning,and the ultrastructure of vocal cords was observed by transmission electron microscopy.In addition,high-throughput sequencing was used to analyze the differences in gene expression between the bilateral vocal cords,and the target genes with significantly different expression were screened out.Results In general morphology,the normal vocal cords were banded and well closed.At 3 weeks af-ter operation,the vocal cords were congested and swollen,with uneven edges and red granulation tissues were seen.At 12 weeks after operation,the vocal cord wound was localized contracture and depression,and scar was formed.HE staining showed obvious thickening of the squamous epithelium of the scarred vocal cords,thickening and disor-dered arrangement of the fiber layer,local clumping or bundle aggregation,and scattered fiber bundles were also seen in the muscle layer.Transmission electron microscopy showed interstitial thickening,uneven density,cell swelling,unclear intercellular boundary,proliferation of nuclei and mitochondria,and cells in an active state.High-throughput sequencing analysis revealed that many gene families were involved in the process of vocal cord scar re-pair,including IL family,CCL and CXCL family,MMPs family and its inhibitor TIMPs family,Wnt family,HSP family,MAPK family and TGF-β family.Conclusion We successfully constructed the canine model of vocal cord scar by low-temperature plasma ablation and screened out the target genes closely related to the formation of vocal cord scar by high-throughput sequencing,which provides certain reference value for exploring the mechanism of vo-cal cord scar.