声带细胞外基质干预和人羊膜上皮细胞移植后声带瘢痕修复再生的研究  

Characterization of scarred vocal fold regeneration after the intervention of extracellular matrix and human amniotic epithelial cells transplanting

作　　者：甄瑞清 王家佳[1] 王方圆[2] 胡爱艳[3] 魏春生[1] 

机构地区：[1]复旦大学附属眼耳鼻喉科医院耳鼻咽喉科,上海200031 [2]上海交通大学附属国际和平妇幼保健院中心实验室 [3]复旦大学附属中山医院病理科

出　　处：《中华耳鼻咽喉头颈外科杂志》2014年第4期311-316,共6页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

基　　金：上海市自然科学基金（11ZR1405900）;上海市科委创新计划项目（12401902300）

摘　　要：目的 应用声带细胞外基质干预（胶原酶和透明质酸注射）和人羊膜上皮细胞（human amniotic epithelial cells,hAEC）在声带瘢痕内的移植,探索其对声带瘢痕修复再生的意义.方法 对20只新西兰兔中的38侧声带行声带锐性损伤,形成声带瘢痕.分离培养实验用hAEC,慢病毒绿色荧光蛋白基因转染作标记.将hAEC移植入10侧瘢痕声带,即hAEC移植组;声带细胞外基质干预（注射胶原酶和透明质酸）10侧瘢痕声带,即基质干预组;hAEC移植同时细胞外基质干预10侧瘢痕声带,即复合组;瘢痕未处理8侧声带,即瘢痕组.2侧正常声带作为对照组.术后1和2个月时示踪观察hAEC在声带内的生长、分布及活性情况;术后1、2、3和6个月采用HE染色、Masson染色及免疫组织化学观察声带形态学变化及声带固有层胶原、纤维连接蛋白的分布及含量变化.结果 hAEC原代培养4d后呈铺路石样生长,移植入瘢痕声带后可在固有层内持续存活2个月;术后1个月和2个月免疫荧光显示角蛋白（上皮细胞标志性蛋白）荧光阳性.术后6个月光镜下观察及Masson染色半定量分析示瘢痕组较正常组胶原量明显增加,排列紊乱;hAEC移植组、基质干预组及复合组胶原量和排列较瘢痕组改善,hAEC移植组、基质干预组二者相似,复合组优于前两组,但尚未及正常;免疫组织化学半定量分析示瘢痕组较正常组纤维连接蛋白平均光密度明显增加,hAEC移植组、基质干预组及复合组的平均光密度介于瘢痕组和正常对照组之间,hAEC移植组与基质干预组相差不明显,复合组优于前两组.结论 声带细胞外基质干预和hAEC注射移植复合作用于兔瘢痕声带后,可更好地促进声带细胞外基质的分泌、合理分布及部分有序化排列,促进声带瘢痕修复再生.Objective To investigate the treatment for scarred vocal folds by transplanting human amniotic epithelial cells （hAECs）and injecting collagenase as well as hyaluronic acid （HA） for the intervention of the extracellular matrix （EMC）,to observe the growth,distribution of hAECs and to assess the abilities of them for scarred vocal fold regeneration.Methods The lamina propria was injured by localized resection in thirty-eight vocal folds of twenty rabbits.hAECs were isolated from human amnion and marked by Lenti-GFP.After the formation of vocal fold scarring,hAECs were transplanted into ten vocal folds,collagenase and HA were injected into ten vocal folds,all three were injected into ten vocal folds,none were injected into eight vocal folds,and two normal vocal folds were used as control.At 1 month and 2 months after the transplanting,the survival,the distribution and the cytoactive of hAECs were examined by immunofluorescence method.Meanwhile,at 1 month,2 months,3 months and 6 months after the operation,HE staining was performed for histopathological research,Masson trichrome staining and immunohistochemical staining were used for collagen and fibronectin respectively.Results After implanted into the scarred vocal folds,hAECs could survive in vocal fold lamina propria for two months.The immunofluorescence analysis showed the cytoactive of hAECs.Six months postoperatively,compared with that in the normal vocal folds,collagen in the untreated scarred vocal folds more increased and disorderly distributed ; the changes in other three groups were between the two groups above,but the group injected with all of hAECs,collagenase and HA was better than other two groups.Besides,the mean density of fibronectin in the scarred untreated control group was more significantly increased than that in the normal vocal folds;the changes in other three groups were between the two groups above,but the group injected with all of hAECs,collagenase and HA was better than other two groups.Conclusion The transplanting of hAECs an

关 键 词：声带 瘢痕 羊膜 上皮细胞 细胞移植 再生 细胞外基质 兔 

分 类 号：R767.91[医药卫生—耳鼻咽喉科]