目的 探讨自体脂肪间充质干细胞移植入损伤声带后的生长分布及对声带固有层及其主要细胞外基质变化的影响特点.方法 对34只实验用兔(68侧声带)的53侧声带进行全麻支撑喉镜下声带锐性损伤.分离培养、鉴定及定向诱导20只实验用兔脂肪间充质干细胞.将自体脂肪间充质干细胞植入20侧损伤声带后,示踪观察脂肪间充质干细胞在声带固有层内的生长分布;同时以单纯支架(胶原或透明质酸凝胶)植入18侧受损声带(胶原10侧,透明质酸8侧)及单纯损伤15侧声带作为对照组.术后15 d～12个月时采用HE染色、Masson染色、Alcian Blue染色及免疫组化染色观察声带形态学变化及声带固有层胶原、透明质酸、纤维连接蛋白的含量及分布变化.结果 脂肪间充质干细胞呈梭形贴壁生长,具有多向分化潜能,植入损伤兔声带后持续分布于声带固有层.损伤声带在脂肪间充质于细胞植入6个月时形态接近正常,12个月时部分组织学结构类似正常;声带细胞外基质中的胶原于植入3个月内含量有增高趋势,无序分布于固有层,后逐渐减少至正常水平附近,12个月时部分分布略显不规则;透明质酸于植入40 d含量亦有增高趋势,分布于固有层各层,6个月含量减至正常水平附近,局限于固有层浅中层;纤维连接蛋白于植入后始终在固有层内散在分布,含量在40 d内有增高趋势,后逐渐减少,12个月时含量接近正常.单纯声带损伤3个月后局部开始出现瘢痕挛缩,以胶原纤维为主的大量纤维组织增生,12个月时仍紊乱分布于声带固有层各层.单纯支架植入组变化介于二者之间.结论 脂肪间充质干细胞植入兔损伤声带后具有促进声带细胞外基质分泌、合理分布及部分有序化排列的功能,具有促进声带修复再生的作用.%Objective To investigate the treatment for injured vocal folds by implanting autologous adipose-derived mesenchymal stem cells ( ADSC ), and to observe the characteristics of lamina propria of the vocal folds and its major extracellular matrix ( ECM ), as well as the growth features of ADSC. Methods The lamina propria was injured by localized resection in fifty-three vocal folds of thirty-four rabbits.Isolation, culture and identification of ADSC were performed in twenty rabbits. Three to five days after vocal folds injury, autogeneic ADSC were implanted into the injured vocal folds. As control, scaffolds ( collagen or hyaluronic acid) and none were injected into eighteen and fifteen vocal folds respectively. Larynges were harvested at 15 days, 40 days and 3, 6, 12 months after operation. The growth and distribution of ADSC were detected by DAPI stain. Meanwhile, HE staining was performed for histopathologic research, Masson trichrome staining, Alcian Blue staining and immunohistochemical staining were used for collagen,hyaluronic acid and fibronection respectively. Results ADSC showed a spindle-shaped adherent growth,with multi-differentiation potential. After implanting into the injured vocal fold, ADSC can survive in vocal fold lamina propria. In ADSC implanting group, the morphology and histologic structure of vocal folds were similar to normal in six and twelve months after ADSC implanting respectively. collagen had an increasing trend in three months, with the disorderly distribution in the vocal fold lamina propria, then it became decreasing until the twelveth month, when concentration closed to normal, however, distribution of collagen was a little irregular. The content of hyaluronic acid increased within forty days and distributed in the lamina propria, then gradually reduced to normal levels in the following twelve months, and limited in the superficial and middle layers of lamina propria, which closed to normal. Fibronectin in the lamina propria continued to scattered at the peak levels at fortieth day, then decreased in the later twelve months when the content became near normal. In the untreated group, vocal fold showed a local scar contracture at third month after injury, histology showed large number of fibrous tissue ( mainly collagen fibers) hyperplasia with a tendency of increasing, disorders was also found in vocal fold lamina propria. In the scaffold implanting group, the changes were between the two groups above. Conclusions ADSC are good source of seed cells for vocal fold tissue engineering, which have the ability for promoting injured rabbit vocal folds ECM secretion, rational distribution and part ordering arrangement. ADSC also play an important role in vocal folds reparation and regeneration.
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