转分化的脂肪间充质干细胞修复损伤声带的研究

Vocal fold injury disrupts the lamina propria of the vocal fold mucosa and modifies the biomechanical properties of the vocal folds by increasing the viscoelastic strength of the mucosa, thus reducing vibration of the folds and causing severe dysphonia. Optimal treatment for vocal fold scarring has not yet been established. Tissue engineering may provide new strategies for the prevention and treatment of vocal fold injuring. To explore the ideal seed cells for vocal fold regeneration is a hotspot. This project intends to transdifferentiate adipose-derived mesenchymal stem cells into fibroblast-like cells in vitro, to study their similarity with vocal fold fibroblasts, and to apply the transdifferentiated adipose-derived mesenchymal stem cells as seed cells to promote vocal fold regeneration. The CM-Dil was used to tracing cells for cell survival, growth and proliferation characteristics study after the implantation. The morphology and pathological changes of the implanted vocal folds are also be studied. The extracellular matrix proteins of vocal fold lamina propria were detected by immunofluorescence and western blot. Furthermore, Using laser confocal immunofluorescence co-localization to investigate the within and outside vocal fold extracellular matrix proteins and transdifferentiated adipose-derived mesenchymal stem cells to explore the mechanism for vocal fold injury therapy.

声带损伤引起固有层细胞外基质无序沉积、紊乱排列，使声带特有的振动功能减弱或丧失，由此引起的不可逆发音障碍是目前世界治疗的难点。探索促进声带修复再生的理想种子细胞是研究的热点。本项目拟将脂肪间充质干细胞于体外转分化为成纤维样细胞，研究其与声带成纤维细胞的相似性，并以这种转分化的脂肪间充质干细胞作为种子细胞，探讨其促进损伤声带的修复再生的特点及机制：应用CM-Dil示踪细胞，研究其植入后的存活、生长及增殖特点；对声带外形及大体病理组织学变化特点进行研究；通过免疫荧光染色及Western Blot检测声带细胞外基质蛋白在固有层内的含量及分布变化特点，并利用激光共聚焦免疫荧光共定位研究声带细胞外基质蛋白与转分化的脂肪间充质干细胞内、外的时空变化关系，以进一步探讨转分化的脂肪间充质干细胞修复损伤声带的作用机制，为声带损伤的治疗提供方法和依据。

由于声带损伤引起的不可逆发音障碍是目前世界治疗的难点。探索促进声带修复再生的理想种子细胞是研究的热点。本项目将脂肪间充质干细胞于体外转分化为成纤维样细胞，发现其形态和细胞表面标记蛋白与声带成纤维细胞类似。将这种转分化的脂肪间充质干细胞作为种子细胞，植入损伤声带后，发现：脂肪间充质干细胞复合物植入后15~40天，细胞外基质中透明质酸、核心蛋白多糖的含量增加最为明显；术后3月各种细胞外基质的含量均有所下降；术后6月各种细胞外基质的含量和分布与正常类似。转分化的脂肪间充质干细胞复合物植入后15~40天，细胞外基质中弹性蛋白的含量增加最为明显，透明质酸、核心蛋白多糖的含量亦有增加；术后3月各种细胞外基质的含量均有所下降；术后6月声带外形及各种细胞外基质的含量和分布与正常类似。声带成纤维细胞复合物植入后15~40天，细胞外基质中弹性蛋白和透明质酸的含量略有增加，术后3月各种细胞外基质含量均有所下降，但胶原含量下降不明显，术后6月细胞外基质中胶原含量高于正常，无序排列。提示脂肪间充质干细胞复合物在植入损伤声带后早期能够分泌大量的透明质酸和核心蛋白多糖，对于抑制胶原的无序沉积、减少瘢痕形成具有重要意义，转分化的脂肪间充质干细胞复合物在植入损伤声带后能够分泌大量的弹性蛋白，并能够维持其在较高水平浓度，有利于声带振动功能的恢复。