兔声带成纤维细胞的体外培养、示踪与声带瘢痕的研究

发布时间：2018-04-30 11:25

本文选题：声带再生 + 成纤维细胞　；参考：《中国人民解放军军医进修学院》2010年硕士论文

【摘要】： 正常声带组织因外伤或疾病损伤后所形成的纤维组织替代产物形成了声带瘢痕。由于瘢痕组织使声带变得坚硬,影响了声带固有层的粘弹性(viscoelastic strength),改变了声带的生物力学特性,从而减弱声带振动黏膜波的产生而影响发声。目前,尚未找到一种理想的预防和治疗声带瘢痕的方法。近年来,组织工程学技术在组织器官重建方面取得了令人瞩目的进展,其主要原理是以少量种子细胞经体外扩增后与生物材料、细胞因子结合,修复较大的组织或器官缺损,重建组织器官的生理功能,最终达到完美的形态甚至功能修复的目的。将组织工程学的技术方法应用于喉科,有望在未来实现声带,甚至喉的再生。人类的声带组织是一个高度分化的分层结构,从组织学结构上可分上皮层、固有层和肌肉层(甲杓肌)。声带固有层又分为固有浅层(Reinke's层),固有中层和固有深层。这种分层在描述声带结构功能方面具有重要意义。固有层是声带最重要的特征性结构,主要包括细胞及细胞外基质成分。组成固有层的细胞为数较少,主要细胞有：成纤维细胞,肌成纤维细胞和巨噬细胞。成纤维细胞是维持固有层结构与功能的重要细胞,主要分布在固有层深层,其调控细胞间质的沉着、降解及重新分布；细胞外基质成分包括纤维蛋白和间隙蛋白。本研究第一部分我们首先制作了兔声带瘢痕模型,并对声带瘢痕进行初步研究。在支撑喉镜下,用喉钳夹取双侧声带前中部组织(未达肌层),未手术者作为正常对照,1个月后处死动物,收获声带组织,采用Masson三色染色观察声带胶原,采用经过透明质酸酶处理的Alcian blue染色观察透明质酸。利用Image-Pro Plus 6.0软件进行对切片染色进行半定量分析,实验结果表明瘢痕声带的胶原成分明显增多,透明质酸含量无明显改变。本研究中,我们对声带固有层的组织工程学再生进行了初步探索。组织工程学的三个要素是种子细胞,支架材料,和细胞因子。本研究第二部分首先进行了种子细胞-兔声带成纤维细胞(vocal fold fibroblasts,VFFs)的体外培养。取兔声带组织,消化法获取成纤维细胞,用含有胎牛血清的高糖DMEM培养基体外培养,摸索出最佳培养条件。在倒置相差显微镜下观察细胞的形态及生长状况,并经过传代使细胞纯化。利用免疫组化方法,检测成纤维细胞的特异性蛋白Vimentin,结果Vimentin阳性率90%以上,证明所培养的细胞为成纤维细胞,且阳性率较高。本研究第三部分观察培养的细胞在异体生物体内相容性及生长情况。我们对培养的细胞进行Ad-EGFP标记。取新西兰兔6只,随机选取2只兔作为无干预组,4只新西兰兔做双侧声带急性损伤模型,左侧声带损伤部位注射104个／ml浓度的Ad-EGFP标记的细胞混悬液,向右侧声带损伤部位注入生理盐水1ml作为对照。声带注射细胞后,兔生长情况良好,均未发生感染、喉水肿,免疫排斥等反应。15天后收获声带组织,荧光显微镜下观察未发现成活的VFFs。说明单纯VFFs注射到异体组织很难存活,为其在动物体内生长提供生长支架可能会利于生长。总之,本研究对声带组织工程学进行了初步探索,制备了种子细胞,探索了种子细胞体内相容性,制备了声带瘢痕动物模型,在后续的研究中,我们将构建细胞-支架材料复合物,移植入声带瘢痕模型,观察声带再生修复情况。
[Abstract]:The normal vocal cord tissue is formed by the replacement of fibrous tissue after injury or injury. The scar tissue makes the vocal band hard and affects the viscoelasticity of the lamina propria (viscoelastic strength), changes the biomechanical properties of the vocal band, and weakens the production of the vocal band vibration mucous membrane and affects the phonation. At present, an ideal method for the prevention and treatment of vocal cord scarring has not been found. In recent years, tissue engineering technology has made remarkable progress in the reconstruction of tissues and organs. The main principle is to combine with a small amount of seed cells in vitro with biomaterials and cytokine, to repair large tissue or organ defects, and to reconstruct the group. The physiological function of the organs of the weave, eventually reaching the perfect form and even the purpose of functional repair. Applying the technical methods of tissue engineering to the larynx, it is expected to realize the vocal cord and even the regeneration of the larynx in the future. The human vocal cord tissue is a highly differentiated stratified structure that can be divided into the cortex, the lamina propria and the muscular layer (a dipper) from the histological structure. The lamina propria of the vocal cord is divided into the inherent shallow layer (Reinke's layer), the inherent middle layer and the inherent deep layer. This stratification is of great significance in describing the structure and function of the vocal cords. The lamina propria is the most important characteristic structure of the vocal cord, mainly including the cell and the extracellular matrix. The number of cells consisting of the solid layer is few, the main cells are fibroblast Vascular cells, myofibroblasts and macrophages. Fibroblasts are important cells to maintain the structure and function of the lamina propria, mainly distributed in the deep lamina of the propria, which regulate the sink, degradation and redistribution of the intercellular substance, and the extracellular matrix consists of fibrin and interstitial egg white.
In the first part of this study, we first made the rabbit SARS scar model and preliminarily studied the vocal cord scar. Under the supporting laryngoscope, we used the laryngeal clamp to take the anterior central tissue of the bilateral vocal cords (without the myometrium) and the non operative subjects as the normal control. 1 months later, the animals were killed and the vocal cord tissue was harvested, and the vocal collagens were observed by Masson tricolor staining. Hyaluronic acid was observed by Alcian blue staining with hyaluronidase. Semi quantitative analysis was carried out with Image-Pro Plus 6 software for slicing staining. The results showed that the collagen composition of the scar tissue was significantly increased and the content of hyaluronic acid was not significantly changed.
In this study, we preliminarily explored the tissue engineering regeneration of the lamina propria. The three elements of tissue engineering are seed cells, scaffold materials, and cytokine. The second part of this study first carried out the culture of vocal fold fibroblasts (VFFs) in the seed cells of the seed cells in vitro. Fibroblasts were obtained and cultured in vitro with high glucose DMEM medium containing fetal bovine serum. The optimum conditions were found. The morphology and growth of the cells were observed under the inverted phase contrast microscope, and the cells were purified by passage. The specific protein Vimentin of the fibroblasts was detected by immunohistochemical method, and the result of Vimentin Yang was detected. The rate of sex was over 90%, which showed that the cultured cells were fibroblasts, and the positive rate was higher.
The third part of this study observed the compatibility and growth of cultured cells in the allogenic organism. We made Ad-EGFP markers for the cultured cells. 6 New Zealand rabbits were taken, 2 rabbits were randomly selected as a dry pregroup, 4 New Zealand rabbits were treated with acute injury model of bilateral vocal cords, and 104 / ml concentrations of Ad-EGFP were injected into the left vocal cord injury site. After the injections of the right vocal cord, 1ml was injected into the right vocal cord at the injury site of the right vocal cord as a control. After the cells were injected with the vocal cord, the growth of the rabbit was good, no infection, larynx edema, immune rejection and other reactions were obtained after.15 days, and the fluorescence microscope observed that the survival of VFFs. showed that the simple VFFs injection to the allogenic tissue was difficult. Survival, providing growth scaffolds for animal growth may be beneficial to growth.
In conclusion, this study conducted a preliminary exploration of vocal cord tissue engineering, prepared the seed cells, explored the compatibility of the seed cells in vivo, and prepared a soundscars model. In the follow-up study, we will construct the cell scaffold composite, transplant the sound scar model of the vocal cord and observe the regenerative repair of the vocal cord.

【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R767.92

【参考文献】