本文選題：聲帶再生 + 成纖維細胞��； 參考：《中國人民解放軍軍醫(yī)進修學院》2010年碩士論文

【摘要】： 正常聲帶組織因外傷或疾病損傷后所形成的纖維組織替代產物形成了聲帶瘢痕。由于瘢痕組織使聲帶變得堅硬,影響了聲帶固有層的粘彈性(viscoelastic strength),改變了聲帶的生物力學特性,從而減弱聲帶振動黏膜波的產生而影響發(fā)聲。目前,尚未找到一種理想的預防和治療聲帶瘢痕的方法。近年來,組織工程學技術在組織器官重建方面取得了令人矚目的進展,其主要原理是以少量種子細胞經體外擴增后與生物材料、細胞因子結合,修復較大的組織或器官缺損,重建組織器官的生理功能,最終達到完美的形態(tài)甚至功能修復的目的。將組織工程學的技術方法應用于喉科,有望在未來實現(xiàn)聲帶,甚至喉的再生。人類的聲帶組織是一個高度分化的分層結構,從組織學結構上可分上皮層、固有層和肌肉層(甲杓肌)。聲帶固有層又分為固有淺層(Reinke's層),固有中層和固有深層。這種分層在描述聲帶結構功能方面具有重要意義。固有層是聲帶最重要的特征性結構,主要包括細胞及細胞外基質成分。組成固有層的細胞為數較少,主要細胞有：成纖維細胞,肌成纖維細胞和巨噬細胞。成纖維細胞是維持固有層結構與功能的重要細胞,主要分布在固有層深層,其調控細胞間質的沉著、降解及重新分布；細胞外基質成分包括纖維蛋白和間隙蛋白。 本研究第一部分我們首先制作了兔聲帶瘢痕模型,并對聲帶瘢痕進行初步研究。在支撐喉鏡下,用喉鉗夾取雙側聲帶前中部組織(未達肌層),未手術者作為正常對照,1個月后處死動物,收獲聲帶組織,采用Masson三色染色觀察聲帶膠原,采用經過透明質酸酶處理的Alcian blue染色觀察透明質酸。利用Image-Pro Plus 6.0軟件進行對切片染色進行半定量分析,實驗結果表明瘢痕聲帶的膠原成分明顯增多,透明質酸含量無明顯改變。 本研究中,我們對聲帶固有層的組織工程學再生進行了初步探索。組織工程學的三個要素是種子細胞,支架材料,和細胞因子。本研究第二部分首先進行了種子細胞-兔聲帶成纖維細胞(vocal fold fibroblasts,VFFs)的體外培養(yǎng)。取兔聲帶組織,消化法獲取成纖維細胞,用含有胎牛血清的高糖DMEM培養(yǎng)基體外培養(yǎng),摸索出最佳培養(yǎng)條件。在倒置相差顯微鏡下觀察細胞的形態(tài)及生長狀況,并經過傳代使細胞純化。利用免疫組化方法,檢測成纖維細胞的特異性蛋白Vimentin,結果Vimentin陽性率90%以上,證明所培養(yǎng)的細胞為成纖維細胞,且陽性率較高。 本研究第三部分觀察培養(yǎng)的細胞在異體生物體內相容性及生長情況。我們對培養(yǎng)的細胞進行Ad-EGFP標記。取新西蘭兔6只,隨機選取2只兔作為無干預組,4只新西蘭兔做雙側聲帶急性損傷模型,左側聲帶損傷部位注射104個／ml濃度的Ad-EGFP標記的細胞混懸液,向右側聲帶損傷部位注入生理鹽水1ml作為對照。聲帶注射細胞后,兔生長情況良好,均未發(fā)生感染、喉水腫,免疫排斥等反應。15天后收獲聲帶組織,熒光顯微鏡下觀察未發(fā)現(xiàn)成活的VFFs。說明單純VFFs注射到異體組織很難存活,為其在動物體內生長提供生長支架可能會利于生長。 總之,本研究對聲帶組織工程學進行了初步探索,制備了種子細胞,探索了種子細胞體內相容性,制備了聲帶瘢痕動物模型,在后續(xù)的研究中,我們將構建細胞-支架材料復合物,移植入聲帶瘢痕模型,觀察聲帶再生修復情況。
[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.

【學位授予單位】：中國人民解放軍軍醫(yī)進修學院
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R767.92

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