本文選題：聲帶再生 + 成纖維細(xì)胞��； 參考：《中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)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(chǎn)物形成了聲帶瘢痕。由于瘢痕組織使聲帶變得堅(jiān)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(xué)特性,從而減弱聲帶振動(dòng)黏膜波的產(chǎn)生而影響發(fā)聲。目前,尚未找到一種理想的預(yù)防和治療聲帶瘢痕的方法。近年來(lái),組織工程學(xué)技術(shù)在組織器官重建方面取得了令人矚目的進(jìn)展,其主要原理是以少量種子細(xì)胞經(jīng)體外擴(kuò)增后與生物材料、細(xì)胞因子結(jié)合,修復(fù)較大的組織或器官缺損,重建組織器官的生理功能,最終達(dá)到完美的形態(tài)甚至功能修復(fù)的目的。將組織工程學(xué)的技術(shù)方法應(yīng)用于喉科,有望在未來(lái)實(shí)現(xiàn)聲帶,甚至喉的再生。人類的聲帶組織是一個(gè)高度分化的分層結(jié)構(gòu),從組織學(xué)結(jié)構(gòu)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(jié)構(gòu)功能方面具有重要意義。固有層是聲帶最重要的特征性結(jié)構(gòu),主要包括細(xì)胞及細(xì)胞外基質(zhì)成分。組成固有層的細(xì)胞為數(shù)較少,主要細(xì)胞有：成纖維細(xì)胞,肌成纖維細(xì)胞和巨噬細(xì)胞。成纖維細(xì)胞是維持固有層結(jié)構(gòu)與功能的重要細(xì)胞,主要分布在固有層深層,其調(diào)控細(xì)胞間質(zhì)的沉著、降解及重新分布；細(xì)胞外基質(zhì)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(duì)聲帶瘢痕進(jìn)行初步研究。在支撐喉鏡下,用喉鉗夾取雙側(cè)聲帶前中部組織(未達(dá)肌層),未手術(shù)者作為正常對(duì)照,1個(gè)月后處死動(dòng)物,收獲聲帶組織,采用Masson三色染色觀察聲帶膠原,采用經(jīng)過(guò)透明質(zhì)酸酶處理的Alcian blue染色觀察透明質(zhì)酸。利用Image-Pro Plus 6.0軟件進(jìn)行對(duì)切片染色進(jìn)行半定量分析,實(shí)驗(yàn)結(jié)果表明瘢痕聲帶的膠原成分明顯增多,透明質(zhì)酸含量無(wú)明顯改變。 本研究中,我們對(duì)聲帶固有層的組織工程學(xué)再生進(jìn)行了初步探索。組織工程學(xué)的三個(gè)要素是種子細(xì)胞,支架材料,和細(xì)胞因子。本研究第二部分首先進(jìn)行了種子細(xì)胞-兔聲帶成纖維細(xì)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(xì)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(xì)胞的形態(tài)及生長(zhǎng)狀況,并經(jīng)過(guò)傳代使細(xì)胞純化。利用免疫組化方法,檢測(cè)成纖維細(xì)胞的特異性蛋白Vimentin,結(jié)果Vimentin陽(yáng)性率90%以上,證明所培養(yǎng)的細(xì)胞為成纖維細(xì)胞,且陽(yáng)性率較高。 本研究第三部分觀察培養(yǎng)的細(xì)胞在異體生物體內(nèi)相容性及生長(zhǎng)情況。我們對(duì)培養(yǎng)的細(xì)胞進(jìn)行Ad-EGFP標(biāo)記。取新西蘭兔6只,隨機(jī)選取2只兔作為無(wú)干預(yù)組,4只新西蘭兔做雙側(cè)聲帶急性損傷模型,左側(cè)聲帶損傷部位注射104個(gè)／ml濃度的Ad-EGFP標(biāo)記的細(xì)胞混懸液,向右側(cè)聲帶損傷部位注入生理鹽水1ml作為對(duì)照。聲帶注射細(xì)胞后,兔生長(zhǎng)情況良好,均未發(fā)生感染、喉水腫,免疫排斥等反應(yīng)。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(shuō)明單純VFFs注射到異體組織很難存活,為其在動(dòng)物體內(nèi)生長(zhǎng)提供生長(zhǎng)支架可能會(huì)利于生長(zhǎng)。 總之,本研究對(duì)聲帶組織工程學(xué)進(jìn)行了初步探索,制備了種子細(xì)胞,探索了種子細(xì)胞體內(nèi)相容性,制備了聲帶瘢痕動(dòng)物模型,在后續(xù)的研究中,我們將構(gòu)建細(xì)胞-支架材料復(fù)合物,移植入聲帶瘢痕模型,觀察聲帶再生修復(fù)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(xué)位授予單位】：中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R767.92

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本文編號(hào)：1824369