本文選題：氣管 + 上皮細(xì)胞　； 參考：《揚(yáng)州大學(xué)》2011年碩士論文

【摘要】：目的： 目前,氣管外傷、腫瘤切除等均可引起氣管缺損,較為理想的治療方法是行一期端端吻合。但當(dāng)氣管缺損長(zhǎng)度超過一定的長(zhǎng)度時(shí),則無法進(jìn)行無張力吻合,必須植入氣管代替物才能重建其連續(xù)性,維持呼吸道通暢。因此,利用組織工程學(xué)的原理和技術(shù)在體外構(gòu)建出具有氣管主要生理功能的生物活性人工氣管作為移植物修復(fù)氣管缺損成為研究熱點(diǎn)。但是人工氣管置換氣管后管腔內(nèi)上皮細(xì)胞的再生依賴于從宿主氣管鄰近部分上皮細(xì)胞的遷移,通常造成靠近吻合口區(qū)域存在完整的上皮細(xì)胞,而管腔內(nèi)表面的中部沒有成熟的上皮細(xì)胞覆蓋。缺乏發(fā)育良好的上皮細(xì)胞層常常導(dǎo)致管腔狹窄和肉芽增生,影響假體移植后管腔的開放而導(dǎo)致失敗。氣管黏膜上皮細(xì)胞的培養(yǎng)技術(shù)是解決該難題的重要工具之一,本研究擬建立氣管黏膜上皮細(xì)胞原代培養(yǎng)模型,觀察細(xì)胞在體外生長(zhǎng)及傳代的規(guī)律,為組織工程氣管假體的上皮化提供技術(shù)支持。 方法： 一、分離培養(yǎng)：(1)從4月齡新西蘭大耳兔取出氣管(甲狀軟骨至氣管分叉),剔除氣管周圍纖維結(jié)締組織,沖洗氣管至蒼白并且無黏液為止。(2)剪開氣管、撕取黏膜,將黏膜置于含有抗生素的PBS液中浸泡除菌。(3)將黏膜剪成約1m×1mm大小的小塊。(4)將黏膜組織小塊移入培養(yǎng)瓶中,置入培養(yǎng)箱培養(yǎng)。(5)對(duì)培養(yǎng)的細(xì)胞采用刮除法結(jié)合消化排除法純化。 二、細(xì)胞鑒定：對(duì)純化后的細(xì)胞通過SABC法免疫組化鑒定、FITC標(biāo)記的熒光抗體免疫熒光鑒定。 三、傳代與凍存：(1)對(duì)純化的細(xì)胞進(jìn)行傳代研究,并觀察細(xì)胞在體外的生長(zhǎng)規(guī)律。(2)對(duì)純化后的細(xì)胞進(jìn)行凍存與復(fù)蘇的研究。 結(jié)果： 一、細(xì)胞培養(yǎng)：(1)培養(yǎng)約5-6d可見組織塊邊緣少量上皮樣細(xì)胞爬出。此后,組織塊周圍上皮樣細(xì)胞覆蓋面積逐漸增大,細(xì)胞呈現(xiàn)三角形、梭形、圓形、多角形、不規(guī)則形等。高倍鏡下可見部分細(xì)胞頂面伸出纖毛,在培養(yǎng)液中不斷擺動(dòng)。10d后去除組織塊,14-15d時(shí),細(xì)胞基本長(zhǎng)滿瓶壁,有纖毛的細(xì)胞無增殖。(2)通過刮除法結(jié)合消化排除法純化后的細(xì)胞純度較高。 二、細(xì)胞鑒定：(1)SABC法染色檢測(cè)細(xì)胞中角蛋白,胞質(zhì)棕黃色,胞核淡藍(lán)色,結(jié)果表明分離培養(yǎng)的細(xì)胞為上皮細(xì)胞。(2)FITC標(biāo)記的熒光抗體檢測(cè)細(xì)胞中的角蛋白,胞質(zhì)亮綠色,可見整個(gè)細(xì)胞形態(tài),表明分離培養(yǎng)的細(xì)胞為上皮細(xì)胞。 三、傳代與凍存：(1)細(xì)胞可以連續(xù)傳代至第5代,傳代后細(xì)胞貼壁及生長(zhǎng)良好,細(xì)胞隨著代數(shù)的增加細(xì)胞體積逐漸變大。傳代后未見有纖毛的細(xì)胞。第2-4代細(xì)胞貼壁及生長(zhǎng)時(shí)問無明顯差異,第5代細(xì)胞貼壁數(shù)量明顯減少,生長(zhǎng)緩慢,細(xì)胞體積與前幾代相比明顯變大。(2)本實(shí)驗(yàn)復(fù)蘇凍存3個(gè)月的細(xì)胞,復(fù)蘇后細(xì)胞存活率、貼壁率較高。經(jīng)過本方法凍存與復(fù)蘇的細(xì)胞,經(jīng)過體外培養(yǎng),細(xì)胞活性、增殖能力仍然能夠恢復(fù)到凍存前水平。 結(jié)論： 本實(shí)驗(yàn)應(yīng)用組織塊法成功構(gòu)建出一種較為經(jīng)濟(jì)、便捷、可傳代、可重復(fù)的兔氣管黏膜上皮細(xì)胞的原代培養(yǎng)模型。成功對(duì)培養(yǎng)細(xì)胞進(jìn)行鑒定、傳代與凍存的研究,為氣管黏膜上皮細(xì)胞培養(yǎng)提供了技術(shù)可行性,為組織工程人工氣管的上皮化提供理論基礎(chǔ)與技術(shù)支持。
[Abstract]:Purpose :

This study is to establish the primary culture model of tracheal mucosa epithelial cells , which is one of the important tools to solve the problem , and to provide technical support for the epithelialization of tracheal prosthesis in tissue engineering .

Method :

The method comprises the following steps : ( 1 ) taking out a trachea ( thyroid cartilage to trachea bifurcation ) from a New Zealand big ear rabbit of 4 months old , removing fibrous connective tissue around the trachea , washing the trachea to be pale and no mucus .

2 . Cell identification : The purified cells were immunohistochemically identified by SABC method , and the FITC - labeled fluorescent antibody was identified by immunofluorescence .

( 3 ) passaging and freezing : ( 1 ) carrying out passage studies on purified cells and observing the growth regulation of the cells in vitro ; ( 2 ) carrying out the research on the freezing and recovery of purified cells .

Results :

1 . Cell culture : ( 1 ) culturing about 5 - 6 days to obtain a small amount of epithelial - like cells from the periphery of the tissue mass . After that , the area of the epithelial - like cells around the tissue mass gradually increases , and the cells appear triangular , fusiform , circular , polygonal , irregular , etc . After 10d , the top surface of the visible part of the cell stretches out of the ciliate , the cells are completely filled with the wall of the bottle after 10d , and the ciliated cells are non - proliferation .

2 . Cell identification : ( 1 ) SABC staining was used to detect the keratin in the cells , the cytoplasm was brown - yellow , and the nucleus was pale blue . The results showed that the isolated cultured cells were epithelial cells . ( 2 ) The FITC - labeled fluorescent antibody detected the keratin in the cells , the cytoplasm was bright green , and the whole cell morphology was seen , indicating that the isolated cultured cells were epithelial cells .

( 2 ) There was no significant difference in the number of cells in the second - fourth generation . The cells were cultured in vitro , cell activity and proliferation ability were still restored to the pre - freezing level .

Conclusion :

In this experiment , the primary culture model of rabbit tracheal mucosa epithelial cells was successfully constructed by using tissue block method . The culture cells were identified , passaged and frozen , which provided the technical feasibility for the culture of tracheal mucosa epithelial cells , which provided theoretical basis and technical support for the epithelialization of the artificial trachea of tissue engineering .
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 鄧三明,汪健,連耀國(guó),倪云峰,盧強(qiáng),楊曄,李小飛;四種氣管上皮細(xì)胞分離培養(yǎng)方法的比較研究[J];醫(yī)學(xué)研究生學(xué)報(bào);2005年10期

2 張龍芳;崔鵬程;陳文弦;劉智;李貴澤;孫永柱;;兔氣管纖毛上皮細(xì)胞原代培養(yǎng)的生物學(xué)特性及其體外生長(zhǎng)規(guī)律[J];中國(guó)臨床康復(fù);2006年01期

3 劉福青;;組織工程化氣管的種子細(xì)胞研究[J];中國(guó)組織工程研究與臨床康復(fù);2008年29期

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本文編號(hào)：1960663