本文選題：氣管 + 上皮細胞��； 參考：《揚州大學》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。但當氣管缺損長度超過一定的長度時,則無法進行無張力吻合,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,利用組織工程學的原理和技術在體外構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氣管缺損成為研究熱點。但是人工氣管置換氣管后管腔內上皮細胞的再生依賴于從宿主氣管鄰近部分上皮細胞的遷移,通常造成靠近吻合口區(qū)域存在完整的上皮細胞,而管腔內表面的中部沒有成熟的上皮細胞覆蓋。缺乏發(fā)育良好的上皮細胞層常常導致管腔狹窄和肉芽增生,影響假體移植后管腔的開放而導致失敗。氣管黏膜上皮細胞的培養(yǎng)技術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胞原代培養(yǎng)模型,觀察細胞在體外生長及傳代的規(guī)律,為組織工程氣管假體的上皮化提供技術支持。 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)對培養(yǎng)的細胞采用刮除法結合消化排除法純化。 二、細胞鑒定：對純化后的細胞通過SABC法免疫組化鑒定、FITC標記的熒光抗體免疫熒光鑒定。 三、傳代與凍存：(1)對純化的細胞進行傳代研究,并觀察細胞在體外的生長規(guī)律。(2)對純化后的細胞進行凍存與復蘇的研究。 結果： 一、細胞培養(yǎng)：(1)培養(yǎng)約5-6d可見組織塊邊緣少量上皮樣細胞爬出。此后,組織塊周圍上皮樣細胞覆蓋面積逐漸增大,細胞呈現(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胞頂面伸出纖毛,在培養(yǎng)液中不斷擺動。10d后去除組織塊,14-15d時,細胞基本長滿瓶壁,有纖毛的細胞無增殖。(2)通過刮除法結合消化排除法純化后的細胞純度較高。 二、細胞鑒定：(1)SABC法染色檢測細胞中角蛋白,胞質棕黃色,胞核淡藍色,結果表明分離培養(yǎng)的細胞為上皮細胞。(2)FITC標記的熒光抗體檢測細胞中的角蛋白,胞質亮綠色,可見整個細胞形態(tài),表明分離培養(yǎng)的細胞為上皮細胞。 三、傳代與凍存：(1)細胞可以連續(xù)傳代至第5代,傳代后細胞貼壁及生長良好,細胞隨著代數(shù)的增加細胞體積逐漸變大。傳代后未見有纖毛的細胞。第2-4代細胞貼壁及生長時問無明顯差異,第5代細胞貼壁數(shù)量明顯減少,生長緩慢,細胞體積與前幾代相比明顯變大。(2)本實驗復蘇凍存3個月的細胞,復蘇后細胞存活率、貼壁率較高。經(jīng)過本方法凍存與復蘇的細胞,經(jīng)過體外培養(yǎng),細胞活性、增殖能力仍然能夠恢復到凍存前水平。 結論： 本實驗應用組織塊法成功構建出一種較為經(jīng)濟、便捷、可傳代、可重復的兔氣管黏膜上皮細胞的原代培養(yǎng)模型。成功對培養(yǎng)細胞進行鑒定、傳代與凍存的研究,為氣管黏膜上皮細胞培養(yǎng)提供了技術可行性,為組織工程人工氣管的上皮化提供理論基礎與技術支持。
[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 .
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329

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相關期刊論文 前3條

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本文編號：1960663