兔气管黏膜上皮细胞原代培养的研究

发布时间：2018-05-31 17:19

本文选题：气管 + 上皮细胞　；参考：《扬州大学》2011年硕士论文

【摘要】：目的：目前,气管外伤、肿瘤切除等均可引起气管缺损,较为理想的治疗方法是行一期端端吻合。但当气管缺损长度超过一定的长度时,则无法进行无张力吻合,必须植入气管代替物才能重建其连续性,维持呼吸道通畅。因此,利用组织工程学的原理和技术在体外构建出具有气管主要生理功能的生物活性人工气管作为移植物修复气管缺损成为研究热点。但是人工气管置换气管后管腔内上皮细胞的再生依赖于从宿主气管邻近部分上皮细胞的迁移,通常造成靠近吻合口区域存在完整的上皮细胞,而管腔内表面的中部没有成熟的上皮细胞覆盖。缺乏发育良好的上皮细胞层常常导致管腔狭窄和肉芽增生,影响假体移植后管腔的开放而导致失败。气管黏膜上皮细胞的培养技术是解决该难题的重要工具之一,本研究拟建立气管黏膜上皮细胞原代培养模型,观察细胞在体外生长及传代的规律,为组织工程气管假体的上皮化提供技术支持。方法：一、分离培养：(1)从4月龄新西兰大耳兔取出气管(甲状软骨至气管分叉),剔除气管周围纤维结缔组织,冲洗气管至苍白并且无黏液为止。(2)剪开气管、撕取黏膜,将黏膜置于含有抗生素的PBS液中浸泡除菌。(3)将黏膜剪成约1m×1mm大小的小块。(4)将黏膜组织小块移入培养瓶中,置入培养箱培养。(5)对培养的细胞采用刮除法结合消化排除法纯化。二、细胞鉴定：对纯化后的细胞通过SABC法免疫组化鉴定、FITC标记的荧光抗体免疫荧光鉴定。三、传代与冻存：(1)对纯化的细胞进行传代研究,并观察细胞在体外的生长规律。(2)对纯化后的细胞进行冻存与复苏的研究。结果：一、细胞培养：(1)培养约5-6d可见组织块边缘少量上皮样细胞爬出。此后,组织块周围上皮样细胞覆盖面积逐渐增大,细胞呈现三角形、梭形、圆形、多角形、不规则形等。高倍镜下可见部分细胞顶面伸出纤毛,在培养液中不断摆动。10d后去除组织块,14-15d时,细胞基本长满瓶壁,有纤毛的细胞无增殖。(2)通过刮除法结合消化排除法纯化后的细胞纯度较高。二、细胞鉴定：(1)SABC法染色检测细胞中角蛋白,胞质棕黄色,胞核淡蓝色,结果表明分离培养的细胞为上皮细胞。(2)FITC标记的荧光抗体检测细胞中的角蛋白,胞质亮绿色,可见整个细胞形态,表明分离培养的细胞为上皮细胞。三、传代与冻存：(1)细胞可以连续传代至第5代,传代后细胞贴壁及生长良好,细胞随着代数的增加细胞体积逐渐变大。传代后未见有纤毛的细胞。第2-4代细胞贴壁及生长时问无明显差异,第5代细胞贴壁数量明显减少,生长缓慢,细胞体积与前几代相比明显变大。(2)本实验复苏冻存3个月的细胞,复苏后细胞存活率、贴壁率较高。经过本方法冻存与复苏的细胞,经过体外培养,细胞活性、增殖能力仍然能够恢复到冻存前水平。结论：本实验应用组织块法成功构建出一种较为经济、便捷、可传代、可重复的兔气管黏膜上皮细胞的原代培养模型。成功对培养细胞进行鉴定、传代与冻存的研究,为气管黏膜上皮细胞培养提供了技术可行性,为组织工程人工气管的上皮化提供理论基础与技术支持。
[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 .
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

【参考文献】