豚鼠鼻黏膜纤毛细胞的手工显微分离

目的：由于呼吸道纤毛取材困难，并且生长在较厚的黏膜上，透光性较差而难以进行直接观察，因此人们尝试用不同的方法获取纤毛细胞。实验拟将手工显微分离技术应用于豚鼠鼻黏膜纤毛细胞的分离，为进一步实验提供纯净的目的细胞。　方法：实验于2007-03/11在解放军总医院耳鼻咽喉头颈外科研究所完成。白色健康豚鼠20只，在显微镜下用显微器械将豚鼠鼻黏膜的纤毛细胞层和黏膜下层进行分离，取手工显微分离的黏膜和未行分离的全层黏膜行苏木精-伊红染色，明确所分离组织层是否为黏膜纤毛细胞层。再将纤毛细胞层酶解，低倍镜下观察单个鼻黏膜纤毛细胞个数，纤毛细胞团大小，杂质细胞多数。高倍镜下观察纤毛细胞的形态。　结果：苏木精-伊红染色见手工显微分离的组织层为纤毛细胞和基底细胞层，较薄，不含有黏膜下层。低倍镜下可见手工显微分离后纤毛细胞较密集，单个鼻黏膜纤毛细胞较多，纤毛细胞团较小，每个细胞团平均有7个纤毛细胞左右，且纤毛细胞团下面无其他细胞。高倍镜下可见手工显微分离的单离纤毛细胞膜光滑，折光性好，纤毛摆动活跃，存活时间明显延长，24 h 仍有50%存活。与传统分离技术相比，手工显微分离技术在目的细胞纯度和活性上有显著优势（P < 0.05）。结论：将手工显微分离技术应用于纤毛细胞分离和纯化降低了杂质细胞干扰，提高了细胞活性，满足了提供纯净目的细胞的实验要求。

Manual microdissection of ciliated cells from guinea pig''s nasal epithelium

AIM: Respiratory tract cilia are difficult to harvest and observe due to weak transparence, which results from their location in thick mucosa, so people always try to obtain ciliated cells with many methods. This study was designed to apply manual microdissection to isolate ciliated cells from guinea pig's nasal epithelium and offer pure cells to further research. METHODS: The experiment was carried out at the Institute of Otolaryngology Head and Neck Surgery in Chinese PLA General Hospital from March of November in 2007. Twenty healthy white guinea pigs were recruited in this study. The ciliated cells layer was isolated from submucous layer by instruments for microsurgery under the light microscope. The isolated mucosa and untreated mucosa layer were stained with hematoxylin-eosin in order to verify the isolated layer is mucous ciliated cells layer. The protease-treated ciliated cells layer was viewed under low magnification microscope, to count number of single ciliated cells and impure cells, as well as size of ciliated cells group. The morphology of ciliated cells was observed under high magnification microscope. RESULTS: Result of hematoxylin-eosin stain confirmed that the cell layer by manual microdissection was ciliated cells and basal cell layer, which were thin without submucous layer. Under low magnification microscope after manual microdissection, the density of isolated ciliated cells was larger, number of single ciliated cells was more and size of ciliated cells group was smaller. There were about 7 ciliated cells in a cell group, and no other cells were observed. Under high magnification microscope, the isolated ciliated cells by manual microdissection were smooth, good in light refraction, and active for cilia beat. The survival time was prolonged obviously, still 50% cells were alive 24 hours later. Compared with conventional isolation technique, manual microdissection was significantly predominant in the purity and activity of cells (P < 0.05). CONCLUSION: Manual microdissection in the isolation and purification of ciliated cells, can reduce the interference of impure cells, increase the cell activity, and is a satisfied technique to offer pure cells.