人中鼻甲来源嗅鞘细胞培养与鉴定的初步研究

doi:10.3877/cma.j.issn.2095-1221.2019.05.002

中华细胞与干细胞杂志(电子版) ›› 2019, Vol. 09 ›› Issue (05) : 262 -266. doi: 10.3877/cma.j.issn.2095-1221.2019.05.002

所属专题：文献；

论著

人中鼻甲来源嗅鞘细胞培养与鉴定的初步研究

陈恒¹, 宋振涛¹, 刘小盾¹, 曲廷瑜¹^,^†(

)

^1. 250012　济南，山东省齐鲁干细胞工程有限公司

收稿日期:2019-05-20 出版日期:2019-10-01
通信作者: 曲廷瑜
基金资助:
泰山学者建设工程专项(tshw201502002); 山东省2016年重点研发计划（创新性产业集群）(2016ZDJQ0104)

A preliminary study on culture and identification of olfactory ensheathing cells from human middle turbinate

Heng Chen¹, Zhentao Song¹, Xiaodun Liu¹, Tingyu Qu¹^,^†()

^1. Shandong Province Qilu Stem Cells Engineering Co., Ltd. Jinan 250021, China

Received:2019-05-20 Published:2019-10-01
Corresponding author: Tingyu Qu
About author:
Corresponding author: Qu Tingyu, Email:tqu2010@hotmail.com

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目的

探索一种从人中鼻甲分离培养嗅鞘细胞的方法并鉴定所获得的细胞。

方法

通过手术获取人中鼻甲黏膜组织，随后进行两步消化并剥离黏膜上皮组织，得到体积较小的组织块，再进行组织块原代（传代）培养并加压筛选，最终得到双极或多极样细胞并对获得的细胞进行免疫荧光鉴定。上皮样细胞比例、S100β和p75阳性细胞比例用±s表示，组间比较采用独立样本t检验。

结果

将不剥离上皮层与剥离上皮层培养的原代细胞中上皮样细胞的比例进行对比，发现上皮层组上皮样细胞比例为（92.23±3.93）%高于剥离上皮层组上皮样细胞的比例（77.63±2.97）%，差异具有统计学意义（t = 5.129，P = 0.007）。采用剥离上皮层法培养原代细胞，经过加压筛选的细胞呈现双极或多极样，符合嗅鞘细胞形态学特点。免疫荧光染色发现，S100β阳性细胞占总细胞量的（8.1±1.7）%，而p75阳性细胞占总细胞量的5%以下，达到国内外研究同等水平。

结论

通过使用两步消化法和加压筛选联合的方法从人中鼻甲粘膜组织中成功获得了人中鼻甲嗅鞘细胞，相比较传统方法，细胞分离培养周期明显缩短。

关键词: 中鼻甲, 嗅鞘细胞, 细胞培养, 鉴定

Objective

To explore a method for isolating, culturing, and characterizing the olfactory ensheathing cells from the human middle turbinate tissues obtained during surgery.

Methods

The mucosa tissues of the middle turbinate was collected during surgery, and the smaller tissue blocks were obtained by two-step digestion and stripping of the mucosal epithelial tissues. The small tissue blocks were then cultured and pressure screening were performed during the culture for isolating the bipolar and multipolar cells. These cells were further identified by a staining with an assay of immunofluorescence. The ratio of epithelial-like cells, S100β as well as p75 positive cells was expressed as ±s; and compared by independent sample t-test.

Results

Comparing the ratio of the epithelial-like cells in the primary cells cultured with or without the epithelial layer, the ratio of epithelial-like cells in the group with the epithelial layer was (92.23±3.93) %, which was higher than that in the group without the epithelial layer (77.63±2.97) % (t = 5.129, P = 0.007) . The primary cells were cultured by the stripping epithelial layer. The cells subjected to culture and pressure screening were bipolar or multipolar-like, which conformed to the morphological characteristics of olfactory ensheathing cells. Immunofluorescence staining confirmed that (8.1±1.7) % cells showed S100β positive and less than 5% cells showed p75 positive in the total population of the cells.

Conclusion

By using a two-step digestion method and a pressure-screening method, the human middle turbinate olfactory ensheathing cells were successfully obtained from the human nasal mucosa tissue, and the cell separation and culture period was significantly shortened compared with the conventional method.

Key words: Middle turbinate, Olfactory ensheathing cells, Cell culture, Identification

图2 原代细胞中上皮样细胞比例对比

图1 光镜下观察培养7 d的原代细胞（×100）

图3 光镜下观察加压筛选2 d后的传代细胞（×100）

图4 间接免疫荧光鉴定的传代细胞（×400）

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