应用酶消化耳蜗基底膜上皮细胞的研究

发布时间：2018-07-07 08:30

本文选题：毛细胞前体细胞 + 细胞培养　；参考：《中华耳科学杂志》2016年02期

【摘要】：目的比较联合应用嗜热菌蛋白酶和I型胶原酶消化对耳蜗基底膜上皮细胞的分离效果。方法分离P0-3天SD大鼠基底膜,并将其分为四组,分别为:A组胰酶消化组;B组嗜热菌蛋白酶消化组;C组I型胶原酶消化组;D组嗜热菌蛋白酶和I型胶原酶消化组。收集各组消化的细胞进行悬浮培养和诱导分化,分别计数各组形成的细胞球数目,应用免疫荧光对获得的细胞来源进行鉴定,并比较各组Cytokeratin-18阳性细胞的百分率。结果从4种方法分离得到的细胞经培养后均可形成细胞球,表达干细胞标记物Nestin和细胞分裂标记物Brd U。获得细胞大部分均表达上皮来标记物E-cadherin和cytokeratin-18,而不表达间质细胞标志物Vimentin,经过诱导分化后可以分化成毛细胞样细胞。应用嗜热菌蛋白酶和I型胶原酶消化获得的细胞球数目显著高于其它组(P0.05);而采用胰酶消化获得的细胞cytokeratin-18阳性率显著低于其它组(P0.05)。结论联合应用嗜热菌蛋白酶和I型胶原酶消化耳蜗基底膜上皮细胞可以显著提高基底膜上皮细胞的分离效果,从而耳蜗前体细胞的研究提供有力研究基础。
[Abstract]:Objective to compare the effects of combined digestion of thermophilic protease and collagenase I on the isolation of cochlear basement membrane epithelial cells. Methods the basement membrane of SD rats was isolated from P0-3 day and divided into four groups: group A, trypsin digestion group, group B, thermophilic protease group, group C, group I, collagenase digestion group, group D, thermophilic protease and type I collagenase digestion group, respectively. The cells digested in each group were collected for suspension culture and induced differentiation. The number of cells formed in each group was counted. The source of the cells was identified by immunofluorescence and the percentage of Cytokeratin-18 positive cells in each group was compared. Results all the cells isolated from the four methods could form cell spheres and express nestin and Brd UU, the marker of stem cell and the marker of cell division. Most of the cells expressed E-cadherin and cytokeratin-18, but not the interstitial cell marker Vimentin, which could differentiate into hair-cell-like cells after induced differentiation. The number of cell balls digested with thermophilic protease and type I collagenase was significantly higher than that of other groups (P0.05), while the positive rate of cytokeratin-18 of cells digested with trypsin was significantly lower than that of other groups (P0.05). Conclusion the combination of thermophilic protease and collagenase I can significantly improve the isolation effect of basal membrane epithelial cells of cochlea and provide an effective basis for the study of cochlear precursor cells.
【作者单位】：第四军医大学第一附属医院耳鼻咽喉头颈外科;
【基金】：国家自然科学基金海外重大合作项目(81120108008);国家自然科学基金(81271069)~~
【分类号】：R764

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