本文選題：毛細(xì)胞前體細(xì)胞 + 細(xì)胞培養(yǎng)　； 參考：《中華耳科學(xué)雜志》2016年02期

【摘要】：目的比較聯(lián)合應(yīng)用嗜熱菌蛋白酶和I型膠原酶消化對耳蝸基底膜上皮細(xì)胞的分離效果。方法分離P0-3天SD大鼠基底膜,并將其分為四組,分別為:A組胰酶消化組;B組嗜熱菌蛋白酶消化組;C組I型膠原酶消化組;D組嗜熱菌蛋白酶和I型膠原酶消化組。收集各組消化的細(xì)胞進行懸浮培養(yǎng)和誘導(dǎo)分化,分別計數(shù)各組形成的細(xì)胞球數(shù)目,應(yīng)用免疫熒光對獲得的細(xì)胞來源進行鑒定,并比較各組Cytokeratin-18陽性細(xì)胞的百分率。結(jié)果從4種方法分離得到的細(xì)胞經(jīng)培養(yǎng)后均可形成細(xì)胞球,表達(dá)干細(xì)胞標(biāo)記物Nestin和細(xì)胞分裂標(biāo)記物Brd U。獲得細(xì)胞大部分均表達(dá)上皮來標(biāo)記物E-cadherin和cytokeratin-18,而不表達(dá)間質(zhì)細(xì)胞標(biāo)志物Vimentin,經(jīng)過誘導(dǎo)分化后可以分化成毛細(xì)胞樣細(xì)胞。應(yīng)用嗜熱菌蛋白酶和I型膠原酶消化獲得的細(xì)胞球數(shù)目顯著高于其它組(P0.05);而采用胰酶消化獲得的細(xì)胞cytokeratin-18陽性率顯著低于其它組(P0.05)。結(jié)論聯(lián)合應(yīng)用嗜熱菌蛋白酶和I型膠原酶消化耳蝸基底膜上皮細(xì)胞可以顯著提高基底膜上皮細(xì)胞的分離效果,從而耳蝸前體細(xì)胞的研究提供有力研究基礎(chǔ)。
[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.
【作者單位】： 第四軍醫(yī)大學(xué)第一附屬醫(yī)院耳鼻咽喉頭頸外科;
【基金】：國家自然科學(xué)基金海外重大合作項目(81120108008);國家自然科學(xué)基金(81271069)~~
【分類號】：R764

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