新生大鼠小肠上皮细胞分离培养研究

STUDIES ON ISOLATION AND PRIMARY CULTURE OF NEONATE RAT INTESTINAL EPITHELIAL CELL

本实验比较了4种分离大鼠IEC的方法,结果显示联合应用粗胶原酶和中性蛋白酶分离效果最好,细胞贴壁生长能力强。胶原涂膜改善玻璃培养瓶或盖玻片表面的性状有利于细胞贴壁生长。细胞的增殖依赖于培养液的质量、成分及细胞间的相互作用。培养细胞一般1～2天贴壁,7～8天明显增殖,10～14天汇合成片。培养细胞细胞角质蛋白、碱性磷酸酶染色阳性,光镜和电镜检查均显示为IEC。本文所建立的新生大鼠IEC体外培养方法为研究IEC生理和病理提供了一个十分有用的实验模型。

Trypsin, collagenase Ⅰ and combined dispase (grade Ⅰ) and collagenase Ⅰ or collagenase Ⅺ were used to isolate neonate rat intestinal epithelial cell (IEC) and the results were compared and the optimal culture conditions were determined. We developed the technique of isolation and primary culture of neonate rat IEC. The results showed that satisfactory isolation of the epithelia and good attachment were achieved by using the collagenase Ⅺ dispase (grade Ⅰ) di-gestion. Coating the glass culture flaskes with bovine dermal collagens or rat tail tendon col-lagens I was beneficial to the attachment of IEC in culture. IEC proliferation in vitro is ini-tially dependent upon the factors produced by heterogeneous mesenchymal celis, also critically dependent upon the quality of the culture medium and constituents used. IEC attached within 1-2 days, proliferated obviously at 7-8 days. Cultures reached conflunce within 10-14 days. Immunocytochemical characterization showed that more than 90% attached cells were positive for cytokeratin and epithelial cell membrane antigen. Histochemical staining for alkaline pho-sphatase (AKP) revealed that AKP activity was expressed in more than 90% attached cells. Electronmicroscopy confirmed apical microvilli in IEC. The results provide a very useful expe-rimental model to study physiology and pathology of IEC.