大鼠结肠上皮细胞分离及培养方法的建立

Establishment of a method for isolation and primary culture of rat colonic epithelial cells

目的:建立大鼠结肠上皮细胞的原代培养方法,为结肠上皮形态及功能研究提供理想的细胞模型.方法:采用6-15日龄乳鼠,取结肠剪碎、反复清洗后,弃上清,加10 mL、0.1%Ⅰ型胶原酶和透明质酸酶联合配制的消化液,37℃消化25 min,分离上皮细胞团;吹打消化液,取上清,加入培养基重悬反复离心3次后,细胞沉淀接种于含100 mL/L胎牛血清的完全培养基中,37℃、50 mL/L CO2细胞培养箱中培养.采用差速贴壁法及相差消化法去除成纤维细胞,细胞贴壁长满瓶80%-90%后用2.5 g/L的胰酶消化液消化并传代.结果:成功分离结肠上皮细胞团且活力较高.24 h后可见部分细胞团贴壁,贴壁细胞为多角形,4-8 d逐渐融合成片,呈现明显的"铺路石"样.细胞经传代处理后成纤维细胞明显减少.免疫荧光染色及透射电镜观察鉴定为上皮细胞.冻存处理复苏后细胞状态较佳.结论:通过该法可建立较稳定的大鼠结肠上皮细胞原代培养体系,为结肠上皮生理、病理及药物作用机制提供了体外研究平台.

AIM： To establish a method for isolation and primary culture of rat colonic epithelial cells which can be used as an ideal cell model for the study of colonic epithelial morphology and function in vitro.METHODS： Colons obtained from suckling rats（6-15 d） were cut into small pieces,washed,and digested with 0.1% collagenase I and hyaluronidase for 25 min at 37 ℃ to separate colonic epithelial cell clusters.After digestion,the supernatant was transferred into a new tube and DMEM medium was added.After centrifuging 3 times,cells were cultured in DMEM solution containing 100 mL/L fetal bovine serum in a CO 2 incubator with a saturated humidity at 37 ℃.Fibroblasts were removed using phase difference digestion and adherence.When 80%-90% of the cells were adherent to culture plates,cells were passaged by trypsin digestion.RESULTS： Colonic epithelial cell clusters were successfully obtained,which showed high viability and became adherent after 24 hours of culture.The cells were typically polygonal in shape and grew into pavestone-like monolayers gradually in 4-8 days,showing excellent proliferative ability.Fibroblasts were significantly decreased after several passages.The colonic epithelial cells were identified by the ways of immunofluorescence staining and TEM observation.The cells were in good condition after being frozen and thawed.CONCLUSION： The above mentioned method allows establishing stable primary colonic epithelial cell lines,which could provide an in vitro platform for the study of colonic epithelial physiology and pathology.