人结肠腺癌细胞系Caco-2单细胞层短期培养法及其评价  

A new protocol for a short-term Caco-2 monolayer culture system and its evaluation

作　　者：祝欣刚[1] 杨青[1] 

机构地区：[1]复旦大学生命科学学院生物化学系,上海200433

出　　处：《复旦学报（医学版）》2012年第1期68-73,共6页Fudan University Journal of Medical Sciences

基　　金：国家自然科学基金项目(30873153);复旦大学-先声药业杯研究生药物创新研究计划~~

摘　　要：目的利用丁酸建立一种短期培养人结肠腺癌细胞系Caco-2(human colon adenocarcinoma cell line)单细胞层的方法。方法使用不同成分的含丁酸培养基培养Caco-2单细胞层,通过显微镜观察细胞形态学特点、测定跨膜电阻(transepithelial electrical resistance,TEER)和荧光黄通透量等指标,评价其完整性;通过检测碱性磷酸酶(alkaline phosphatase,AKP)活性,评价其细胞极化程度;通过RT-PCR评价其对主要药物转运蛋白表达的影响。结果使用短期培养法获得的Caco-2单细胞层形态完整,TEER>500Ω.cm2,漏出标志物荧光黄表观分配系数(apparent permeability,Papp)<5×10-5 cm/s,表明单细胞层的完整性良好。同时肠腔侧(apical,AP)AKP活性显著升高,表明单细胞层出现明显的极性分化。短期培养与21天培养所得的Caco-2单细胞层中,药物转运蛋白P-糖蛋白(P-glycoprotein,P-gp)和多药耐药相关蛋白2(multidrug resistance-associated protein 2,MRP2)的RNA表达水平基本一致。结论含丁酸的无血清培养基可促进Caco-2单细胞层的快速形成,利用该法建立的Caco-2细胞模型符合各项指标的要求,可用于研究口服药物转运的吸收机制。Objective To develop and evaluate a new protocol for a short-term Caco-2（human colon adenocarcinoma cell line） monolayer culture system with butyratic acid. Methods Caco-2 cell monolayer model was established with different culture systems and evaluated by morphology feature using inverted microscope and transepithelial electrical resistance（TEER） assay.Additionally,the model was further tested for the activity of alkaline phosphatase（AKP） and the apparent permeability（Papp） of standard compound fluorescein.Meanwhile,the RNA level of transporters P-glycoprotein（P-gp） and multidrug resistance-associated protein 2（MRP2） was examined by RFPCR. Results Caco-2 cell monolayer established with the short-term culture system showed good integrality of cellular morphology and cell monolayer reflected by TEER500 Ω·cm2,and Papp value of standard compound fluorescein 5×10-5cm/s and obvious polar differentiation of cell monolayer reflected by enhanced AKP activity on the apical（AP） side.Short-term culture had the similar RNA level of P-gp and MRP2 to 21-day duture. Conclusions The short-term established Caco-2 mono-layer can be used to study the intestinal absorption of orally administrated chemical components and their absorption mechanism.

关 键 词：CACO-2 单细胞层 短期培养 丁酸 药物转运 分化 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学] R965.1[医药卫生—基础医学]