Caco-2细胞单层模型的建立与验证

Establishment and Validation of Caco-2 Cell Lines for Lntestinal Epithelial Permeability

目的研究建立Caco-2细胞模型的方法,并确认Caco-2细胞模型的正常生长特性,以在日常培养中进行监控。方法常规培养条件下,Caco-2细胞以每1 mL含8×104个的接种密度接种于Millicell插入式培养皿中,培养21 d后用光镜、电镜、细胞密度测定等方法检查细胞的形态学及生长特点,测定碱性磷酸酶活性、跨膜电阻、荧光素钠透过量以检验细胞单层的极化现象和致密程度。结果Caco-2细胞单层在生长10 d以后均匀、致密,21 d后微绒毛、碱性磷酸酶、紧密连接等呈不对称分布,跨膜电阻达到600Ω.cm2,荧光素钠透过量为4.16μg.h-1.cm-2,而同样条件下空白膜的透过量大于240μg.h-1.cm-2。结论本培养条件下,Caco-2细胞单层形态与小肠上皮细胞类似,跨膜电阻、标志物透过量均达到要求,可以作为研究小肠药物转运过程的体外模型。

OBJECTIVE To investigate the establishment of Caeo-2 cell monolayer model,and to identify Caeo-2＇s natural growth in order to monitor the daily cultivation. METHODS Caco-2 cell was cultivated in Millicell plate inserts with 8 × 104cells· mL^-1 cell density in standard procedures for 21 d. The morphological and growth characters of the monolayer were examined with optical microscope, electron microscope and cell density determination. The polarization and compactification of the monolayer was determined by the alkaline phosphatase activity, transepithelial electrical resistance（TEER） and apical-to-basolateral of sodium fluorescein across cell monolayers.RESULTS Caco-2 cell monolayer became well-proportioned and compact after 10 d growth. After 21 d, microvillus, alkaline phosphatase and tight conjunctions were asymmetrically distributed, TEER value reached 600Ω·cm^-2, and the transportation of sodium fluorescein was 4.16μg·h^-1·cm^-2 the transportation across non-cell membrane was more than 240 μg·h^-1·cm^-2 in the same conditions. CONCLUSION In these cultivation conditions, the Caco-2 cell monolayer had become an analogy of small intestinal epithelium. The value of TEER and transportation of sodium lluorescein all reached the criterions, which meant that the monolayer became an valuable model system for intestinal epithelial permeability. In this research, Caco-2 cell monolayer model was established and evaluated.