Caco-2建立肠黏膜屏障模型及其在通透性研究中的应用

Establishment of Caco-2 cell monolayer model and barrier permeability

目的利用Caco-2细胞建立体外肠黏膜屏障模型，系统评价并初步探讨其在炎症损伤后黏膜通透性改变中的应用。方法体外培养Caco-2细胞，接种于Transwell板上，每日观察细胞形态；自培养第5天起，隔日测细胞膜的跨上皮电阻（transepithelialelectricalresistance，TEER）；对于TEER值达标准的孔测定荧光黄透过率，进行透射电镜的完整性验证。应用培养21d的Caco-2细胞屏障，加入不同浓度（0、50、100、200nmoWL）的血小板活化因子（platelet—activatingfactor，PAF）孵育24h，光镜、电镜观察形态学改变，检测TEER和荧光黄透过率，应用间接免疫荧光和Westernblot观察ZO-1蛋白的分布及表达。结果Caco-2细胞单层TEER从第5～15天逐渐增加，在第15天已经达到600Ω·cm2，平台期保持至第21天；在此期间，细胞形成紧密单层，电镜下细胞呈高分化，细胞间形成紧密连接，绒毛整齐，极性形成；荧光黄透过量极低，体外肠上皮细胞屏障形成。加入PAF后，以100nmoVL对黏膜屏障通透性影响最大：电镜下见紧密连接结构破坏、断裂，细胞表面微绒毛脱落、稀疏；免疫荧光可见紧密连接的标志蛋白ZO-1荧光信号减弱，ZO-1环断裂，胞浆内可见阳性染色，提示其向膜下转移。此时，TEER值下降，荧光黄透过量明显增加，与对照组比较差异有统计学意义（P〈0．01），与形态学改变规律一致；ZO—1蛋白表达亦降至最低，与对照组相比差异有统计学意义（P〈0．01）。结论经形态学及细胞通透性验证，培养2～3周的Caco-2细胞可形成肠屏障模型，用于体外肠黏膜屏障的研究；PAF影响紧密连接相关蛋白的表达，破坏紧密连接结构，从而影响肠黏膜屏障通透性。

Objective To establish and evaluate intestinal epithelial barrier model using Caco-2 cell so as to play a foundation for next study of barrier permeability. Methods Caco-2 cells were cultured in vitro then seeded into Transwell cell culture inserts. The permeability of the intestinal epithelial barrier was detected by transepithelial electrical resistance （TEER） and lucifer yellow flux, and verified by transmission electron microscope. Different concentrations of PAF （0,50,100, and 200 nmol/L） were exposed for 24 hours to Caco-2 monolayer when cultured 21 days. The tight junction was observed under transmission electron microscope. Assessment of ZO-1 protein localization and expression were detected by immunofluorescence and Western blot analysis. Results Cultured Caco-2 cell confluencd as monolayer with time passed. From 5th day, TEER increased, then reached 600Ω. cm2 at 15th day and lasted to 21st day, there was little flux of lucifer yellow, transmission electron microscopy also found cells differentiated better,had well-arranged villi and polarity alined as monolayer, forming completed tight junction which was the marker of intestinal epithelial barrier model in vitro. TEER decreased and lucifer yellow flux increased in cells exposed to PAF. The permeability reached the peak when exposed to 100 nmol/L PAF（P 〈 0. 01 ）, tight junction disrupted, ZO-1 protein expression downregulated, abnormal localization and distribution was assessed by immunofluorescence staining. Conclusion Cultured Caco-2 cells for 2-3w can be used to study intestinal epithelial barrier as a model in vitro. PAF increased intestinal epi- thelial permeability, which would correlate to the decreased protein expression and abnormal distribution of ZO-1.