体外肝细胞三明治培养动态变化及其在药源性肝损伤中的应用

Dynamic changes in sandwich-cultured hepatocytes and applications for drug-induced injury

目的:本文采用三明治构型培养原代大鼠肝细胞,综合观测其体外极性的动态形成过程,为体外药物肝毒性研究建立合适的评价模型。方法:三明治构型培养Wistar大鼠肝细胞,选取不同培养时间细胞检测肝细胞分泌功能,同时采用PCR、免疫荧光及跨上皮细胞电阻实验观测肝细胞极性结构基础紧密连接的动态变化过程。结果:肝细胞在"三明治夹层"中培养72 h后具有胆小管分泌功能,同时紧密连接处分子ZO-1与Occludin结构为促使极性形成,其mRNA表达与通透性均产生一个先升高后降低的变化趋势;ZO-1免疫荧光定位发现不同培养时间随着极性形成其结构分布也有所不同。结论:肝细胞三维培养极性形成是一个动态变化的过程,了解这一动态变化过程对药物肝毒性检测及其毒性机制探讨具有重要的指导意义。

AIM： To establish a suitable drug- induced hepatotoxicity evaluation model in vitro, and observe the dynamic changes of hepatic polarity in sandwich-cultured rat primary hepatocytes as time progresses. METHODS： Bile excretory function was measured in sandwich-cultured rat primary hep- atocytes with time elapsing. Besides, PCR , Transepithelial Electrical Resistance and Immunoflu- orescent staining were used to detect the expression, permeability and localization of tight junctions in re- polarized hepatocytes. RESULTS：At 72 h of sand- wich culture, the FDA fluorescence concentrated mainly at the canaliculi, which means the formation of hepatic polarity. The mRNA expression and elec-trical resistance were first up to the highest levels at culuture and then decreased as time progresses. Localization of Z0-1 also appeared a translocation during the cell culture. CONCLUSION： A series of changes have taken place in the formation of hepatic polarity, which play an important role in the drug toxicity testing and its underlying mechanisms.