Construction Of Functional Cllulare Model And The Application In The Research Of Chinese Herbs

Extracts and different fractions of natural products were screened and analyzed on the functionalTCRP-based cell models, followed by the interpretation with the utility of biochemical techniques such asimmunofluorescence, western blot, et al. This method can not only supply a good platform for activityscreening and functional prediction of natural products, but also direct its development and applicationincluding identification of components. However, because of vast variety of species and complexities, moredifferent functional cell models are requiered. Therefore, we constructed a cell model based on theintestinal epithelial barrier function in this study. The main contents are as follows:1. The principle of the TCRP technique and a revieew of its application in celular researchReal-time cell analyzers (xCELLigence system) with microelectronic impendance sensors couldrecord the time/dose-dependent cell response pattern (TCRP). The principle and its applicationresearch in cell biology were briefly introduced.2. Potential anti-cancer activity of Rhizome Dioscorea disclosed by EGF/EGFR based TCRPsPlenty of Chinese herbs have been screened on the EGF/EGFR model and Rhizome Dioscorea isone of the Chinese herbs which partially repress the cellular response to EGF stimulation. Variousfractions were also screened on this model and the petroleum ether extraction contains effectivecomponents which was further demonstrated by its role in downregulation of the phosphorylated ofERK, an important protein in EGF signal pathway. Morover, the petroleum ether extraction decreasesthe IC50of cis-platinum to A549-DDP cells. So the petroleum ether phase of Rhizome Dioscorea isthought to be the active moiety. 3. Construction of TCRPs to real-time monitor intestinal epithelial barrier function.The behavior of intestinal epithelial cell Caco-2disturbed by ethanol at a non-cytotoxicconcentration was recorded and the curve of CI (cell index) was consistent with the curve obtained bythe standard trans epithelial resistance (TER) assay. The immunofluorescent experiment showed thelocation of cytoskeletal protein F-actin and tight junction protein ZO-1were changed synchronouslywhen Caco-2was stimulated by ethanol. In addition, the protection of epidermal growth factor (EGF),or inducible nitric oxide synthase (iNOS) inhibitor on epithelial barrier has been demonstrated andcould be reflected by the TCRP-based method, which further supported the feasibility of theapplication of the novel system. Compared with traditional assay method, the new system is label-free,dynamic monitoring and high-throughput, which supplies a good platform for screening drugs that canprotect the intestinal epithelial barrier from injuries caused by ethanol or other factors.