To Establish Caco-2 Cell Model And Application Of Caco-2 Cell Model In Research Of Absorption

Caco-2 cell came from human adenocarcinoma of colon cells and form polarity monolayer spontaneously in condition of cultivation,which have the differentiation characteristic of microvillus and tight junction which was similar to human small intestine epithelium striated border. The cell can differentiate side of enteric cavity(Apical ,AP ) and intestine intine(Basolate ral ,BL ). AP side contain typical intestinal microvillus hydratase, CYP1A1,β-glucuronidase(β-G) and various kinds nutrient substance conveying vehicles such as P-gp,which may educe effetive of active transfer material. And caco-2 cell reserve characteristics of high expressing P-gp.P-glycoprotein(P-gp) is an important ATP-dependent membrane transporter which is expressed in many normal tissues, including liver, brain, kidney, adrenal gland and intestinal epithelial cells. It is involved in the absorption, distribution and elimination of a variety of clinically important drugs. Now, Caco-2 cell model was though as effective tool on studying the interaction of oral administration medicne in absorption process.As one of the rapid screen tool of researching drug absorption, Caco-2 cell model was used in drug absorption, distribution, metabolism, elimination and toxicity research. This kind of cell have the same features of cell polarity and tight junction compared to enterocytes, which can imitate the small intestine epithelium absorption. This model is the best absorption moded in vitro and the powerful tool to illuminate mechanism of absorption. In this research, we established the Caco-2 cell model and screen in vitro many kinds of adjuvants in order to search probalble P-gp inhibitor. We definited the innocuity high performance adjuvant was absorbed by Ganciclovir which can improve the low biological availability.1. Establishment and evaluation the Caco-2 cell modelObjective: establishment the Caco-2 cell monolayer model mimicing small intestine epithelium and screen the high performance adjuvant was absorbed by Ganciclovir which can improve the low biological availability; Evaluation the normal growth characteristics of Caco-2 cells and monitor the system in daily culture.Methods:Caco-2 cells were pursursed from cell culture center of Wuhan university. Passage number in experiment was between 40 and 60. Culture solution was DMEM( 4.5g/lD-glucose ) ,which contain 1% nonessential amino acid, 1% L-glutamine, 100U/ml Benzylpenicillin, 100ug/ml Streptomycin, 10% fetal calf serum(FBS), 0.25%Trypsin solution, Millicell apparatus for culture.The formation of functional epithelial monolayers was monitored by measuring the transepithelial electrical resistance (TEER) of cell monolayers with a Millicell-ERS meter,observing cellular morphology of Cell monolayers with a electron microscope,determining the permiability of cell monolayers with phenol red as a marker, and detecting the activityof alkaline phosphatase by biological analysis.Results: Light microscope, electron microscope, phenol red permeation, transmembrane resistance and alkaline phosphatase activities were used to verified in this experiment. The Caco-2 monolayer cell cultures have microvillus in the cultivation condition of our lab. And phenol red permeation tight junction was less than 5%. The transmembrane resistance value was 220?/cm2 and the alkali phosphatase were mainly focus on the side of camisia foetus, which was simlar to small intestine. So it canb be used as the model of small intestine drug transportin vitro.2 Study the accessories -PVPK30 influencing on absorbing Ganciclovir using the Caco-2 cell model.When we study the cytotoxicity of PVPK30 and found that PVPK30 didn,t show any cytotoxicity when the density was less than 30mg/ml. So we definited 30, 30 and 3mg/ml were the high, middle and low density in transmembrane transport experiment. P-gp pump of substrate-Verapamil was used as positive control and Celiprolol as probe medicine. We found PVPK30 in different condition of density all can inhibit the founction of drug pump P-gp, decrease Celiprolol transport from the single cellular membrane BL side of Caco-2 to side of AP and improve the transmembrane transport of Celiprolol in the Caco-2 cell monolayer model transport experiment. We also discoved PVPK30 can promote drug transport in the method of concentration dependent. So we confirm PVPK30 is one kind of P-gp pump inhibitor.We further study the PVPK30 promoting absorbtion of low biological availability drug-Ganciclovir. After 2 hours of transmembrane transport, the density of side of BL in all different density group of PVPK30+ Ganciclovir were 1.7990, 2.0023 and 2.0416ug/ml, reseptively, which were all high than the density(1.4978 ug/ml) of Ganciclovir transmembrane transport after 2 hours. The density of BL side in positive control group which were given Ganciclovir and Verapamil were 1.7392 ug/ml.Conclusion: The results indicate that PVPk30 could inhibit drug efflux pump and could enhance the oral bioavailability of Ganciclovir which has low bioavailability at concentrations that could be achieved in vivo. Use of surfactants in excipients may increase the intestinal absorption of some drugs through P-gp inhibition and thus improve drug bioavailability for P-gp substrate.