Effect of cell-substratum interaction on hemicyst formation by MDCK cells

On impermeable substrata MDCK cells, a cell line derived from normal dog kidney, forms a confluent monolayer that is studded with numerous hemicysts. Previous studies with this cell line suggest that thes hemicysts develop as a result of active fluid accumulation between cell sheet and substratum. However, the formation of hemicysts as a multifocal phenomenon is still unexplained. The results presented here show that the hemicysts are not only expressions of active transport of solutes and water, but also of cell-substratum interaction. The increase in number and size of the hemicyst produced by dbcAMP may be explained by a decrease in the adhesive strength to substrata produced by this compound. Moreover, when the strength of the cell-substratum adhesion was increased the number of hemicysts was reduced or abolished. On the contrary, when this strength was reduced, larger hemicysts occurred, covering practically all the area available for growth. Results from cinematographic time lapse studies, showing that 90% of the area of the monolayer is able to produce hemicysts, also suggest that hemicyst formation as a multifocal phenomenon is more an expression of local variations in cell-substratum interaction than of regional changes in transepithelial active transport.     

Distribution and characteristics of the occluding junctions in a monolayer of a cell line (MDCK) derived from canine kidney

Summary On solid substrates MDCK, a cell line derived from normal dog kidney, forms a confluent monolayer that is studded with blisters. Previous studies with this cell line suggest that these hemicysts develop as a result of active fluid accumulation between cell sheet and substratum. One factor that may determine when and how hemicysts appear only in localized sites is the interruption of occluding junctions in nonhemicyst areas. To study this possibility, we compared the permeability characteristics of the occluding junctions in hemicysts and in an uninterrupted monolayer of MDCK grown on a permeable support of collagen-coated nucleopore filter. The spontaneous electrical potential differences were small, without statistical differences between them. Relative ionic permeability coefficients were evaluated from the voltage deflections to imposed salt gradients or to a single ion substitution across both structures. The results showed that the relative permeability ratios for Na⁺, K⁺, choline⁺, and Cl^– were the same in hemicysts and the uninterrupted monolayer. These and other results indicate that the junctional complex encircling the apical surface of a sheet of MDCK cells can provide an effective permeability barrier constituting a true occluding junction with the same properties in hemicyst and nonhemicyst areas.     

Serum-independent modulation of hemicyst formation by dissolved oxygen in postconfluent epithelial monolayers

Summary Hemicyst formation is considered a manifestation of either transepithelial solute and fluid movement or secretory activity in culture. This study shows that hemicyst formation in postconfluent monolayers of rhesus monkey kidney (LLC-MK₂) cells is modulated by the dissolved oxygen concentration (P_O2) of the culture medium. Either daily replacement of serum-free medium or displacement of the gas phase with 18% vol/vol O₂ (initial medium P_O2=125 to 135 mm Hg) enhances formation of hemicysts. Use of 30% O₂ (medium P_O2≊175 mm Hg) does not further increase the incidence, but neither 10% O₂ (medium P_O2=90 to 95 mm Hg) nor 1% O₂ (medium P_O2=35 to 50 mm Hg), the approximate range of dissolved oxygen values in blood, supports hemicyst formation unless cultures are gently rocked to disrupt diffusion gradients. Phase photomicrography of living cultures shows that the surface of a turgid hemicyst is furrowed, and cinephoto-micrography reveals that the walls vibrate subtly. When hypoxic conditions (0 to 1% O₂) are introduced this vibration ceases within 2 to 3 h, whereas collapse and disappearance of turgid hemicysts requires 18 to 20 h, seems virtually synchronous, and is reversible. Hemicysts form in a broad osmotic range, and increased electrolyte concentration increases the incidence. Hemicysts persist in localyy dense areas when cell-free strips are etched in the postconfluent monolayer; no DNA synthesis is detected under these conditions, but two-dimensional cell spreading into the denuded area is seen along the edge of the wound. We conclude that the dissolved oxygen supply in the cellular microenvironment modulates functional expression by differentiated kidney epithelial cells in culture and that increased electrolyte concentration also enhances expression of this phenotypic marker.     

Hemicyst formation stimulated by cyclic AMP in dog kidney cell line MDCK.

Certain epithelial cell lines have morphologic, physiologic, biochemical and pharmacologic characteristics of transporting epithelia from intact organs. In this paper we show that dibutyryl cyclic AMP, 5' AMP, adenosine and cyclic AMP phosphodiesterase inhibitors stimulate hemicyst formation by the dog kidney cell line MDCK. It is suggested that this effect is explained by elevation of intracellular cyclic AMP levels by means of an exogenous non-metabolizable source of cyclic AMP, phosphodiesterase inhibition or adenyl cyclase stimulation. Since hemicyst formation is in part due to transepithelial fluid transport, these findings raise the possibility that this fraction might be modulated by cAMP in an established cell line. We believe that cultured epithelial cells may provide an exploitable model system to investigate at the cellular and subcellular levels, the mechanism by which cyclic AMP modifies water and solute movements across epithelia.     

Comparison of the ability of basement membranes produced by corneal endothelial and mouse-derived Endodermal PF-HR-9 cells to support the proliferation and differentiation of bovine kidney tubule epithelial cells in vitro

The proliferation and morphological differentiation of bovine kidney collecting-tubule epithelial cells has been examined as a function of substrata and plasma factors. Collecting kidney tubule explant maintained in vitro gave rise to two distinct cell populations; one was composed mostly of fibroblastic cells whereas the other was epithelioid (EP cells). The proliferation of fibroblastic cells when exposed to serum-supplemented medium was best expressed when cells were maintained on a basement membrane produced by bovine corneal endothelial cells. This basement membrane has a composition, which in previous studies has been shown to favor the proliferation of mesenchymal cells. In contrast, the proliferation of EP cells was best expressed when cells were maintained on a basement membrane produced by the mouse-derived endodermal cell line PF-HR-9 (HR-9-BM). This basement membrane has a biochemical composition very similar to the basement membrane underlying the kidney tubules. Although the fibroblast confluent monolayer maintained on bovine corneal endothelial cell extracellular matrix did not undergo morphogenesis, the confluent monolayer of EP cells maintained on HR-9-BM shows hemicyst formation, suggesting that they were capable of vectorial fluid transport. They also built a complex three-dimensional kidney tubulelike network. Some tubules became grossly visible and floated into the tissue culture medium, remaining tethered to the cell monolayer at either end of the tubule. On an ultrastructural level, the tubules consisted of cells held together with junctional complexes arranged so as to form a lumen. The smallest lumen were bordered by 2-3 cells, and the largest ones by 8-15 cells. The lumens of the larger tubules did contain granular fibrillar and amorphous debris. Low-density EP cell cultures maintained on HR-9-BM could be induced to proliferate at a rate approaching that of cultures exposed to serum when they were exposed to medium supplemented with high-density lipoprotein (HDL, 750 micrograms protein/ml) and transferrin (50 micrograms/ml). When exposed to HDL concentrations equal or lower than 250 micrograms protein/ml, low-density cultures proliferated at a slow rate and readily formed tubulelike structures. This observation indicates that EP cells do not need to reach confluence to undergo morphogenesis, and that HDL, which in the presence of transferrin supports the cell proliferation, can favor their differentiation into tubulelike structures once its concentration becomes limiting for mitogenesis.     

Cell-substratum adhesion strength as a determinant of hepatocyte aggregate morphology

Cultured hepatocytes typically form multicellular aggregates which are either monolayered or spheroidal in morphology. We propose that the aggregate morphology resulting from a particular cell-substratum interaction has a biophysical basis: when cell contractile forces are greater than cell-substratum adhesion forces, spheroidal aggregates form; when cell contractile forces are weaker than cell-substratum adhesion forces, cells remain essentially spread and form monolayered aggregates. We tested this hypothesis by systematically varying the morphology of hepatocellular aggregates formed on substrata coated with a series of different concentrations of Matrigel, and correlating aggregate morphology with the cell-substratum adhesion strength measured in a shear flow detachment assay. Aggregate morphology was binary-spheroidal aggregates formed at low Matrigel concentrations and monolayered aggregates formed at high Matrigel concentrations. Cell-substratum adhesion strength was similarly binary, with low adhesion strengths correlated with spheroidal aggregates and high adhesion strengths correlated with formation of monolayered aggregates. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 415-426, 1997.     

Suramin-induced differentiation of the human colic adenocarcinoma cell clone HT29-D4 in serum-free medium

The clonal cell line HT29-D4 was able to grow in a completely defined medium containing EGF, selenous acid, and transferrin in the presence of the anti-helminthic drug suramin. In the absence of suramin, the kinetics of cell growth and the cell density obtained were dependent on the external EGF concentration. In the presence of suramin, cell density reached a plateau independent of EGF concentration above 50 ng/ml. At the morphological level, suramin allowed hemicyst formation in the cell monolayer. The cells were polarized with a well-ordered brush border facing the culture medium and mature junctional complexes that divided the cell membrane in two distinct domains. The carcinoembryonic antigen was found to be restricted to the apical membrane domain while the major histocompatibility molecules HLA-ABC were segregated within the basolateral domain. The electrical parameters of suramin-treated cells grown on permeable filters were measured and demonstrated that the cell monolayer was electrically active. These properties were never found in the absence of the drug. Moreover, the vasoactive intestinal polypeptide (VIP) was able to induce a dramatic increase in cAMP only when it was added, in agreement with the localization of the VIP receptor, in the lower compartment of the culture chamber. In conclusion we described for the first time conditions allowing the growth of functionally differentiated human colic cell monolayers in chemically defined medium. This model will contribute to a better understanding of suramin action and of the mechanisms involved in cell polarization.     

Continuous growth of proximal tubular kidney epithelial cells in hormone-supplemented serum-free medium

An epithelial cell line from pig kidney (LLC-PK1) with properties of proximal tubular cells can be maintained indefinitely in hormone-supplemented serum-free medium. Continuous growth requires the presence of seven factors: transferrin, insulin, selenium, hydrocortisone, triiodothyronine, vasopressin, and cholesterol. The hormone-defined medium (a) supports growth of LLC-PK1 cells at a rate of approaching that observed in serum-supplemented medium; (b) allows vectorial transepithelial salt and fluid transport as measured by hemicyst formation; and (c) influences cell morphology. The vasopressin dependency for growth and morphology can be partially replaced by isobutylmethylxanthine or dibutyryl cyclic AMP. The medium has been used to isolate rabbit proximal tubular kidney epithelial cells free of fibroblasts.     

Interaction exists between matriptase inhibitors and intestinal epithelial cells

The type II trypsin-like transmembrane serine protease matriptase, is mainly expressed in epithelial cells and one of the key regulators in the formation and maintenance of epithelial barrier integrity. Therefore, we have studied the inhibition of matriptase in a non-transformed porcine intestinal IPEC-J2 cell monolayer cultured on polyester membrane inserts by the non-selective 4-(2-aminoethyl)-benzosulphonylfluoride (AEBSF) and four more selective 3-amidinophenylalanine-derived matriptase inhibitors. It was found that suppression of matriptase activity by MI-432 and MI-460 led to decreased transepithelial electrical resistance (TER) of the cell monolayer and to an enhanced transport of fluorescently labelled dextran, a marker for paracellular transport between apical and basolateral compartments. To this date this is the first report in which the inhibition of matriptase activity by synthetic inhibitors has been correlated to a reduced barrier integrity of a non-cancerous IPEC-J2 epithelial cell monolayer in order to describe interaction between matriptase activity and intestinal epithelium in vitro.     

Phospholipase C-gamma1 potentiates integrin-dependent cell spreading and migration through Pyk2/paxillin activation

Phospholipase C-gamma1 (PLC-gamma1), which generates two second messengers, namely, inositol-1, 4, 5-trisphosphate and diacylglycerol, is implicated in growth factor-mediated chemotaxis. However, the exact role of PLC-gamma1 in integrin-mediated cell adhesion and migration remains poorly understood. In this study, we demonstrate that PLC-gamma1 is required for actin cytoskeletal organization and cell motility through the regulation of Pyk2 and paxillin activation. After fibronectin stimulation, PLC-gamma1 directly interacted with the cytoplasmic tail of integrin beta1. In PLC-gamma1-silenced cells, integrin-induced Pyk2 and paxillin phosphorylation were significantly reduced and PLC-gamma1 potentiated the integrin-induced Pyk2/paxillin activation in its enzymatic activity-dependent manner. In addition, specific knock-down of PLC-gamma1 resulted in a failure to form focal adhesions dependent on fibronectin stimulation, which appeared to be caused by the suppression of Pyk2 and paxillin phosphorylation. Interestingly, PLC-gamma1 potentiated the activations of Rac, thus integrin-induced lamellipodia formation was up-regulated. Consequently, the strength of cell-substratum interaction and cell motility were profoundly up-regulated by PLC-gamma1. Taken together, these results suggest that PLC-gamma1 is a key player in integrin-mediated cell spreading and motility achieved by the activation of Pyk2/paxillin/Rac signaling.     

Cell-substratum and cell-cell adhesion forces and single-cell mechanical properties in mono- and multilayer assemblies from robotic fluidic force microscopy

The epithelium covers, protects, and actively regulates various formations and cavities of the human body. During embryonic development the assembly of the epithelium is crucial to the organoid formation, and the invasion of the epithelium is an essential step in cancer metastasis. Live cell mechanical properties and associated forces presumably play an important role in these biological processes. However, the direct measurement of cellular forces in a precise and high-throughput manner is still challenging. We studied the cellular adhesion maturation of epithelial Vero monolayers by measuring single-cell force-spectra with high-throughput fluidic force microscopy (robotic FluidFM). Vero cells were grown on gelatin-covered plates in different seeding concentrations, and cell detachment forces were recorded from the single-cell state, through clustered island formation, to their complete assembly into a sparse and then into a tight monolayer. A methodology was proposed to separate cell-substratum and cell-cell adhesion force and energy (work of adhesion) contributions based on the recorded force-distance curves. For comparison, cancerous HeLa cells were also measured in the same settings. During Vero monolayer formation, a significantly strengthening adhesive tendency was found, showing the development of cell-cell contacts. Interestingly, this type of step-by-step maturation was absent in HeLa cells. The attachment of cancerous HeLa cells to the assembled epithelial monolayers was also measured, proposing a new high-throughput method to investigate the biomechanics of cancer cell invasion. We found that HeLa cells adhere significantly stronger to the tight Vero monolayer than cells of the same origin. Moreover, the mechanical characteristics of Vero monolayers upon cancerous HeLa cell influence were recorded and analyzed. All these results provide insight into the qualitative assessment of cell-substratum and cell-cell mechanical contacts in mono- and multilayered assemblies and demonstrate the robustness and speed of the robotic FluidFM technology to reveal biomechanical properties of live cell assemblies with statistical significances.     

Adhesion of L1210 cells to sulfonated styrene copolymer surfaces: imaging of F-actin AND alpha-actinin

The static adhesion of living L1210 cells to sulfonated copolymer surfaces of different sulfonic group content and the actin cytoskeleton organization in the adhering cells were studied. The strength of the cell-substratum interaction was estimated by determining the relative number of cells remaining adherent despite experiencing a shearing force equal to 1.25 x 10(-11) N caused by the laminar flow of the medium. The cell-substratum interaction took place in a medium with or without serum. The distribution of F-actin and alpha-actinin in the adhering cells was determined in sequences of fluorescent images of cell optical slices with the use of a computer method of cell image analysis. It was shown that the surface sulfonic groups affect not only the rate and strength of cell-substratum adhesion but also the F-actin and alpha-actinin distribution (in the cell regions near the substratum surface) in cells adhering in the medium containing serum. These proteins, concentrated in the tips of microvilli, were observed as dots. The distinctness (discernibleness) and sizes of these dots depend on the surface content of sulfonic groups. F-actin is located at the periphery of the cells in cells adhering in the medium without serum and alpha-actinin is concentrated in small dots at the periphery and in the central part of the cells.     

Electrical currents flow out of domes formed by cultured epithelial cells

Domes are localized areas of fluid accumulation between a cultured epithelial cell monolayer and the impermeable substratum on which the cells are cultured in vitro. Dome formation has been documented in a variety of epithelial cell lines that retain their transepithelial transport properties in vitro. However, it is not known whether domes are predominantly areas of specific active transport, or, alternatively, are predominantly areas of relative weak attachment to the culture surface. In the present study we adapted a vibrating microelectrode, which can detect small currents flowing in extracellular fluid, to determine if current was flowing into or out of domes and thereby to determine if domes were specialized areas of active transport. We used alveolar type II cells as the main epithelial cell type because they readily form domes in vitro and because they transport sodium from the apical to the basal surface. We found that electrical current flowed out of domes. The direction of the current was independent of the size of a dome, of the age of an individual dome, and of the number of days in primary culture for alveolar epithelial cells. This current was inhibited by amiloride and ouabain and was dependent on sodium in the medium. We made similar observations (outward current from domes which is blocked by amiloride and by sodium substitution) with domes formed by the Madin-Darby canine kidney cell line. The data support the hypothesis that sodium is transported across the entire monolayer and leaks back mainly through the domes. We conclude that domes in epithelial monolayers are not predominantly special sites of active transport but are more likely simply areas of weak attachment to the substratum.     

Inhibitory effects of tetradecanoylphorbol acetate and diacylglycerol on erythropoietin production in human renal carcinoma cell cultures

A human renal carcinoma from a patient with an erythrocytosis, serially transplanted into athymic nude mice, was grown in primary monolayer cell cultures. After reaching confluency the cultured cells formed multicellular hemicysts (domes) which became more abundant as the cultures approached saturation density. Erythropoietin (Ep) production by this renal carcinoma in culture was only slightly increased at the time of semiconfluency but showed a marked increase in Ep levels in the culture medium after the cultures reached confluency, in parallel with an increase in dome formation. The phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) showed a significant dose-related inhibitory effect on Ep production and dome formation in the renal carcinoma cell cultures, suggesting an important role of protein kinase C, the only known receptor for TPA, in inhibiting the expression of differentiated phenotypes in the renal carcinoma cells. TPA also suppressed Ep secretion over a period of 96 h, indicating a time course of suppression of this differentiated function of the renal carcinoma cells in culture. This hypothesis was further supported by the observation that diacylglycerol, the endogenous activator of protein kinase C, likewise inhibited Ep production and dome formation in the renal carcinoma cell cultures. These studies suggest a role of the inositol-lipid second messenger path and protein kinase C in the regulation of Ep production.     

Early stages of endothelial wound repair: conversion of quiescent to migrating endothelial cells involves tyrosine phosphorylation and actin microfilament reorganization

Endothelial repair to reestablish structural integrity following wounding is a complex process. Since the actin cytoskeleton undergoes specific changes in distribution as quiescent endothelial cells switch to activated migrating cells over a 6-h period following wounding (Lee et al. 1996), we studied tyrosine phosphorylation in association with actin microfilaments and adhesion proteins using double immunofluorescent confocal microscopy. We showed that in a confluent monolayer phosphotyrosine localized at the periphery of the cell at vinculin cell-cell adhesion sites within the actin-dense peripheral band (DPB) and centrally at talin/vinculin cell-substratum adhesion sites at the ends of central microfilaments. Over a period of 6 h following in vitro wounding there was a reduction of peripheral phosphotyrosine associated with the loss of both cell-cell adhesion sites and the DPB (stage I). Concomitantly, an increase in central phosphotyrosine was associated with an increase in cell-substratum adhesion sites and central microfilaments parallel to the wound edge (stage II), which subsequently redistributed perpendicular to the wound edge (stage III). We also localized FAK and paxillin at the ends of parallel and perpendicular central microfilaments. Immunoprecipitation of paxillin showed increased phosphotyrosine and protein levels when prominent central microfilaments were present and underwent remodeling. Inhibition of tyrosine kinases by genistein and tyrosine phosphatases by sodium orthovanadate resulted in reduced endothelial repair associated with disruption of adhesion site formation and central microfilament formation/redistribution in each stage of repair. We suggest that tyrosine phosphorylation of adhesion proteins, such as paxillin, may be important in regulating the early stages of endothelial wound repair. Received: 22 March 1999 / Accepted: 24 March 1999     

Redistribution of fibronectin and cytoskeletal proteins during the differentiation of rat mammary tumor cells in vitro

The tumorigenic mammary epithelial stem cell line, Rama 25, is capable of synthesizing and secreting fibronectin but incorporates only small amounts of fibronectin into pericellular material localised in regions of cell-cell and cell-substratum contact. Under certain culture conditions, Rama 25 differentiates into a non-tumorigenic myoepithelial-like cell line, Rama 29, which is capable of retaining fibronectin on the cell surface in characteristic fibrillar formation. The redistribution of fibronectin is accompanied by a reorientation of the cytoskeleton from circular bundles in Rama 25 to parallel arrays of filaments in Rama 29. In vivo, fibronectin is found in the basement membrane of the mammary gland and our in vitro studies lead us to suggest that the mammary myoepithelial cell in vivo synthesizes much of the basement membrane fibronectin.     

3D Single Molecule Tracking with Multifocal Plane Microscopy Reveals Rapid Intercellular Transferrin Transport at Epithelial Cell Barriers

The study of intracellular transport pathways at epithelial cell barriers that line diverse tissue sites is fundamental to understanding tissue homeostasis. A major impediment to investigating such processes at the subcellular level has been the lack of imaging approaches that support fast three-dimensional (3D) tracking of cellular dynamics in thick cellular specimens. Here, we report significant advances in multifocal plane microscopy and demonstrate 3D single molecule tracking of rapid protein dynamics in a 10 micron thick live epithelial cell monolayer. We have investigated the transferrin receptor (TfR) pathway, which is not only essential for iron delivery but is also of importance for targeted drug delivery across cellular barriers at specific body sites, such as the brain that is impermeable to blood-borne substances. Using multifocal plane microscopy, we have discovered a cellular process of intercellular transfer involving rapid exchange of Tf molecules between two adjacent cells in the monolayer. Furthermore, 3D tracking of Tf molecules at the lateral plasma membrane has led to the identification of different modes of endocytosis and exocytosis, which exhibit distinct temporal and intracellular spatial trajectories. These results reveal the complexity of the 3D trafficking pathways in epithelial cell barriers. The methods and approaches reported here can enable the study of fast 3D cellular dynamics in other cell systems and models, and underscore the importance of developing advanced imaging technologies to study such processes.     

Restricted localization of functional vasoactive intestinal peptide (VIP) receptors in in vitro differentiated human colonic adenocarcinoma cells (HT29-D4)

HT29-D4, a clone of the human colonic adenocarcinoma cell line (HT29), possesses at its cell surface specific binding sites for the vasoactive intestinal peptide (VIP) (KD = 0.5 nM). Their molecular weight was previously estimated to 117 kDa and 64 kDa. This clone underwent functional and structural differentiation when grown in a glucose-free galactose-containing medium. The [125I]VIP binding capacity of cells grown in this medium gradually declined while the cell density increased and reached a value close to zero when cell monolayer was able to form hemicysts. At this time, cells presented numerous tight junctions and desmosomes and a well organized brush border. Binding capacity could be recovered when the post-confluent monolayers were previously disaggregated with EDTA. Neither the affinity for VIP nor the molecular weight of the [125I]VIP cross-linked polypeptides were modified in these cells compared to cells grown in glucose-containing medium. However, surface receptor number of differentiated cells was twice that of undifferentiated cells. Leakproof differentiated cell monolayers grown on permeable substratum produced cAMP in response to VIP only when the peptide was present in the lower chamber of the culture wells. Taking these data altogether, we conclude that the localization of functional VIP receptors is restricted to the basolateral domain in differentiated post-confluent HT29-D4 cells.     

Growth characteristics of CHO cells in culture

The dependence of the growth characteristics and monolayer formation on the initial cell plating concentration were studied on a permanent CHO cell line. The cells were cultivated under standard conditions on plastic substrate. Initial plating concentrations were varied as 1000, 2000, 3000, 4000, 5000, and 6000 cells/cm². It was shown that the cell growth can be formally described by a standard S-shaped dependence. However, a more detailed analysis revealed inconsistency of the experimental and expected data. Specifically, the cell growth termination produced by monolayer formation does not coincide with the time when the theoretical curves approach a plateau. It is concluded that cell proliferation and monolayer formation are independent processes (at least in CHO cells). Both processes may be considered as analogs of proliferation and morphogenesis in metazoa. In addition, it is shown that the cessation of cell division is induced by reduction in the cell size to some limiting dimension and increasing of the cell polarization rather than contact inhibition of proliferation after the monolayer formation.     

药物跨膜转运细胞模型影响因素及应用

本文详细介绍了Caco-2细胞系和MDCK细胞系的特点、跨膜转运细胞模型的建立及其影响因素,包括细胞模型的选择、细胞接种密度、细胞单层的紧密性等细胞因素和Transwell多微孔膜的性质等环境因素。概述了国内外关于利用Caco-2和MDCK细胞系作为模型进行药物筛选、药物相互作用和研究药物吸收转运机制等方面的内容及MDCK细胞模型作为肠道模型、肾脏模型及血脑屏障模型的应用。比较了Caco-2细胞和MDCK细胞在肠道模型方面的差别,MDCK细胞主要用于选择性研究药物在小肠吸收及转运机制,特别用于细胞旁被动转运药物的研究,而Caco-2细胞用于双向转运或能量依赖主动转运研究。MDCK细胞模型可在体外培养条件下平稳转染人类MDR1基因,因此可高表达P-gp基因,可作为可用于评估肾脏药物相互作用、快速进行候选药物筛选及研究药物转运机制的理想模型。