Met behulp van Caco-2 cellen te Lipid Transport studie van de Darm
Summary
Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.
Abstract
Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).
Introduction
Studies van intestinale absorptie van dieetvet en vetoplosbare geneesmiddelen en vitaminen kunnen in vivo worden uitgevoerd door toepassing van een model lymfe fistel 1-4. De chirurgische technieken noodzakelijk zijn niet alleen moeilijk maar ook duur. Hoewel in vivo benade- fecal analyse kan worden toegepast, worden zij vooral gebruikt om het percentage opname bepaald door het maagdarmkanaal 2,5. De in vitro beschreven in dit document model is meer kosteneffectief, en de betrokken technieken zijn misschien wel minder uitdagend. Genetische modificatie studies ook economischer en minder tijdrovend wanneer ze worden uitgevoerd in deze in vitro model.
Aangezien vetoplosbare stoffen die door enterocyten genomen worden verpakt in lipoproteïnen 6,7, de doeltreffendheid van deze in vitro model lipoproteïnen produceren cruciaal. De twee belangrijkste intestinal lipoproteïnen zijn chylomicronen en lipoproteïnen zeer lage dichtheid (VLDL). Chylomicronen, gedefinieerd als lipoproteïnen met 80 nm of meer in diameter, strikt door de dunne darm wanneer lipiden zijn overvloedig aanwezig in de maag lumen. Omdat ze de grootste lipoproteïnen, chylomicronen zijn denkbaar de meest efficiënte lipide transporteurs. Deze in vitro model dat kan produceren chylomicronen 8, kan worden gebruikt om voeding vetabsorptie, vet-oplosbare vitamine absorptie door de darm en orale biologische beschikbaarheid lipofiele geneesmiddelen te bestuderen. De aanwezigheid van lipide oplosbare moleculen, vitamines of drugs in de lipoproteïne fractie is een indicator van de absorptie door de dunne darm. Zoals eerder besproken, kan dit model worden gebruikt om orale biobeschikbaarheid lipofiel geneesmiddel 6 verbeteren.
Dit artikel beschrijft hoe Caco-2 cellen permeabel membraan of vaste weefselkweekplaten, hoe de lipide mi te handhavenxture voor het stimuleren lipoproteïne produktie moet worden voorbereid, hoe lentivirus expressiesysteem kan worden toegepast om effectief overexpressie halen en hoe de lipoproteïnen die moeten worden geanalyseerd.
Protocol
Representative Results
Discussion
In dit document kunnen de twee systemen die kunnen worden gebruikt voor het onderhoud Caco-2 cellen zijn beschreven, namelijk de normale weefselkweek schaal en de permeabele membraan. De voordelen van het permeabele membraan systeem omvatten de scheiding van de apicale en basolaterale compartimenten en de mogelijkheid om het lipide mengsel incubeer en laat het lipoproteïne secretie tegelijk. De permeabele membraan inserts zijn duur en hun polycarbonaatmembraan laat geen goede mobiele zicht. Een van de voordelen van dez…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Seed Grant Award from California Northstate University College of Pharmacy (to AMN). The authors would like to thank California Northstate University College of Pharmacy for covering the publication cost of this article, and George Talbott for his help in editing this manuscript.
Materials
| DMEM | VWR | 16750-112 | Pre-warm the growth media in individual tissue culture dish before adding cells |
| FBS | Fisher | 3600511 | We do not heat inactivate our serum |
| Trypsin | VWR | 45000-660 | Cells can be washed with PBS prior to trypsin treatment |
| Permeable membrane system | Fisher | 7200173 | 10-cm dish, 3-mm pore size, polycarbonate membrane |
| 10 cm dish | Fisher | 08-772-E | Tissue culture dish |
| 15 cm dish | Fisher | 08-772-24 | Tissue culture dish |
| 24 well plate | Fisher | 12565163 | Tissue culture plate |
| Lipid-based transfection reagent | Fisher | PRE2691 | Can be substituted with other transfection reagent |
| Reduced serum media | Invitrogen | 11058021 | For transfection |
| pLL3.7 eGFP | Addgene | 11795 | https://www.addgene.org/11795/ |
| Bottle-top filter | Fisher | 9761120 | 0.45 mm pore |
| Polybrene | Fisher | NC9840454 | 10 mg/mL |
| Oleic acid | Sigma | 01383-5G | Prevent freeze-thaw cycle |
| Lecithin | Fisher | IC10214625 | Egg lecithin |
| Sodium taurocholate | Fisher | NC9620276 | Product discontinued; alternative catalog number: 50-121-7956 |
| Protease inhibitor cocktail tablet (EDTA-free) | Fisher | 5892791001 | Used mainly for samples that need TEM analysis |
| Polycarbonate ultracentrifuge tube | Fisher | NC9696153 | Reusing it multiple times will collapse the tube |
| Lid for ultracentrifuge tube | Fisher | NC9796914 | A tool is required to remove the tube/lid from rotor |
| Syringe | Fisher | 50-949-261 | Disposable |
| Syringe filter | Fisher | 09-719C | Pore size = 0.2 mm; nylon |
| Phosphotungstic acid | Fisher | AC208310250 | For preparing 2% phosphotungstic acid, pH 6.0 |
| Tweezer | Fisher | 50-238-62 | Extra fine and strong tips |
| Formvar/carbon grid | Fisher | 50-260-34 | Formvar/carbon film square grid 400 Copper |
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