采用Caco-2细胞研究脂质运输由肠
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
4 –的饮食脂肪，和脂溶性药物和维生素肠吸收的研究可以在体内通过使用淋巴瘘模型1进行。然而，所涉及的手术技术不仅具有挑战性的，而且成本很高。虽然体内方法的基础上的粪便分析可以利用，它们被用于主要由胃肠道2,5，以确定百分摄取。本文所述的体外模型更符合成本效益，以及所涉及的技术，可以说是不太具有挑战性。基因修饰的研究也更经济和耗时更少时，他们利用这种体外模型进行的。
因为这是采取由肠细胞的脂溶性物质被打包成脂蛋白6,7，该体外模型的有效性，以产生脂蛋白是至关重要的。两个主要intestina升脂蛋白是乳糜微粒和极低密度脂蛋白（VLDL）。乳糜微粒，其定义为具有80nm以上脂蛋白直径，严格生产由小肠时的脂质是大量存在于胃肠内腔。因为它们是最大的脂蛋白，乳糜微粒是可以想象的最有效的脂质转运。此体外模型，其能够产生乳糜微粒8，可用于研究饮食脂肪吸收，脂溶性维生素的吸收由肠道和口服脂溶性药物的生物利用度。脂溶性分子，维生素，或药物中的脂蛋白组分的存在是其吸收由小肠的指标。如先前讨论的，该模型可以用于改善口腔脂溶性药物的生物利用度6。
本文介绍Caco-2细胞如何应保持在渗透膜或定期组织培养皿，如何脂质英里夹具用于刺激脂蛋白生产应准备，如何慢病毒表达系统可以被用来实现有效的过表达，而分离出的脂蛋白应如何进行分析。
Protocol
Representative Results
Discussion
在本文中，可以使用两个系统，以保持Caco-2细胞进行了描述，即，常规的组织培养皿和渗透膜。利用渗透膜系统的优点包括心尖的和分离基底外侧室，并孵育所述脂质混合物并同时收集所述脂蛋白的分泌的能力。然而，渗透膜刀片是昂贵的，并且它们的聚碳酸酯膜不允许良好的细胞可见性。一个这种体外模型的一个优点是，遗传操作的研究将是更经济的和耗时少。为了获得更好的效果?…
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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