腸によって脂質輸送を研究するためのCaco-2細胞を用いました
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 –食事脂肪の腸管吸収に関する研究、および脂溶性薬物とビタミンは、リンパ瘻モデル1を用いて、in vivoで行うことができます。しかし、関連する外科技術は、困難でなく、高価なだけでなく、です。糞便分析に基づいて、in vivoでのアプローチを利用することができるが、それらは、胃腸管2,5パーセントの取り込みを決定するために主に使用されています。この論文に記載のin vitroモデルは、よりコスト効果的であり、関係する技術は、おそらくあまり挑 戦しています。遺伝的修飾研究は、時間がかかり、それらは、このin vitroモデルを使用して行われる場合に、より経済的かつ少ないです。
腸細胞によって取り込まれる脂溶性物質は、リポタンパク質6,7にパッケージ化されているので、リポタンパク質を生成するために、このin vitroモデルの有効性が重要です。二つの主要なintestinaLのリポタンパク質は、カイロミクロン及び超低密度リポタンパク質（VLDL）です。脂質は、胃腸内腔に豊富に存在する場合、直径が80nm以上とリポタンパク質として定義されるキロミクロンは、小腸により厳密に製造されています。彼らは最大のリポタンパク質であるため、カイロミクロンは考えられる最も効率的な脂質トランスポーターです。カイロミクロン8を生成することができる。このインビトロモデルは、食事の脂肪吸収、消化管による脂溶性ビタミンの吸収、および親油性薬物の経口バイオアベイラビリティを研究するために使用することができます。リポタンパク質画分中の脂溶性分子、ビタミン、または薬物の存在は、小腸によるその吸収の指標です。前述のように、このモデルは、経口バイオアベイラビリティ、親油性薬剤6を改善するために使用することができます。
本稿では、Caco-2細胞はどのように脂質マイル、透過膜または通常の組織培養皿で維持されるべきである方法を説明しますリポタンパク質の産生を刺激するための固定装置は、レンチウイルス発現系は、効果的な過剰発現を達成するために使用する方法、調製方法、および単離されたリポタンパク質を分析するべきであるべきです。
Protocol
Representative Results
Discussion
この論文では、使用できる2つのシステムは、Caco-2細胞は、すなわち、正規の組織培養皿や透過膜に記載されている維持します。透過膜システムを使用する利点は、頂端の分離および基底外側コンパートメント、および脂質混合物をインキュベートし、同時にリポタンパク質分泌を収集する能力を含みます。しかし、透過膜インサートは高価であり、それらのポリカーボネート膜は、良好…
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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