隔离和鼠标主动脉内皮细胞原代培养

Summary

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Abstract

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

Introduction

血管内皮不仅是分隔的血液和组织的屏障层，它被认为是一个巨大的内分泌腺，绵延在整个血管树具有400平方米¹的表面积。内皮的福祉是血管稳态的关键。的内皮机能障碍参与各种心血管疾病，包括动脉粥样硬化，血管炎和缺血/再灌注损伤， 等 ^2-4。迄今为止，涉及这些疾病的设置特定的细胞和分子机制尚未很好地理解，由于内皮的漫射解剖性质。

鼠标是研究的重要模型，因为遗传操作技术在小鼠比较发达的比任何其他哺乳动物物种。然而，由于主动脉的小尺寸使得enzymat原代鼠主动脉内皮细胞的分离被认为是特别困难内皮不切实际的IC消化。一些报道的方法分离和纯化的EC要求^5-7。

该协议的目标是用一个简单的方法来从鼠标主动脉隔离和扩大血管内皮细胞，而无需使用任何特殊的设备。在这个协议中，新鲜分离的主动脉切成小片段，并接种到朝下以允许内皮出芽内皮的基质。段被删除后，内皮细胞在血管内皮青睐中等扩大，准备好两三个代后实验。所描述的方法的优点是：1）相当高数量的内皮细胞来自单一主动脉收获的; 2）细胞存活率保存良好; 3）不需要特殊的设备或技术。它提供了鉴定内皮细胞病理生理学特异性细胞和分子机制的一种有效手段。对于那些有兴趣谁在学习公关从任一基因敲除小鼠imary培养的内皮细胞，基因敲除小鼠中，或鼠疾病模型中，该协议是非常有用和易于实践。

Protocol

1.主动脉的小鼠中分离这里所描述的所有程序均通过的韦恩州立大学的机构动物护理和使用委员会批准。 把鼠标放到麻醉机的感应室。设置异氟烷流至4％结合25％的新鲜空气和75％ 的 O 2。 通过感应腔随后由专用的鼻锥异氟醚（诱导4％，±1.0％用于保养）用25％的新鲜空气和75％的O 2一起吸入麻醉鼠标。注：麻醉用异氟烷可以在75％氧气/ …

Representative Results

内皮细胞发芽 自发的内皮细胞出芽来自小鼠主动脉段开始。鼠标主动脉段被允许在低生长因子基质生长然后在内皮细胞生长培养基中4天。内皮细胞出芽通常出现在2 – 4天。显微照片拍摄第4天（ 图1）。正如图片所示，无数的内皮细胞迁移从段路程。新成立的豆芽继续从段和分支延伸。 内皮细胞表型 <p class="jove_…

Discussion

这项研究表明了一个简单的方法来分离，培养内皮细胞从鼠标主动脉细胞无任何特殊设备。免疫荧光染色显示，大部分细胞是第二通道后内皮细胞。有人建议，细胞在第二至第三通道适于在体外 和体内实验来研究内皮生物学。

从本议定书的重要注意事项

有在步骤五个关键点。首先，血管腔与含肝素的PBS冲洗，以尽量减少内皮?…

Materials

4- or 6-week-old mice (Jackson Laboratory, #000664).

Sterile 1X phosphate-buffered saline (PBS, Gibco, #10010-023).

Sterile 1X PBS containing 1,000 U/ml of heparin (Sigma Aldrich, H3149).

Endothelial cell growth medium (Dulbeccos’ Modified Eagle’s Medium[DMEM] with 25mM HEPES[Gibco, #12320-032 ], supplemented with 100μg/ml endothelial cell growth supplement from bovine neural tissue [ECGS, Sigma,#2759], 10% fetal bovine serum [FBS, Gibco, #10082-147], 1,000 U/ml heparin [Sigma Aldrich, H3149], 10,000U/ml penicillin and 10mg/ml streptomycin [Gibco, #15140-122]).

Growth factor reduced matrix (BD Biosciences, #356231).

Neutral proteinase (Dispase, 1U/ml, Fisher Scientific #CB-40235) and D-Val medium (D-Valine, 0.034g/L[Sigma, #1255], in Dulbeccos’ Modified Eagle’s Medium, low glucose[Gibco, #12320-032 ]).

Gelatin (100‑200 μg/cm2, Sigma, #G1393).

1,1`-dioctadecyl-3,3,3`,3`- tetramethylindo- carbocyanine perchlorate-labeled acetylated LDL (Dil-ac-LDL, Life Technologies, #L3484),  FITC-labeled Ulex europeus agglutinin (Ulex-Lectin, Sigma, #L9006),  Anti-mouse CD31-FITC conjugated antibody (BD Biosciences, # 553372).

Anti-mouse vascular endothelial growth factor receptor 2 antibody (Cell signaling, #9698), anti-mouse endothelial nitric oxide synthase (Abcam, #ab5589), anti-mouse vascular endothelium-cadherin (Abcam, #33168), anti-mouse calponin (Abcam, #700), FITC-conjugated anti-rabbit IgG (Sigma, #F6005)

One ml syringe fitted with 25-G needle (Fisher Scientific, #50-900-04222).

100mm Peri dishes (Fisher Scientific,  #07-202-516).

Six-well cell culture plates (Fisher Scientific, #08-772-1B).

T12.5 cultuer flask (Fisher Scientific, #50-202-076)

Scissors, forceps, microdissection scissors and forceps, Scalpel blade (Fine Science Tools, Inc)

Anesthesia machine with isoflurane (Webster Veterianary Supply, #07-806-3204), heating lamp

Centrifuge machine.

Inverted phase-contrast microscope.

inverted fluorescence microscope.