İzolasyon ve Fare aort endotel hücreleri İlköğretim Kültür
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
Vasküler endotelyum, sadece kan ve doku ayıran bir bariyer tabakası, bir 400 metre kare 1 kadar bir yüzey alanı ile damar ağacı boyunca uzanır geniş bir endokrin bezi olarak kabul edilir. endotel refahını damar homeostazının için gereklidir. Disfonksiyonel endotel ateroskleroz, vaskülit ve iskemi / reperfüzyon yaralanması gibi 2-4 dahil olmak üzere çeşitli kardiyovasküler bozuklukların, katılır. Bugüne kadar, bu hastalık ayarlarında yer alan spesifik hücresel ve moleküler mekanizmalar iyi sonucu endotel dağınık anatomik yapısına anlaşılamamıştır.
Genetik manipülasyon teknikleri, diğer herhangi bir memeli türlerde daha farelerde geliştirilmektedir, çünkü, fare araştırmaları için önemli bir modeldir. Aort küçük boyutlu enzymat yapar çünkü Ancak, birincil fare aort endotel hücreleri izolasyonu özellikle zor olarak kabul edilirpratik endotel ic sindirimi. Bazı izole ve EC 5-7 gerektirir arındırmak için prosedürler bildirdi.
Bu protokolün amacı, herhangi bir özel ekipman kullanmadan fare aorta endotel hücreleri izole ve genişletmek için basit bir yöntem kullanmaktır. Bu protokol, yeni izole edilmiş aort küçük parçalar halinde kesilir ve endotel filizlenmesi için izin vermek için aşağı doğru dönük endotel ile bir matris üzerine tohumlandı. segmentler kaldırıldıktan sonra endotel hücreleri endotel tercih ortamda genişletildi ve iki ya da üç geçişten sonra deneyler için hazırdır. tarif edilen metodun avantajları: 1), tek bir aort hasat endotel hücrelerinin oldukça yüksek sayılar; 2) hücre canlılığı iyi korunmuştur; ve 3) hiçbir özel ekipman veya teknik gereklidir. Bu endotel hücre patofizyolojisinde spesifik hücresel ve moleküler mekanizmaların tanımlanması için etkili bir araç sağlar. pr okuyan ilgilenen olanlar içinHer iki gen knock-out farelerinden alınan imary kültürlenmiş endotel hücreleri, gen knock-fareler ya da bir fare hastalık modeli, bu protokol, çok kullanışlı ve pratik kolaydır.
Protocol
Representative Results
Discussion
Bu çalışma, herhangi bir özel ekipman olmadan bir fare aort hücreleri izole ve kültür endotel için basit bir yöntemi gösterir. immünofloresan boyama hücrelerin çoğunluğu, ikinci geçiş sonra endotel hücreleri olduğunu göstermiştir. Üçüncü geçişine saniyede hücreleri, in vitro ve endotelyal biyolojisi okumak için in vivo deneyler uygun olduğu ileri sürülmektedir.
Mevcut Protokolü Önemli Notlar
pro…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This study is supported by American Heart Association Scientist Development Grant 13SDG16930098 and the National Science Foundation of China Youth Award 81300240 (PI: Wang). We thank Roberto Mendez from Wayne State University for assisting in the preparation of the manuscript.
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. |
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