Isolering og primær kultur af muse aortaendotelceller
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
Det vaskulære endotel er ikke kun et barrierelag, der adskiller blod og væv, anses det for et stort endokrine kirtel, der strækker sig over hele vaskulære træ med et overfladeareal på 400 kvadratmeter 1. Den trivsel endotelet er afgørende for vaskulær homeostase. Den dysfunktionelle endotel deltager i forskellige kardiovaskulære lidelser, herunder åreforkalkning, vasculitis og iskæmi / reperfusionsskader osv 2-4. Til dato er de specifikke cellulære og molekylære mekanismer, der er involveret i dette sygdomsstadie ikke godt forstået på grund af den spredte anatomiske karakter endotel.
Musen er en vigtig model for forskning, fordi genetiske manipulation teknikker er mere udviklet i mus end i nogen anden pattedyrarter. Imidlertid er isolering af primære murine aortaendotelceller betragtes særlig vanskelig, fordi den lille størrelse af aorta gør enzymatic fordøjelse af endotel upraktisk. Nogle rapporterede procedurer til at isolere og oprense EC'er kræver 5-7.
Målet med denne protokol er at bruge en simpel metode til at isolere og udvide endotelceller fra muse aorta uden brug af særligt udstyr. I denne protokol, er det frisk isolerede aorta skæres i små segmenter og podet på en matrix med endotel nedad for at give mulighed for endotel spiring. Efter segmenter er fjernet, er endotelceller ekspanderes i endotel stillede medium og er klar til forsøg efter to eller tre passager. Fordelene ved den beskrevne fremgangsmåde er, at: 1) betydeligt højt antal endotelceller høstes fra en enkelt aorta; 2) cellernes levedygtighed er velbevaret; og 3) ikke er behov for særligt udstyr eller teknik. Det giver et effektivt middel til at identificere specifikke cellulære og molekylære mekanismer i endotelceller patofysiologi. For dem der er interesseret i at studere PRimary dyrkede endotelceller fra enten gen-knock-out-mus, gen knock-i mus, eller en murin sygdomsmodel, denne protokol er meget nyttig og let at praktisere.
Protocol
Representative Results
Discussion
Denne undersøgelse viser en simpel metode til at isolere og kultur endotel celler fra en mus aorta uden særligt udstyr. Den immunfluorescensfarvning viste, at størstedelen af cellerne var endotelceller efter den anden passage. Det foreslås, at celler ved andet til tredje passage er egnede til in vitro og in vivo forsøg for at studere endotel biologi.
De Hovednotater fra denne protokol
Der er fem kritiske punkter i pro…
Disclosures
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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