सात कदम कक्ष तारामय
Summary
यहाँ हम माउस जिगर से यकृत तारामय कोशिकाओं के अलगाव के लिए एक विधि का वर्णन. तारामय सेल शुद्धि के लिए, माउस यकृत को पचा रहे हैं<em> बगल में</em> और<em> इन विट्रो में</em> Pronase – collagenase घनत्व ढाल centrifugation के लिए पहले उपचार के द्वारा. इस तकनीक अत्यधिक शुद्ध यकृत तारामय कोशिकाओं पैदावार.
Abstract
Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5.
Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets.
Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.
Protocol
Discussion
यकृत तारामय कोशिकाओं को जिगर में आवश्यक शारीरिक और pathophysiological प्रक्रियाओं को विनियमित. इसके अलावा, तारामय कोशिकाओं प्रतिजन पेश गुणों के अधिकारी, उन्हें यकृत प्रतिरक्षा प्रतिक्रिया का एक महत्वपूर्ण घटक …
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
इस काम बायोमेडिकल अनुसंधान और NIH AI083426 01-RO1 (परिवार कल्याण) में उत्कृष्टता के लिए स्मिथ परिवार पुरस्कार द्वारा वित्त पोषित किया गया था. पैट्रिक Maschmeyer और मेलनी Flach Boehringer Ingelheim फाउंडेशन से पीएचडी फैलोशिप द्वारा समर्थित हैं.
Materials
| Name Of Reagent | Company | Catalog No. |
|---|---|---|
| 70μm Cell Strainer | BD | 352350 |
| CaCL2 x 2H2O | Sigma-Aldrich | C3306 |
| Collagenase P | Roche | 11249002001 |
| DMEM | Gibco | 11960 |
| DNase I | Roche | 10104159001 |
| DPBS | Cellgro | 21-031-cv |
| EGTA | Fluka | 3777 |
| Fetal Bovine Serum | Gibco | 16000 |
| Glucose | Sigma-Aldrich | G7528 |
| Gradient Centrifugation Tubes | Greiner Bio-One | 163160 |
| HEPES | Gibco | 15630 |
| KCL | Sigma-Aldrich | P9541 |
| KH2PO4 | Sigma-Aldrich | P9791 |
| L-Glutamine | Gibco | 25030 |
| MgCl2 x 6H2O | Fluka | 63068 |
| MgSO4 x 7H2O | Sigma-Aldrich | M2773 |
| Na2HPO4 x 2H2O | Fluka | 71643 |
| NaCl | Sigma-Aldrich | S3014 |
| NaH2PO4 x H2O | Fluka | 71507 |
| NaHCO3 | Fluka | 71628 |
| Nycodenz | Accudenz AG | AN7050/BLK |
| Penicillin/Streptomycin | Gibco | 15140 |
| Peristaltic Pump | Cole Parmer | HV-7523-70 |
| Phenol Red | Sigma-Aldrich | P4633 |
| Pronase E | Calbiochem | 7433-2 |
| Silicone Tube | Masterflex | HV-96440-14 |
| Sodium Pyruvate | Gibco | 11360 |
| Tissue culture flask (25cm2) | BD | 353108 |
| Winged Infusion Set | Terumo | 1SV27EL |
Riferimenti
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- Gressner, A. M. &. a. m. p. ;. a. m. p., Weiskirchen, R. Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets. J Cell Mol Med. 10, 76-99 (2006).
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- Minicis, S. D. e. Gene expression profiles during hepatic stellate cell activation in culture and in vivo. Gastroenterology. 132, 1937-1946 (2007).
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