Yalıtım, yayma ve insan diş Pulp kök hücrelerin Nöronal Farklılaşma sırasında Prion Protein ifade
Summary
Burada insan diş Pulp kök hücre izolasyon ve yayılması için bir protokol Nöronal Farklılaşma süreci sırasında prion protein ifade değerlendirmek için mevcut.
Abstract
Embriyonik kök hücre manipülasyon ilgili Biyoetik sorunları tıbbi araştırma alanındaki gelişmeler engelliyordu. Bu nedenle, farklı dokuların yağ, göbek kordonu, kemik iliği ve kan gibi yetişkin kök hücre elde etmek çok önemlidir. Biyoetik konuları ile ilgili olarak elde etmek kolaydır çünkü olası kaynaklar arasında diş pulp özellikle ilginçtir. Nitekim, insan diş Pulp kök hücreleri (hDPSCs) erişkin kök hücreleri nöronal benzeri hücrelerde ayırt etmek güçlü bir türüdür ve sağlıklı hastalar (13-19 yaş) üçüncü molar elde edilebilir. Özellikle, diş hamuru bir ekskavatör ile kaldırıldı, küçük dilimler halinde kesip, IV collagenase ile tedavi ve bir şişeye kültürlü. Nöronal Farklılaşma ikna etmek için hDPSCs 2 hafta boyunca EGF/bFGF ile teşvik. Daha önce ayırt etme işlemi sırasında Protein hDPSCs (PrPC) hücresel prion içeriğini artış göstermiştir. Cytofluorimetric analiz artmış Nöronal Farklılaşma süreci sonrasında PrPC erken ifadesi gösterdi. PrPC ablasyon siRNA tarafından PrP Nöronal Farklılaşma EGF/bFGF tarafından indüklenen engelledi. Bu yazıda, biz gibi biz yalıtım, ayrılık ve vitro yetiştirme yöntemleri hDPSCs birkaç kolay yordamlar ile gelişmiş, daha verimli Hücre klonlar Mezenkimal kök hücrelerin (MSCs) elde edilen ve büyük ölçekli genişleme olduğunu göstermek gözlenmiştir. Biz de nasıl iletişim kuralında, ayrıntılı yöntemleri ile elde edilen hDPSCs, MSCs Nöronal Farklılaşma süreci ve sonraki hücresel ve moleküler eğitim için mükemmel bir deneysel model olduğunu göster.
Introduction
Mezenkimal kök hücrelerin kemik iliği, göbek kordon kanı, insan diş pulp, yağ dokusu ve kan1,2,3,4,5 de dahil olmak üzere birkaç dokulardan izole edilmiştir , 6. çeşitli yazarlar tarafından bildirildiği gibi hDPSCs plastik bağlılık, tipik bir fibroblast benzeri Morfoloji göster. Bunlar farklı klonlar ve proliferatif ve farklılaştırıcı kapasitesi7,8farklılıkları ile son derece heterojen bir nüfusu temsil etmektedir. hDPSCs hızlı Mezenkimal Kök hücre (yani CD44, CD90, CD73, CD105, STRO-1) için belirli işaretleri, onlar bazı hematopoetik işaretleri (örneğin CD14 ve CD19) negatif ve yeteneğine sahip tüp bebek multilineage farklılaşma9/, 10,11.
Birden çok yazar bu hücreler NGF, bFGF, EGF12belirli medya ve takviyeleri7,ile birlikte ek içeren farklı protokolleri kullanarak nöron benzeri hücrelerine ayırt etmek mümkün olduğunu göstermiştir. Ayrıca, birçok protein Nöronal Farklılaşma işlemi sırasında söz konusu ve bunlar arasında bir ilgili rolü ve önemli ifade hücresel prion protein (PrPC), embriyonik ve yetişkin kök hücreleri13, her ikisi de birkaç belgelerini göster 14. PrPC temsil eden bir pleiotropic molekül hücrelere farklı işlevler bakır metabolizması, Apoptozis, sahip ve direnç oksidatif stres15,16,17 , 18 , 19 , 20 , 21 , 22.
Bizim önceki kağıt23‘ te, biz PrPC hDPSCs Nöronal Farklılaşma sürecindeki rolü araştırdık. Aslında, hDPSCs tanımışlar PrPC hızlı ve Nöronal Farklılaşma sonra ek artış gözlemlemek mümkün. Diğer yazarlar PrPC kök hücrelerin Nöronal Farklılaşma süreçlerde olası bir rol olan. Nitekim, PrPC insan embriyonik kök hücre farklılaşma nöronlar, oligodendrocytes ve astrocytes24götürmek. Bu çalışmada kök hücre diş pulp, onun farklılaşma süreci ve PrPC rolünü Nöronal Farklılaşma sırasında elde etmek için metodoloji vurgulamak oldu.
Protocol
Representative Results
Discussion
Bu çalışmada, yalıtım ve hDPSCs Nöronal Farklılaşma için metodoloji üzerinde duruldu; Ayrıca, PrPC bu süreçte rol değerlendirildi. Yalıtmak ve hDPSCs nöron benzeri hücreler ve kritik adımları işlemi sırasında ayırt etmek için birkaç yöntem vardır. hDPSCs chondroblasts, adipositler, dokusunu ve sinir hücreleri gibi birkaç soy ayırt edebiliyoruz. Bizim gazetede Nöronal Farklılaşma mekanizmaları ve PrPCvarlığı araştırıldı. Yukarıda da açıklandığı gibi bu h…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Bu eser “Fondazione Varrone” ve Rieti Üniversitesi Hub Vincenzo Mattei için “Sabina Universitas” tarafından desteklendi.
Şekil 5 (A, B) Taylor & Francis Ltd yayımcı izni ile yayımlanmaktadır: rol Prion protein-EGFR multimolecular karmaşık insan diş hamuru elde edilen kök hücreleri Nöronal Farklılaşma sırasında. Martellucci, S., Manganelli V., Santacroce C., Santilli F., Piccoli L., Sorice M., Mattei V. deli dana. 2018 4 Mart. Taylor & Francis Ltd.
Materials
| Amphotericin B solution | Sigma-Aldrich | A2942 | It is use to supplement cell culture media, it is a polyene antifungal antibiotic from Streptomyce |
| Anti-B3tubulin | Cell Signaling Technology | #4466 | One of six B-tubulin isoform, it is expressed highly during fetal and postnatal development, remaining high in the peripheral nervous system |
| Anti-CD105 | BD Biosciences | 611314 | Endoglin (CD105), a major glycoprotein of human vascular endothelium, is a type I integral membrane protein with a large extracellular region, a hydrophobic transmembrane region, and a short cytoplasmic tail |
| Anti-CD44 | Millipore | CBL154-20ul | Positive cell markers antibodies directed against mesenchymal stem cells |
| Anti-CD73 | Cell Signaling Technology | 13160 | CD73 is a 70 kDa glycosyl phosphatidylinositol-anchored, membrane-bound glycoprotein that catalyzes the hydrolysis of extracellular nucleoside monophosphates into bioactive nucleosides |
| Anti-CD90 | Millipore | CBL415-20ul | Positive cell markers antibodies directed against mesenchymal stem cells |
| Anti-GAP43 | Cell Signaling Technology | #8945 | Is a nervous system specific, growth-associated protein in growth cones and areas of high plasticity |
| Anti-mouse PE | Abcam | ab7003 | Is an antibody used in in flow cytometry or FACS analysis |
| Anti-NFH | Cell Signaling Technology | #2836 | Is an antibody that detects endogenous levels of total Neurofilament-H protein |
| Anti-PrP mAb EP1802Y | Abcam | ab52604 | Rabbit monoclonal [EP 1802Y] to Prion protein PrP |
| Anti-rabbit CY5 | Abcam | ab6564 | Is an antibody used in in flow cytometry or FACS analysis |
| Anti-STRO 1 | Millipore | MAB4315-20ul | Positive cell markers antibodies directed against mesenchymal stem cells |
| B27 Supp XF CTS | Gibco by life technologies | A14867-01 | B-27 can be used to support induction of human neural stem cells (hNSCs) from pluripotent stem cells (PSCs), expansion of hNSCs, differentiation of hNSCs, and maintenance of mature differentiated neurons in culture |
| BD Accuri C6 flow cytometer | BD Biosciences | AC6531180187 | Flow cytometer equipped with a blue laser (488 nm) and a red laser (640 nm) |
| BD Accuri C6 Software | BD Biosciences | Controls the BD Accuri C6 flow cytometer system in order to acquire data, generate statistics, and analyze results | |
| bFGF | PeproThec, DBA | 100-18B | basic Fibroblast Growth Factor |
| Centrifuge CL30R | Termo fisher Scientific | 11210908 | it is a device that is used for the separation of fluids,gas or liquid, based on density |
| CO2 Incubator 3541 | Termo fisher Scientific | 317527-185 | it ensures optimal and reproducible growth conditions for cell cultures |
| Collagenase, type IV | Life Technologies | 17104019 | Collagenase is a protease that cleaves the bond between a neutral amino acid (X) and glycine in the sequence Pro-X-Glyc-Pro, which is found with high frequency in collagen |
| Disposable scalpel | Swann-Morton | 501 | It is use to cut tissues |
| DMEM-L | Euroclone | ECM0060L | Dulbecco's Modified Eagle's Medium Low Glucose with L-Glutamine with Sodium Pyruvate |
| EGF | PeproThec, DBA | AF-100-15 | Epidermal Growth Factor |
| Fetal Bovine Serum | Gibco by life technologies | 10270-106 | FBS is a popular media supplement because it provides a wide array of functions in cell culture. FBS delivers nutrients, growth and attachment factors and protects cells from oxidative damage and apoptosis by mechanisms that are difficult to reproduce in serum-free media (SFM) systems |
| Filtropur BT50 0.2,500ml Bottle top filter | Sarstedt | 831,823,101 | it is a device that is used for filtration of solutions |
| Flexitube GeneSolution for PRNP | Qiagen | GS5621 | 4 siRNAs for Entrez gene 5621. Target sequence N.1 TAGAGATTTCATAGCTATTTA N.2 CAGCAAATAACCATTGGTTAA N.3. CTGAATCGTTTCATGTAAGAA N.4 CAGTGACTATGAGGACCGTTA |
| Hank's solution 1x | Gibco by life technologies | 240200083 | The essential function of Hanks′ Balanced Salt solution is to maintain pH as well as osmotic balance. It also provides water and essential inorganic ions to cells |
| HiPerFect Transfection Reagent | Qiagen | 301705 | HiPerFect Transfection Reagent is a unique blend of cationic and neutral lipids that enables effective siRNA uptake and efficient release of siRNA inside cells, resulting in high gene knockdown even when using low siRNA concentrations |
| Neurobasal A | Gibco by life technologies | 10888022 | Neurobasal-A Medium is a basal medium designed for long-term maintenance and maturation of pure post-natal and adult brain neurons |
| Paraformaldehyde | Sigma-Aldrich | 30525-89-4 | Paraformaldehyde has been used for fixing of cells and tissue sections during staining procedures |
| penicillin/streptomycin | Euroclone | ECB3001D | It is use to supplement cell culture media to control bacterial contamination |
| Phosphate buffered saline (PBS) | Euroclone | ECB4004LX10 | PBS is a balanced salt solution used for the handling and culturing of mammalian cells. PBS is used to to irrigate, wash, and dilute mammalian cells. Phosphate buffering maintains the pH in the physiological range |
| TC-Platte 6 well, Cell+,F | Sarstedt | 833,920,300 | It is a growth surface for adherent cells |
| Tissue culture flask T-25,Cell+,Vented Cap | Sarstedt | 833,910,302 | Tissue culture flask T-25, polystyrene, Cell+ growth surface for sensitive adherent cells, e.g. primary cells, canted neck, ventilation cap, yellow, sterile, Pyrogen-free, non-cytotoxic, 10 pcs./bag |
| Triton X-100 | Sigma-Aldrich | 9002-93-1 | Widely used non-ionic surfactant for recovery of membrane components under mild non-denaturing conditions |
| Trypsin-EDTA | Euroclone | ECB3052D | Trypsin will cleave peptides on the C-terminal side of lysine and arginine amino acid residues. Trypsin is used to remove adherent cells from a culture surface |
| Tube | Sarstedt | 62,554,502 | Tube 15ml, 120x17mm, PP |
| VBH 36 C2 Compact | Steril | ST-003009000 | Offers totally protection for the enviroment and worker |
| ZEISS Axio Vert.A1 – Inverted Microscope | Zeiss | 3849000962 | ZEISS Axio Vert.A1 provides a unique entry level price and can provide all contrasting techniques, including brightfield, phase contrast, PlasDIC, VAREL, improved Hoffman Modulation Contrast (iHMC), DIC and fluorescence. Incorporate LED illumination for gentle imaging for fluorescently-labeled cells. Axio Vert.A1 is ergonomically designed for routine work and compact enough to sit inside tissue culture hoods. |
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