分離、伝達、およびプリオン蛋白質発現ひと歯髄幹細胞の神経分化
Summary
ここでプリオン蛋白発現神経細胞の分化過程を評価するために人間の歯髄幹細胞の分離と伝搬するためのプロトコルを提案する.
Abstract
胚性幹細胞の操作に関連する生命倫理の問題は、医学研究の分野での進歩を妨げています。このため、脂肪、臍帯、骨髄血などさまざまな組織から成体幹細胞を得ることが非常に重要です。可能なソースの間で倫理的配慮に関して入手が容易だから、歯髄は特に興味深いです。確かに、ひと歯髄幹細胞 (hDPSCs) は成体幹細胞の神経様細胞に区別することの一種で、健康な患者 (13-19 歳) の第 3 大臼歯から取得することができます。特に、歯髄除去掘削機と、小さなスライスにカット、IV コラゲナーゼで処理し、フラスコで培養します。神経細胞の分化を誘導して、2 週間 hDPSCs EGF/bFGF と刺激を受けました。以前は、その分化過程プリオンの内容 hDPSCs の蛋白質 (PrPC) 増加を確認しました。Cytofluorimetric 分析は、神経細胞の分化プロセス後に増加した PrPCの早期発現を示した。SiRNA による prpCアブレーション PrP EGF/bfgf 投与による神経細胞の分化を防止しました。本稿で我々 は分離、分離およびいくつかの簡単な手順で hDPSCs 栽培体外を強化より効率的な細胞クローンされた間葉系幹細胞 (MSCs) の取得と大規模な拡大を説明します。観察されました。また、どのプロトコルでは、詳細な方法で得られた、hDPSCs、MSCs の神経分化過程とその後の細胞および分子プロセスを研究する優秀な実験モデルを示す.
Introduction
間葉系幹細胞は、骨髄、臍帯血、歯髄、脂肪組織、血液1,2,3,4,5など、いくつかの組織から分離されています。,6。 hDPSCs がプラスチック付着、典型的な線維芽細胞様形態を示す何人かの著者によって報告されるようです。これらは異なるクローン増殖と分化能力7,8の違いと非常に異種の人口を表しています。hDPSCs 間葉系幹細胞 (すなわち CD44、CD90、CD73, CD105、STRO 1) 特定のマーカーの表現、(CD14 CD19 など) いくつかの造血のマーカーは陰性、血液分化9、ことができる10,11。
何人かの著者は、これらの細胞は神経成長因子、bFGF、特定メディアとサプリメント7,12との組み合わせで EGF を添加し、異なるプロトコルを使用して神経細胞のような細胞に分化することが示されています。また、神経細胞の分化のプロセス中に多くのタンパク質が関与しているし、これらのうち、いくつかの論文を表示関連する役割と重要な発現細胞プリオンタンパク質 (PrPC)、胎児および成体幹細胞13,の両方14., 分子として銅代謝、アポトーシス、細胞内の異なる機能を実行できるを表し、耐酸化ストレス15,16,17 PrPC,18,19,20,21,22。
私たち以前紙23、hDPSCs 神経分化過程における PrPCの役割を検討しました。実際には、hDPSCs エクスプレス早熟 PrPCと、神経細胞の分化後追加の増加を観察することが可能だった。他の著者は、幹細胞の神経分化過程における PrPCの可能な役割を仮定しました。確かに、PrPCはアストロ サイト、オリゴデンドロ サイト、ニューロンの24にヒト胚性幹細胞の分化をドライブします。本研究の目的は、神経細胞の分化の間に歯髄、その分化過程と PrPCの役割から幹細胞を得るための方法論を強調していた。
Protocol
研究で使用されている第三大臼歯 (13-19 歳) 薬剤またはアルコールの消費のない前の歴史、すべて禁煙と適切な口腔衛生の患者から摘出し.科学歯科で「サピエンツァ」ローマ大学の顎顔面の説明の日に患者または両親からインフォームド コンセントを得た。倫理的な考察、倫理委員会の承認に基づくインフォームド コンセントを得た。 1. 歯や歯髄の抽出 ?…
Representative Results
第 3 大臼歯から得られる歯髄から hDPSCs の分離と分離のプロシージャは、小さな変化が破滅的な結果を導くことができる複雑なプロセスです。本稿でのアーサー et al.プロトコルを使用してください。12いくつかの改善をします。プロシージャの代表的な方式を図 1に示します。 <p class="jove_content" fo:keep-together.within-page="1…
Discussion
この作品での分離と hDPSCs; 神経分化の方法論に着目さらに、このプロセスで PrPCの役割を評価しました。分離し、プロセス中に神経細胞のような細胞で重要なステップ hDPSCs を区別するいくつかの方法があります。hDPSCs は、軟骨細胞、脂肪細胞、骨芽細胞、神経細胞などいくつかの系統に区別することができます。私たちの論文では神経細胞の分化のメカニズムと PrPCの存在?…
Declarações
The authors have nothing to disclose.
Acknowledgements
この作品は、「Fondazione Varrone」と Rieti 大学ハブ ヴィンチェンツォ ・ マッテイを”サビーナ大学」によって支持されました。
テイラー & フランシス ・株式会社から、図 5 (a, B) は出版社の許可を得て転載: 人間歯科パルプ由来幹細胞の神経分化の間に役割のプリオン蛋白質 EGFR multimolecular 複合体。Martellucci、s.、マンジャネッリ V. マッテイ V.プリオンSorice m ・ ピコリ L. Santilli F. Santacroce C.。2018 3 月 4日。テイラー ・ フランシス (株)
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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Citar este artigo
Martellucci, S., Santacroce, C., Manganelli, V., Santilli, F., Piccoli, L., Cassetta, M., Misasi, R., Sorice, M., Mattei, V. Isolation, Propagation, and Prion Protein Expression During Neuronal Differentiation of Human Dental Pulp Stem Cells. J. Vis. Exp. (145), e59282, doi:10.3791/59282 (2019).