人牙髓干细胞神经分化过程中的分离、繁殖及普利翁蛋白表达
Summary
本文提出了一种人类牙髓干细胞分离和繁殖的方案, 以评估神经元分化过程中的 prion 蛋白表达。
Abstract
与操纵胚胎干细胞有关的生物伦理问题阻碍了医学研究领域的进展。因此, 从脂肪、脐带、骨髓和血液等不同组织中获得成人干细胞是非常重要的。在可能的来源中, 牙髓特别有趣, 因为在生物伦理方面很容易获得。事实上, 人类牙髓干细胞 (Hdpsc) 是一种成人干细胞, 能够在神经元样细胞中进行分化, 可以从健康患者 (13-19) 的第三磨牙中获得。特别是, 牙髓被删除与挖掘机, 切成小片, 治疗胶原酶 IV 和培养在一个烧瓶。为诱导神经元分化, 用 Egf/bfgf 对 Hdpsc 进行了2周的刺激。此前, 我们已经证明, 在分化过程中, hDPSCs 中细胞蛋白 (prpc) 的含量增加。细胞荧光分析显示, 在神经元分化过程中, Prpc的早期表达增加。siRNA PrP 消融 PrP可防止 Egf/bfgf 诱导的神经元分化。本文说明, 随着我们通过几个简单的程序加强了 hDPSCs 的分离、分离和体外培养方法, 获得了更有效的细胞克隆, 并大规模扩展了间充质干细胞 (mscs)。被观察到。我们还展示了用协议中详细介绍的方法获得的 hDPSCs 是研究骨髓间充质干细胞神经元分化过程以及随后的细胞和分子过程的一个很好的实验模型。
Introduction
间充质干细胞已从几个组织中分离出来, 包括骨髓、脐带血、人牙髓、脂肪组织和血液1、2、 3、4、5,6. 正如一些作者所报告的, hDPSCs 显示出塑料粘附, 这是一种典型的成纤维细胞样形态。这些代表了一个高度异质的群体与独特的克隆和差异的增殖和分化能力7,8。Hdpsc 表达间充质干细胞的特定标记物 (即 CD44、CD90、CD73、CD105、STRO-1), 对某些造血标志物 (如 CD14 和 CD19) 呈阴性, 并能够在体外多系分化9, 10,11。
一些作者已经证明, 这些细胞能够分化为神经元样的细胞使用不同的协议, 其中包括添加 ngf, bfgf, egf 结合特定的媒体和补充7,12.此外, 许多蛋白质参与神经元分化过程中, 其中一些论文显示了相关的作用和重要的表达细胞 prion 蛋白 (Prpc), 无论是在胚胎和成人干细胞13,14. prpc是一种多向异性分子, 能够在细胞内发挥不同的功能, 如铜代谢、凋亡和抗氧化应激 15、16、17,18,19,20,21,22岁
在前面的论文23中, 我们研究了 Prpc在 Hdpsc 神经元分化过程中的作用。事实上, Hdpsc 在早熟的情况下表达 Prpc, 在神经元分化后, 有可能观察到额外的增加.其他作者推测Prpc在干细胞神经元分化过程中可能起到的作用。事实上, Prpc 推动人类胚胎干细胞分化为神经元、少突胶质细胞和星形胶质细胞24.本研究的目的是强调牙髓干细胞的获取方法、分化过程以及 Prpc 在神经元分化中的作用.
Protocol
研究中使用的第三磨牙被切除给以前没有吸毒或饮酒史、所有不吸烟和适当口腔卫生的患者 (13-19)。在解释当天, 在罗马 “Sapienza” 大学科学牙科和颌面系, 获得了患者或家长的知情同意。知情的同意是根据道德考虑和道德操守委员会的批准获得的。 1. 牙齿和牙齿纸浆提取 为养护或运输准备适当的介质。 用 l-谷氨酰胺 (494.5 mL) 制备杜尔贝科的修正鹰?…
Representative Results
从第三磨牙中获得的 hDPSCs 从牙髓中分离和分离的过程是复杂的过程, 在这个过程中, 微小的变化会导致毁灭性的结果。在本文中, 我们使用了亚瑟等人的协议.12与几个改进。图 1显示了一个具有代表性的过程方案。 hdpsc 代表了具有明显克隆和增殖和分化能力差异的异质细胞<sup class…
Discussion
在这项工作中, 我们集中在分离和神经元分化的方法 hDPSCs;此外, 我们还评估了 Prpc在这一过程中的作用。有几种方法可以分离和区分神经元样细胞中的 Hdpsc, 也有几个方法可以在此过程中采取关键步骤。Hdpsc 能够在几个谱系中进行分化, 如软骨细胞、脂肪细胞、成骨细胞和神经元。本文研究了神经元分化的机制和 Prpc 的存在.如上文所述, 这些细胞表达典型的间充质结构特异性表面抗…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了 “Fondazione Varrone” 和 Rieti 大学枢纽 “Sabina 大学” 对 Vincenzo Mattei 的支持。
图 5 (A, b) 经出版商泰勒 & Francis Ltd 的许可转载, 来自: Prion 蛋白 egfr 多分子复合体在人类牙齿浆源性干细胞神经元分化过程中的作用。Martellucci, S., Manganelli V., Santacroce C., Santellli f., Piccoli l., Sorice M., Mattei v. prion. 2018年3月4日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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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).