使用Y-27632的原发性人类金吉瓦尔表皮细胞的隔离与培养
1Department of Tissue Engineering and Regeneration, School and Hospital of Stomatology, Cheeloo College of Medicine,Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration, 2Department of Orthodontics, School and Hospital of Stomatology,Cheeloo College of Medicine, Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration, 3Department of Stomatology,Shengli Oilfield Central Hospital, 4Department of Pediatric Surgery,Shengli Oilfield Central Hospital, 5Ningbo Stomatology Hospital of Savaid Stomatology School,Hangzhou Medical College
Summary
在这里，我们提出了一个改进的方法，通过添加岩石抑制剂，Y-27632，以传统的方法隔离和培养人类金吉瓦尔上皮细胞。这种方法更容易，更省时，提高干细胞的特性，并产生更多的高潜力上皮细胞，无论是为实验室和临床应用。
Abstract
牙周组织是保护牙周组织的第一个结构，在许多口腔功能中起着有意义的作用。金银核酸上皮是金银组织的重要结构，特别是在牙周组织的修复和再生中。研究金吉瓦尔上皮细胞的功能具有重要的科学价值，如修复口腔缺陷和检测生物材料的兼容性。由于人类上皮细胞是高度分化的角蛋白细胞，其寿命短，难以通过。到目前为止，只有两种方法可以分离和培养金吉瓦尔上皮细胞，一种是直接的外生方法，另一种是酶。然而，使用直接外生法获得上皮细胞所需的时间较长，酶法的细胞存活率较低。临床上，金银组织采集有限，因此需要一个稳定、高效、简单的 体外 隔离培养系统。我们通过添加Y-27632（一种与Rho相关的激酶（ROCK）抑制剂，改进了传统的酶方法，这种抑制剂可以选择性地促进上皮细胞的生长。我们改良的酶法简化了传统酶方法的步数，提高了上皮细胞的培养效率，比直接除菌法和酶法具有显著优势。
Introduction
人牙石是保护牙周组织的第一道防线结构，它不仅是物理和化学屏障1，而且分泌不同类别的炎症调解员参与免疫反应，构成免疫屏障2、3。金银上皮在牙周组织的修复和再生中起着重要的作用。因此，研究牙周炎的防御和免疫，对于了解牙周炎的发生、诊断和治疗具有重要意义。从人类金银杏组织中分离和培养金吉瓦尔上皮细胞是研究金吉瓦尔上皮的第一步。这种程序需要基本操作，如为组织工程生产种子细胞、牙周相关疾病的体外模型以及修复牙周缺陷的材料。
原发性金银体上皮细胞的特点是体外4的分化率低，几十年来，研究人员一直在寻找最佳的分离和培养方法。迄今为止，实验室中通常使用两种不同的技术，即直接外生法和酶法，在体外获得原发性金吉瓦尔上皮细胞。直接除种方法具有组织标本量低、隔离程序简单等优点，但具有培养时间长、易受污染等优点。虽然酶方法缩短了所需的培养时间，但效率相对较低，并且因使用的酶和介质而异。Kedjarune等人6表明，与酶方法相比，直接外植方法在亚文化（2周）之前需要更多的时间，在培养金吉瓦尔上皮细胞方面似乎更为成功。然而，比较这两种方法，Klingbeil等人发现，这种酶方法对口腔上皮细胞的原发培养效果最好，并且有可能在最短的时间内（11.9天对14.2天）获得最佳细胞产量。
因此，开发一种更方便、更有效的口腔上皮细胞分离与培养方法非常重要。我们之前报告说，添加Y-27632，Rho相关蛋白激酶（ROCK）的抑制剂，简化了人类原发性表皮细胞和角蛋白细胞从成人皮肤组织8，9，10的分离程序。我们开发了G-media，一种新的条件接种介质，自发地将表皮与皮肤细胞分离，支持原发性表皮细胞8、9、10的生长和产量。在本研究中，我们通过将G-中型细胞与Y-27632结合，开发出一种新的无血清分离培养技术。从本质上讲，我们的方法是基于对传统两步酶方法的简化，因此我们比较了新方法和直接外生方法。这种改良的酶方法大大缩短了将金吉瓦尔上皮细胞与金银杏组织分离所需的时间，并提高了培养金吉瓦尔上皮细胞的效率。
Protocol
根据该学会人类研究伦理委员会的指导方针，本协议中使用的人体组织是从Maxillo面部外科拔出受影响的牙齿中丢弃的新鲜成人金银杏组织。GR201711，日期：02-27-2017）。 1. 准备工作 在含有 10 mL 磷酸盐缓冲盐水 （PBS） 的 15 mL 管中收集新鲜的成人金银杏组织，辅以 3% 青霉素/链霉素 （P/S），并将组织保持在 4 °C。注：切除后24小时内将下面的组织详细处?…
Representative Results
图1 显示了直接外分法和改性酶法的示意图。在整个过程中，直接外生方法不需要任何消化酶。相比之下，传统的酶方法通常需要两套消化酶，脱口而出和拼贴，将上皮板从底层成纤维细胞层分离，然后尝试将上皮细胞释放到悬浮中。我们的新方法省略了分离的步骤，是一种简化的酶方法。此外，在G-中型中加入Y-27632可有效促进上皮细胞生长。直接外植方法通常需要大约2周?…
Discussion
牙周组织是维持牙周完整性和健康的关键结构。金吉瓦尔上皮细胞在牙周组织的修复和再生中起着重要作用，可用于科学研究和临床应用及相关领域，包括口腔生物学、药理学、毒理学和口腔粘膜缺乏症18。因此，有必要开发一种稳定而有效的方法来收获口腔上皮细胞19。原发性上皮细胞是一种完全分化的细胞，通道少，寿命短。培育上皮细胞已被证明是比培养…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了山东省自然科学基金重点项目（ZR2019ZD36）和山东省重点研究发展计划（2019GSF108107）的支持。山东省重点研发计划（2018GSF118240）向J.G.：山东省医疗卫生科技发展项目（2018WS163）至Z.X.，山东省医疗卫生科技发展项目（2019WS045）至J.S.
Materials
| Names | Abbreviations & Comments | ||
| Countess automated cell counter | Shanghai Ruiyu Bio-science&Technology Co.Ltd. | BBA0218AC | Automatic cell counting |
| CO2 Incubator | Thermo Scientific | 51026333 | For cell incubation |
| Sorvall ST 16R Centrifuge | Thermo Scientific | 75004380 | Cell centrifuge |
| Cell Culture Dish | Eppendorf | 30702115 | For cell culture |
| 50 ml Centrifuge Tube | KIRGEN | 171003 | For cell centrifugation |
| 1.5 ml microcentrifuge Tubes | KIRGEN | 190691J | For cell digestion |
| Cell Strainer | Corning incorporated | 431792 | Cell filtration |
| Phosphate buffered solution | Solarbio Life Science | P1020-500 | Washing solution |
| DMEM | Thermo Scientific | C11995500 | Component of neutralization medium |
| Defined K-SFM | Life Technologies | 10785-012 | Gingival epithelial cells culture medium |
| Penicillin Streptomycin | Thermo Scientific | 15140-122 | Antibiotics |
| Fetal Bovine Serum | Biological Industries | 04-001-1AC5 | Component of neutralization medium |
| 0.05% Trypsin | Life Technologies | 25300-062 | For HGGEPCs dissociation |
| Dilution Medium | Life Technologies | 50-9701 | For coating matrix |
| Dispase | Gibco | 17105-041 | For HGGEPCs isolation |
| Collagenase Type I | Life Technologies | 17100-017 | For HGGEPCs isolation |
| F12 Nutrient Mix, Hams | Life Technologies | 31765035 | Component of G-medium |
| B27 Supplement | Life Technologies | 17504044 | Growth factor in G-medium |
| FGF-2 Millipore | Merck Biosciences | 341595 | Growth factor in G-medium |
| Y-27632 | Gene Operation | IAD1011 | ROCK inhibitor |
| Fungizone | Gibco | 15290026 | Preparation for G-medium |
| EGF Recombinant Human Protein | Gibco | PHG0311 | Growth factor in G-medium |
| Cell Counting Kit-8 | Dojindo Molecular Technologies | CK04 | For Cell proliferation assay |
| Rabbit Anti-Human CK18 | Abcam | ab82254 | For immunofluorescence staining to check differentiation marker of HGGEPCs |
| Rabbit Anti-Human Cytokeratin10 | Abcam | ab76318 | For immunofluorescence staining to check differentiation marker of HGGEPCs |
| Mouse anti-human Vimentin | Cell Signaling Technology | 3390 | For immunofluorescence staining of Gingival fibroblasts |
| Rabbit Anti-Human pan-ck | BD | 550951 | For immunofluorescence staining to check differentiation marker of HGGEPCs |
| rabbit anti-Ki67 | Abcam | 15580 | For immunofluorescence staining to check differentiation marker of HGGEPCs |
| rabbit anti-p63 | Biolegend | 619002 | For immunofluorescence staining to check differentiation marker of HGGEPCs |
| rabbit anti-p75NGFR | Abcam | ab52987 | For immunofluorescence staining to check differentiation marker of HGGEPCs |
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Cite This Article
Xie, Z., Shi, J., Zong, M., Xu, Q., Liu, C., Wen, J., Zhang, Q., Liu, P., Liu, G., Guo, J., Wu, X. Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632. J. Vis. Exp. (177), e62978, doi:10.3791/62978 (2021).