牙龈三维炎症人体组织当量
Summary
该协议的目标是建立一个炎症的人牙龈模型在体外。该组织模型共培养三种类型的人细胞, HaCaT 角质形成细胞, 牙龈成纤维细胞, THP-1 巨噬细胞, 在三维条件下。该模型可用于牙周疾病的研究, 如牙龈炎和牙周炎。
Abstract
牙周疾病 (如牙龈炎和牙周炎) 是成人牙齿丢失的主要原因。牙龈炎症是牙周疾病的基本生理病理。目前在各种类型的动物中建立了牙周疾病的实验模型。然而, 动物模型的生理病理与人类不同, 因此很难分析细胞和分子机制, 并对牙周疾病新药进行评价。在这里, 我们提出了一个详细的协议, 以重建牙龈炎症组织等值的龈 (iGTE)在体外。我们首先使用两种类型的人类细胞, 包括人牙龈成纤维细胞 (HGF) 和人表皮角质细胞 (HaCaT), 在三维条件下, 构建牙龈 (GTE) 的人体组织当量。我们创建一个伤口模型, 使用组织冲床打孔在 GTE。然后, 将人 THP-1 单核细胞与胶原凝胶混合, 注入 GTE 的孔内。通过 adimistration 10 佛波 12-酯 13-醋酸盐 (PMA) 为72小时, THP-1 细胞分化成巨细胞形成炎症灶在 GTE (iGTE) (iGTE 也可以 stumilated 与2µg/毫升脂多糖 (LPS) 48 h 启动炎症).IGTE 是第一个体外模型的炎症牙龈使用人类细胞与三维体系结构。IGTE 反映了牙周疾病的主要病理变化 (免疫 activition、fibryoblasts、上皮细胞、单核和巨噬细胞之间的细胞间相互作用)。GTE, 受伤的 GTE 和 iGTE 可作为多功能工具, 研究伤口愈合, 组织再生, 炎症, 细胞相互作用, 和屏幕潜在药物的牙周疾病。
Introduction
牙周疾病是成人牙齿丢失的主要原因。牙龈炎和牙周炎是最常见的牙周疾病。牙龈中存在生物膜介导的急性或慢性炎症变化。牙龈炎的特点是急性炎症, 而牙周炎通常呈现为慢性炎症。在组织学层面上, 细菌成分会触发免疫细胞的活化, 如巨噬细胞、淋巴细胞、血浆细胞和桅杆单元1,2。这些免疫细胞, 特别是巨细胞, 与局部细胞 (包括牙龈上皮细胞、成纤维细胞、血管内皮和成骨细胞) 相互作用, 导致牙周组织的炎症性病变3,4。实验模型的牙周疾病已建立在各种类型的动物, 如大鼠, 仓鼠, 兔, 雪貂, 犬, 和灵长类动物。然而, 动物模型的生理病理与人类不同, 因此很难分析细胞和分子机制, 并评估牙周病的新药 5.利用牙周细菌和单层人口腔上皮细胞的共同培养, 研究了牙周感染的机制6。然而, 口腔细胞的单层培养缺乏完整组织的三维 (3D) 细胞结构;因此, 它们无法模拟体外情况。
在这里, 3D 炎症人的牙龈组织等效物 (iGTE) 是用来表示牙周疾病的在体外。这种3D 模型的牙周疾病在单层细胞培养和动物模型之间占据中间位置。三种类型的人类细胞, 包括 HaCaT 角质形成细胞, 牙龈成纤维细胞, 和 THP-1 巨噬细胞, 是由胶原凝胶共同培养, 并刺激炎症启动器, 以建立 iGTE。IGTE 严密模拟了牙龈中细胞分化、细胞相互作用和巨噬细胞活化的体内条件。该模型有许多可能的应用, 药物筛选和测试新的药理方法, 牙周疾病, 以及分析细胞和分子机制的伤口愈合, 炎症, 和组织再生。
Protocol
本协议旨在创建人牙龈组织当量, 牙龈伤口模型, 牙龈炎模型。人皮肤表皮角质细胞 (HaCaT) 是亲切提供的 Fusenig 教授 e. 德意志 Krebsforschungszentrum (德国海德堡)7。根据先前发布的协议8, 人牙龈成纤维细胞 (HGFs) 与牙龈组织分离。事先获得知情同意, 并根据道德委员会制定的指导方针, 东京牙科大学生活牙科学院 (授权号码: NDU-T2012-35) 批准了这项研究。协议步骤1–3应…
Representative Results
HaCaT 细胞在相对比显微镜观察下显示典型的角质形成形态 (图 2A)。扫描电镜 (SEM) 图像表明, HaCaT 细胞表面被许多微绒毛覆盖。HaCaT 细胞之间的细胞间连接由膜过程介导 (图 2B)。HaCaT 细胞表达牙龈上皮标记 K8/1814, 表明 HaCaT 细胞适合牙龈重建 (图 2C)。 <p class="jove_content" fo:keep-together.within-…
Discussion
该协议是基于创建牙龈组织当量和皮下脂肪组织等价的方法, 以前的报告中描述的8,21,22。虽然这是一个简单而简单的方法, 但有些步骤需要特别注意。例如, 胶原混合物应保持在冰上, 直到使用, 以避免凝胶形成的解决方案。当将胶原蛋白混合物添加到区域性插入中时, 请确保将该溶液注入插入中心, 以避免形成有偏见的凝胶 (如<…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了日本促进科学促进会 (26861689 和 17K11813) 资助的部分支持。作者感谢纳撒尼尔. 格林先生校对。
Materials
| Collagen type I-A | Nitta Gelatin Inc | For making dermis of GTE | |
| MEM-alpha | Thermo Fisher Scientific | 11900073 | Cell culture medium |
| Cell Culture Insert (for 24-well plate), pore size 3.0 μm | Corning, Inc. | 353096 | For tissue culture |
| GlutaMAX | Thermo Fisher Scientific | 35050061 | Cell culture reagent |
| DMEM | Thermo Fisher Scientific | 31600034 | Cell culture medium |
| KnockOut Serum Replacement | Thermo Fisher Scientific | 10828028 | Cell culture reagent |
| Tissue puncher | Shibata system service co., LTD | SP-703 | For punching holes in GTE |
| RPMI 1640 | Thermo Fisher Scientific | 31800022 | Cell culture medium |
| BSA | Sigma-Aldrich | A3294 | For immunostaining |
| Hoechst 33342 (NucBlue Live Cell stain) | Thermo Fisher Scientific | R37605 | For labeling nuclei |
| Fluorescence mount medium | Dako | For mounting samples after immunostaining | |
| Anti-Cytokeratin 8+18 antibody [5D3] | abcam | ab17139 | For identifying epithelium |
| Scaning electron microscope | Hitachi, Ltd. | HITACHI S-4000 | For observing samples' surface topography and composition |
| Confocal laser scanning microscopy | LSM 700; Carl Zeiss Microscopy Co., Ltd. | LSM 700 | For observing samples' immunofluorescence staining |
| Anti-Cytokeratin 19 antibody | abcam | ab52625 | For identifying epithelium |
| Anti-vimentin antibody | abcam | ab92547 | For identifying fibroblasts and activated macrophages |
| Anti-TE-7 antibody | Millipore | CBL271 | For identifying fibroblasts in the dermis |
| Anti-CD68 antibody | Sigma-Aldrich | SAB2700244 | For identifying macrophages |
| Human CD14 Antibody | R&D Systems | MAB3832-SP | For identifying macrophages |
| Alexa Fluor 594-conjugated secondary goat anti-rabbit antibody | Thermo Fisher Scientific | A11012 | For immunofluorescence staining |
| Alexa Fluor 488-conjugated secondary goat anti-mouse antibody | Thermo Fisher Scientific | A11001 | For immunofluorescence staining |
| EVOS FL Cell Imaging System | Thermo Fisher Scientific | For observing the sample's immunofluorescence staining | |
| THP-1 cells | Riken BRC cell bank | RCB1189 | For making iGTE |
| PMA(Phorbol 12-myristate 13-acetate) | Sigma-Aldrich | P8139 | For differentiatting THP-1 cells |
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Citer Cet Article
Xiao, L., Okamura, H., Kumazawa, Y. Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva. J. Vis. Exp. (134), e57157, doi:10.3791/57157 (2018).