| 分化可控的人类鼻粘膜类器官模型的建立 |
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| 引用本文： | 汪 珂,于 言,韩 日,王显文,赵云腾,唐浩程,李 刚. 分化可控的人类鼻粘膜类器官模型的建立[J]. 南方医科大学学报, 2022, 42(6): 868-877. DOI: 10.12122/j.issn.1673-4254.2022.06.10 |
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| 作者姓名： | 汪 珂 于 言 韩 日 王显文 赵云腾 唐浩程 李 刚 |
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| 作者单位： | 南方医科大学南方医院耳鼻喉科，广东 广州 510515 |
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| 摘 要： | 目的 建立分化程度可控、能够重现来源组织结构和功能的人类鼻粘膜类器官模型。方法 采集手术切除的新鲜中鼻甲和鼻息肉组织，将消化过滤的鼻粘膜上皮细胞分为连续“扩增”培养组（EO组）及“扩增-分化”分段培养组（DO组）。分别在基于气液界面进行体外3D类器官培养，通过STR鉴定、电镜及免疫组化染色分别对两组鼻粘膜类器官的结构、细胞组成及纤毛功能进行鉴定。再通过PAS染色对DO组中分化后的鼻粘膜类器官分泌功能进行鉴定。结果 整个培养期间，均能生长出直径逐渐增大的空泡状或实心球状3D类器官。培养第16天，DO组多为空泡状类器官，EO组多为实心球状类器官，DO组空泡数比例大于EO组（[ 21.67±8.57）% vs（54.67±13.26）%，P<0.05]。将鼻粘膜类器官与来源组织同时进行STR检测，匹配度为100%。培养第21天，DO组鼻粘膜类器官扫描及透射电镜显示纤毛超微结构，而EO组多显示出短绒毛结构。免疫组化染色结果显示，呼吸区粘膜主要包含P63（基底细胞）、β-tubulin（纤毛柱状细胞）、MUC5AC（杯状细胞）。相比EO组，DO组类器官纤毛细胞数[(7.95± 1.81)% vs (27.04±5.91)%，P<0.05]及杯状细胞数[(14.46±0.93)% vs (39.85±5.43)%，P<0.05）占比更大，基底细胞占比差异无统计学意义（P>0.05）。DO组中分化后的鼻粘膜类器官糖原染色呈阳性表达。结论 本研究首次采用“扩增-分化”分段式培养法，培养出能够在体外长期稳定生长、高度拟似来源组织形态结构和功能（纤毛功能及分泌功能），且分化程度可控的鼻粘膜类器官。
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| 关 键 词： | 类器官；鼻粘膜；气液界面；分化培养；3D细胞模型 |
Establishment of a culture system for human nasal mucosa organoids with controllable differentiation |
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| WANG Ke,YU Yan,HAN Ri,WANG Xianwen,ZHAO Yunteng,TANG Haocheng,LI Gang. Establishment of a culture system for human nasal mucosa organoids with controllable differentiation[J]. Journal of Southern Medical University, 2022, 42(6): 868-877. DOI: 10.12122/j.issn.1673-4254.2022.06.10 |
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| Authors: | WANG Ke YU Yan HAN Ri WANG Xianwen ZHAO Yunteng TANG Haocheng LI Gang |
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| Affiliation: | Department of Otolaryngology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China |
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| Abstract: | Objective To establish a culture system for human nasal mucosal organoids with controllable differentiation to reproduce the structure and function of the source tissue through staged expansion-differentiation culture. Methods Fresh samples of surgically resected middle turbinate and nasal polyp tissues were collected, from which the nasal mucosa epithelial cells were isolated by enzymatic digestion and filtration for continuous culture at the air-liquid interface for expansion (EO group) or staged culture for expansion and differentiation (DO group). Immunohistochemical staining was used to characterize the structure, cellular composition and ciliary function of nasal mucosal organoids in the two groups. The secretion function of the differentiated nasal mucosal organoids in DO group was evaluated using PAS staining. Results Both of the two organoid culture systems yielded vacuolar or solid spherical 3D organoids, and their diameters increased progressively with time. On day 16 of culture, more vacuolar organoids occurred in DO group, while more solid spherical organoids were seen in EO group, and the proportion of vacuoles was significantly greater in DO group than in EO group [(54.67±13.26)% vs (21.67±8.57)%, P<0.05]. Short tandem repeat (STR) test of the nasal mucosal organoids and the source tissue showed a 100% match between them. On day 21 of culture, scanning and transmission electron microscopy of the nasal mucosal organoids identified ultrastructure of cilia in DO group and short villi structure in most of the organoids in EO group. Immunohistochemical staining showed positivity for P63 (basal cells), β-tubulin (ciliated columnar cells), and MUC5AC (goblet cells) in the organoids. Compared with those in EO group, the organoids in DO group showed significantly greater percentages of ciliated cells [(7.95±1.81)% vs (27.04±5.91)%, P<0.05] and goblet cells [(14.46±0.93)% vs (39.85±5.43)%, P<0.05) with a similar percentage of basal cells [(56.91±14.12)% vs (53.42±15.77)%, P>0.05]. The differentiated nasal mucosal organoids in DO group were positively stained for glycogen. Conclusion The staged expansion-differentiation culture method allows more stable and prolonged growth of the cultured cells in vitro to produce organoids with controllable differentiation closely resembling the morphological structure and functions (ciliary function and secretory function) of the source tissue. |
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| Keywords: | organoids nasal mucosa air-liquid interface differentiation culture 3D cell model, |
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