人鼻粘膜类器官EB病毒感染模型的建立

doi:10.12122/j.issn.1673-4254.2026.02.12

摘要/Abstract

摘要：

目的开发一种经济、简便的人鼻粘膜类器官培养方法，用于建立EB病毒（EBV）体外感染模型。方法从手术获得的鼻息肉组织，经清洗、剪切、消化、过滤后得到细胞团，在无基质胶的条件下经动态悬浮培养扩增为未分化的鼻粘膜类器官，再通过14 d的促分化处理以形成分化的鼻粘膜类器官，通过免疫荧光、免疫组化鉴定类器官主要细胞组成。加入EBV体外感染鼻粘膜类器官，通过RT-qPCR检测病毒特异性基因EBNA1、BALF5的表达，并通过EBNA1免疫荧光染色确认病毒感染。结果动态悬浮培养法可在无基质胶的培养体系中，成功培养出由基底细胞、粘液细胞、纤毛细胞构成的鼻粘膜类器官。分化的鼻粘膜类器官高表达EBV相关受体EphA2、NRP1、NMHCⅡ-A。未分化和分化的鼻粘膜类器官均可被EBV感染，类器官中病毒复制随病毒暴露载量的增加而增加，分化类器官可能更有利于病毒复制（P<0.001）。结论无基质胶的动态悬浮培养法是一种经济、简便的鼻粘膜类器官建模方法，人鼻粘膜类器官能够作为EBV感染模型，为上皮EBV感染相关研究提供一个良好的平台。

关键词: 类器官, EBV, 鼻粘膜

Abstract:

Objective To develop an cost-effective and convenient method for culturing human nasal organoids to establish an in vitro Epstein-Barr virus (EBV) infection model. Methods Nasal polyp tissue obtained from surgery was routinely washed, cut, digested and filtered to obtain cell clusters. The cell clusters were then expanded into undifferentiated nasal organoids through dynamic suspension culture under matrix-free conditions, followed by a 14-day differentiation induction treatment to obtain differentiated nasal organoids. The major cellular components of the organoids were identified by immunofluorescence staining and immunohistochemistry. The nasal organoids were infected by EBV in vitro, and viral replication was verified by detecting the expressions of the virus-specific genes EBNA1 and BALF5 using RT-qPCR and immunofluorescence staining. Results Nasal organoids consisting of basal cells, mucous cells, and ciliated cells in a martigel-free system were obtained successfully by dynamic suspension culture. The differentiated nasal organoids expressed high levels of EBV-associated receptors EphA2, NRP1, and NMHCII-A. Both the undifferentiated and differentiated nasal organoids could be infected by EBV. Viral replication in the organoids increased with the viral exposure load, and the differentiated organoids appeared more permissive to viral replication. Conclusion The matrigel-free dynamic suspension culture method is economical and simple for constructing nasal organoids, which can be used as a model of EBV infection for studies of epithelial EBV infection.

Key words: organoids, Epstein-Barr virus, nasal mucosa

魏金燕, 郑海锐, 赵云腾, 于言, 徐莹莹, 李刚. 人鼻粘膜类器官EB病毒感染模型的建立[J]. 南方医科大学学报, 2026, 46(2): 345-352.

Jinyan WEI, Hairui ZHENG, Yunteng ZHAO, Yan YU, Yingying XU, Gang LI. Establishment of an Epstein-Barr virus infection model using human nasal organoids[J]. Journal of Southern Medical University, 2026, 46(2): 345-352.

图/表 9

表1 鼻粘膜样本信息

Tab.1 Information of the nasal mucosa samples

Sample number	Source	Reason for surgery
ZZH-9580	Nasal polyps	CRSwNP
LHX-4635	Nasal polyps	CRSwNP
XWJ-4024	Nasal polyps	CRSwNP
XCY-8640	Nasal polyps	CRSwNP
LJL-3202	Nasal polyps	CRSwNP
LHB-4635	Nasal polyps	CRSwNP
TRJ-8166	Nasal polyps	CRSwNP

表2 免疫荧光和免疫组化抗体信息

Tab.2 Antibodies for immunofluorescence assay and immunohistochemistry

Antibody	Brand	Cat No.	dilution ratio
Anti-Cytokeratin 5 antibody	Abcam	ab75869	1:100
Anti-Cytokeratin 14 antibody	Abcam	ab119695	1:100
Anti-p63 antibody	Abcam	ab124762	1:100
Anti-MUC5AC antibody	Abcam	ab198294	1:200
Anti-beta Ⅳ tubulin antibody	Abcam	ab179509	1:200
Anti-FoxJ1 antibody	Abcam	ab235445	1:2000
COL4A2 Polyclonal antibody	Proteintech	55131-1-AP	1:300
MYH9 Monoclonal antibody	Proteintech	60233-1-IG	1:200
EPHA2 Monoclonal antibody	Proteintech	66736-1-IG	1:200
Neuropilin 1/CD304 Recombinant antibody	Proteintech	84429-5-RR	1:300
Anti-EBNA1 antibody	Abcam	ab316860	1:2000
Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488	Invitrogen	A11034	1:500
Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488	Invitrogen	A11029	1:500
HRP-conjugated Goat Anti-Rabbit/mouse IgG for IHC (ready to use)	Proteintech	PR30009	No dilution needed

表3 RT-qPCR引物序列

Tab.3 Primer sequence for RT-qPCR

Gene	Foward (5'→3')	Reverse (5'→3')
GAPDH	GGAGCGAGATCCCTCCAAAAT	GGCTGTTGTCATACTTCTCATGG
EPHA2	CCCGATGAGATCACCGTCAG	GGCACCGATATCCTGGAAGG
NMHC-ⅡA	GAGCAAATGGGCCTGCT	TGTTGTCGGGCATGGA
NRP1	ACCCAAGTGAAAAATGCGAATG	CCTCCAAATCGAAGTGAGGGTT
EBNA1	GGTCGTGGACGTGGAGAAAA	GGTGGAGACCCGGATGATG
BALF5	GAGCGATCTTGGCAATCTCT	TGGTCATGGATCTGCTAAACC

图1 鼻粘膜类器官培养光镜图

Fig.1 Phase-contrast microscopy of nasal organoids in culture. A: Representative images at different time points during the expansion of nasal organoids. B: Representative images of the nasal organoids on days 3, 7, and 14 during differentiation induction.

图2 鼻粘膜类器官HE染色结果及基底膜标志物染色

Fig.2 HE staining of the nasal organoids and immunofluorescence staining for the basal lamina marker collagen IV. A: HE staining of undifferentiated and differentiated nasal organoids. The differentiated nasal organoids exhibit a higher density of cilia on their apical surface. B: Immunofluorescence staining for collagen IV in differentiated nasal organoids.

图3 未分化和分化的鼻粘膜类器官组成细胞免疫荧光

Fig.3 Immunofluorescence analysis of cellular composition of undifferentiated and differentiated nasal organoids. A: Representative images of undifferentiated organoids showing positive expressions of epithelial basal stem cell markers CK5, CK14, and p63. B: Representative images of differentiated organoids consisting of basal cells (CK5), goblet cells (MUC5AC), and ciliated cells (β-tubulin IV).

图 4 鼻粘膜组织和类器官组成细胞免疫组化

Fig.4 Immunohistochemical analysis of cellular composition of nasal mucosal tissue and organoids. Representative images of nasal mucosal tissue are shown in the upper panel and differentiated nasal organoids in the lower panel. The organoids exhibit comparable expression patterns to nasal mucosal tissue for P63 (basal cells), MUC5AC (goblet cells), and FOXJ-1 (ciliated cells).

图5 分化的鼻粘膜类器官高表达EBV感染受体

Fig.5 Differentiated nasal organoids exhibit high expressions of Epstein-Barr virus (EBV) receptors. A: Relative mRNA expressions of EBV receptors NMHC-IIA, EPHA2 and NRP1 in undifferentiated and differentiated nasal organoids. ***P<0.001 vs undifferentiated group. B: Immunofluorescence staining for EBV receptors NMHC-IIA, EPHA2 and NRP1 in differentiated nasal organoids (n=3).

图6 鼻粘膜类器官EBV感染模型的建立

Fig.6 Establishment of EBV-infected nasal organoids. A, B: Relative mRNA expression levels of EBV-specific genes EBNA1 and BALF5 in differentiated organoids harvested at 72 h post-EBV infection at MOI=250 and 2500 (A; *P<0.05, ***P<0.001, ****P<0.0001 vs Mock group) and in both undifferentiated and differentiated organoids harvested 72 h after EBV infection at MOI=2500 (B; ***P<0.001 vs undifferentiated group). C, D: Immunofluorescence staining for EBNA1 undifferentiated (C) and differentiated (D) organoids with EBV infection at MOI=2500 (n=3).

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