人鼻粘膜类器官冠状病毒感染模型可用于抗病毒药物的筛选和评价

doi:10.12122/j.issn.1673-4254.2024.11.20

摘要/Abstract

摘要：

目的利用人鼻粘膜类器官建立冠状病毒（CoV）感染模型，并用于药物抗病毒作用体外评价，探讨人类鼻粘膜类器官病毒感染模型用于病毒研究和抗病毒药物开发的可行性。方法使用人源鼻粘膜类器官，测试其对SARS-CoV-2和HCoV-OC43假病毒的易感性。在P3实验室中，评估鼻粘膜类器官对SARS-CoV-2原始株及4种变异株的感染情况，优化感染条件。通过荧光定量PCR检测不同时间点培养基中病毒含量，并利用免疫荧光法定位新冠病毒衣壳蛋白，以确定病毒感染细胞的类型。在优化后的鼻粘膜病毒感染模型中，分析已知具有抑制新冠病毒作用的化合物，如卡莫司他和佛手柑素，单因素方差分析推断其在类器官中的抑制效果并预测其机制。结果通过培养体系和感染条件的优化，使用SARS-CoV-2不同亚型感染鼻粘膜类器官，结果显示，2~24 h受感染鼻粘膜类器官上清液中的病毒载量明显增加，鼻粘膜类器官可被SARS-CoV-2，HCoV-OC43假病毒感染（P<0.001）。真病毒感染实验证实，SARS-CoV-2原始株及4种变异株，均能稳定感染鼻粘膜类器官，证实病毒感染模型的建立。卡莫司他和佛手柑素，在鼻粘膜类器官中均剂量依赖性抗病毒作用（P<0.0001）。结论人鼻粘膜类器官能够作为新冠病毒感染模型，用于抗病毒药物筛选和评价，特别是经鼻腔给药方式的提供了理想的体外研究模型。

关键词: 类器官, 鼻粘膜, 病毒感染, 疾病模型

Abstract:

Objective To establish a coronavirus (CoV) infection model using human nasal mucosa organoids for testing antiviral drugs and evaluate the feasibility of using human nasal mucosa organoids with viral infection as platforms for viral research and antiviral drug development. Methods Human nasal mucosa organoids were tested for susceptibility to SARS-CoV-2 and HCoV-OC43 pseudoviruses. In a P3 laboratory, nasal mucosa organoids were infected with the original strain of SARS-CoV-2 and 4 variant strains, and the infection conditions were optimized. The viral loads in the culture supernatants were measured at different time points using RT-qPCR, and immunofluorescence assay was employed to localize SARS-CoV-2 nucleocapsid protein to determine the type of the infected cells. In the optimized nasal mucosa viral infection model, the antiviral effects of camostat and bergamot extract (which were known to inhibit SARS-CoV-2) were tested and the underlying molecular mechanisms were explored. Results In the optimized nasal mucosa organoid models infected with SARS-CoV-2 and HCoV-OC43 pseudoviruses, the viral load in the culture supernatants increased significantly during the period of 2 to 24 h following the infection, which confirmed infection of the organoids by both of the pseudoviruses. The nasal mucosa organoids could be stably infected by the original SARS-CoV-2 strain and its 4 variant strains, validating successful establishment of the viral infection model, in which both camostat and bergamot extract exhibited dose-dependent antiviral effects. Conclusions Human nasal mucosa organoids with SARS-CoV-2 infection can serve as platforms for screening and testing antiviral drugs, particularly those intended for nasal administration.

Key words: organoids, nasal mucosa, virus infection, disease model

于言, 曹浚垣, 刘蓉, 周旻旻, 魏金燕, 郑海锐, 王薇, 李刚. 人鼻粘膜类器官冠状病毒感染模型可用于抗病毒药物的筛选和评价[J]. 南方医科大学学报, 2024, 44(11): 2227-2234.

Yan YU, Junyuan CAO, Rong LIU, Minmin ZHOU, Jinyan WEI, Hairui ZHENG, Wei WANG, Gang LI. Development of a new platform for testing antiviral drugs using coronavirus-infected human nasal mucosa organoids[J]. Journal of Southern Medical University, 2024, 44(11): 2227-2234.

图/表 7

表1 鼻粘膜样本信息

Tab.1 Nasal mucosa sample information

Sample number	Source	Passage number	Days	Cryopreservation date	Culture status
RNM-0126	Respiratory region	P1	4	2021-12-17	Success
RNM-0179	Respiratory region	P1	6	2021-12-28	Success
ONM-0325	Olfactoryactory region	P0	4	2022-01-08	Success
RNM-0160	Respiratory region	P2	10	2022-03-10	Success
RNM-0234	Respiratory region	P1	8	2022-03-10	Success
RNM-0370	Respiratory region	P0	0	2022-03-10	Failure
RNM-0328	Respiratory region	P1	6	2022-03-14	Success
RNM-0344	Respiratory region	P0	8	2022-03-16	Success
RNM-0350	Respiratory region	P0	7	2022-03-16	Success
RNM-0351	Respiratory region	P0	7	2022-03-16	Success
ONM-0375	Olfactoryactory region	P0	10	2022-03-18	Success
RNM-0359	Respiratory region	P1	5	2022-03-21	Failure
RNM-0408	Respiratory region	P0	8	2022-03-23	Success
ONM-0489	Olfactoryactory region	P0	8	2022-03-23	Success

图1 鼻粘膜类器官的生长动态图

Fig.1 Observation of growth of nasal mucosal organoids under inverted microscope at different time points and HE staining of the organoids on Day 5, P25 (Scale bar=100 μm).

图2 假病毒对鼻粘膜类器官感染情况

Fig.2 SARS-CoV-2 (A) and HCoV-OC43 (B) pseudovirus infection of nasal mucosal organoids. ***P<0.001, ****P<0.0001.

图3 鼻粘膜类器官新冠病毒感染模型建立

Fig.3 Modeling of SARS-CoV-2 infection in nasal mucosal organoids. A: Virus content in the supernatant at different time points following infection ay MOI of 1 and 10. B: Detection of viral content in the supernatant following infection with different viruses. *P<0.05, ***P<0.001, ****P<0.0001.

图4 免疫组化检测冠状病毒感染受体, 免疫荧光检测新冠病毒感染杯状细胞

Fig.4 Immunohistochemistry for detecting coronavirus receptors and immunofluorescence staining for detecting SARS-CoV-2-infected goblet cells (scale bar=50 μm). A: Immunohistochemistry analysis of ACE2 and TMPRSS2, the key proteins for coronavirus infection (scale bar: 50 μm). B: Immunofluorescence staining of goblet cells infected by SARS-CoV-2 showing DAPI staining of the cell nuclei, MUC5AC staining of goblet cells, and viral nucleoprotein.

图5 药物在人鼻粘膜类器官中对新型冠状病毒的抑制效果检测

Fig.5 Inhibitory effect of the drugs for testing on SARS-CoV-2 in human nasal mucosal organoids. A: Camostat dose-dependently inhibits SARS-CoV-2 infection in human nasal mucosal organoids. B: Bergamottin dose-dependently inhibits SARS-CoV-2 infection in human nasal mucosal organoids. **P<0.01, ***P<0.001, ****P<0.0001 vs DMSO.

图6 佛手柑素与卡莫司他通过ACE2结合抑制冠状病毒感染鼻粘膜细胞

Fig.6 Bergamottin and camostat interact with ACE2 to block coronavirus infection. A: Docking position of bergamottin with human ACE2. B: Docking position of camostat with human ACE2. C: Binding position of lisinopril with human ACE2. D: Binding details of lisinopril with human ACE2. E: Docking energy of bergamottin, camostat and lisinopril against human ACE2.

参考文献 33

1	Ballard DH, Boyer CJ, Alexander JS. Organoids-Preclinical Models of Human Disease[J]. N Engl J Med, 2019, 380(20): 1981-2.
2	Kondo J, Inoue M. Application of cancer organoid model for drug screening and personalized therapy[J]. Cells, 2019, 8(5): 470.
3	Cleary SJ, Pitchford SC, Amison RT, et al. Animal models of mechanisms of SARS-CoV-2 infection and COVID-19 pathology[J]. Br J Pharmacol, 2020, 177(21): 4851-65.
4	Papazian D, Wurtzen PA, Hansen SW. Polarized airway epithelial models for immunological Co-Culture Studies[J]. Int Arch Allergy Immunol, 2016, 170(1): 1-21.
5	Qin X, Sufi J, Vlckova P, et al. Cell-type-specific signaling networks in heterocellular organoids[J]. Nat Methods, 2020, 17(3): 335-42.
6	Charles DD, Fisher JR, Hoskinson SM, et al. Development of a novel ex vivo nasal epithelial cell model supporting colonization with human nasal microbiota[J]. Front Cell Infect Microbiol, 2019, 9 165.
7	Antoni D, Burckel H, Josset E, et al. Three-dimensional cell culture: a breakthrough in vivo [J]. Int J Mol Sci, 2015, 16(3): 5517-27.
8	Li C, Chiu MC, Yu Y, et al. Establishing human lung organoids and proximal differentiation to generate mature airway organoids[J]. J Vis Exp, 2022, (181).
9	Zou L, Ruan F, Huang M, et al. SARS-CoV-2 viral load in upper respiratory specimens of infected patients[J]. N Engl J Med, 2020, 382(12): 1177-9.
10	Wang XW, Xia TL, Tang HC, et al. Establishment of a patient-derived organoid model and living biobank for nasopharyngeal carcinoma[J]. Ann Transl Med, 2022, 10(9): 526.
11	Liu Y, Zhou Y, Chen P. Lung cancer organoids: models for preclinical research and precision medicine[J]. Front Oncol, 2023, 13 1293441.
12	Schutgens F, Clevers H. Human organoids: tools for understanding biology and treating diseases[J]. Annu Rev Pathol, 2020, 15, 211-34.
13	Tsang JO, Zhou J, Zhao X, et al. Development of three-dimensional human intestinal organoids as a physiologically relevant model for characterizing the viral replication kinetics and antiviral susceptibility of enteroviruses[J]. Biomedicines, 2021, 9(1).
14	Bredenoord AL, Clevers H, Knoblich JA. Human tissues in a dish: The research and ethical implications of organoid technology[J]. Science, 2017, 355(6322).
15	Li M, Izpisua Belmonte JC. Organoids - Preclinical Models of Human Disease. Reply. N Engl J Med, 2019, 380(20): 1982.
16	Shapira T, Monreal IA, Dion SP, et al. A TMPRSS2 inhibitor acts as a pan-SARS-CoV-2 prophylactic and therapeutic[J]. Nature, 2022, 605(7909): 340-8.
17	Elbadawy M, Kato Y, Saito N, et al. Establishment of intestinal organoid from rousettus leschenaultii and the susceptibility to bat-associated viruses, SARS-CoV-2 and Pteropine Orthoreovirus[J]. Int J Mol Sci, 2021, 22(19).
18	Lamers MM, Beumer J, van der Vaart J, et al. SARS-CoV-2 productively infects human gut enterocytes[J]. Science, 2020, 369(6499): 50-4.
19	Han Y, Duan X, Yang L, et al. Identification of SARS-CoV-2 inhibitors using lung and colonic organoids[J]. Nature, 2021, 589(7841): 270-5.
20	Chiu MC, Li C, Liu X, et al. A bipotential organoid model of respiratory epithelium recapitulates high infectivity of SARS-CoV-2 Omicron variant[J]. Cell Discov, 2022, 8(1): 57.
21	汪珂, 于言, 韩日. 可用于新冠病毒研究的鼻粘膜类器官模型的建立[J]. 南方医科大学学报, 2022, 42(6): 868-77.
22	Hsu KC, Chen YF, Lin SR, et al. iGEMDOCK: a graphical environment of enhancing GEMDOCK using pharmacological interactions and post-screening analysis[J]. BMC Bioinformatics, 2011, 12(Suppl 1): S33.
23	Zhang L, Li Q, Liu Q, et al. A bioluminescent imaging mouse model for Marburg virus based on a pseudovirus system[J]. Hum Vaccin Immunother, 2017, 13(8): 1811-7.
24	Su S, Wong G, Shi W, et al. Epidemiology, Genetic recombination, and pathogenesis of coronaviruses[J]. Trends Microbiol, 2016, 24(6): 490-502.
25	Lednicky JA, Waltzek TB, McGeehan E, et al. Isolation and genetic characterization of human coronavirus NL63 in primary human renal proximal tubular epithelial cells obtained from a commercial supplier, and confirmation of its replication in two different types of human primary kidney cells. Virol J, 2013, 10 213.
26	Sungnak W, Huang N, Becavin C, et al. SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes. Nat Med, 2020, 26(5): 681-7.
27	Wu CT, Lidsky PV, Xiao Y, et al. SARS-CoV-2 replication in airway epithelia requires motile cilia and microvillar reprogramming. Cell, 2023, 186(1): 112-30. e20.
28	Hoffmann M, Kleine-Weber H, Schroeder S, et al. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell, 2020, 181(2): 271-80. e8.
29	Zhou M, Liu Y, Cao J, et al. Bergamottin, a bioactive component of bergamot, inhibits SARS-CoV-2 infection in golden Syrian hamsters. Antiviral Res, 2022, 204 105365.
30	Antunes JL, Amado J, Veiga F, et al. Nanosystems, drug molecule functionalization and intranasal delivery: an update on the most promising strategies for increasing the therapeutic efficacy of antidepressant and anxiolytic drugs[J]. Pharmaceutics, 2023, 15(3).
31	Ferreira MD, Duarte J, Veiga F, et al. Nanosystems for brain targeting of antipsychotic drugs: an update on the most promising nanocarriers for increased bioavailability and therapeutic efficacy[J]. Pharmaceutics, 2023, 15(2).
32	Alberto M, Paiva-Santos AC, Veiga F, et al. Lipid and polymeric nanoparticles: successful strategies for nose-to-brain drug delivery in the treatment of depression and anxiety disorders[J]. Pharmaceutics, 2022, 14(12).
33	Ulusoy S, Bayar Muluk N, Karpischenko S, et al. Mechanisms and solutions for nasal drug delivery-a narrative review[J]. Eur Rev Med Pharmacol Sci, 2022, 26(2 ): 72-81.

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