气道类器官显微注射及极性反转模型的构建

doi:10.11958/20231469

天津医药 ›› 2024, Vol. 52 ›› Issue (1): 4-10.doi: 10.11958/20231469

• 专题研究·类器官与器官芯片（主编·陈怀永） • 上一篇下一篇

气道类器官显微注射及极性反转模型的构建

宋立成¹(), 张宇涵², 余忠阔¹, 解立新¹^,^△()

1.中国人民解放军总医院第八医学中心呼吸与危重症医学部（邮编100091）
2.上海交通大学医学院附属瑞金医院呼吸与危重症医学科

收稿日期:2023-11-26 出版日期:2024-01-15 发布日期:2024-01-18
通讯作者: ^△E-mail：xielx301@126.com
作者简介:宋立成（1989），男，主治医师，主要从事呼吸危重症及肺损伤修复方面研究。E-mail：songlicheng@tom.com
基金资助:
国家自然科学基金面上资助项目(82172109);国家自然科学基金面上资助项目(82070001)

Construction of airway organoid microinjection and polarity reversal model

SONG Licheng¹(), ZHANG Yuhan², YU Zhongkuo¹, XIE Lixin¹^,^△()

1. College of Pulmonary and Critical Care Medicine, 8th Medical Center of Chinese PLA General Hospital, Beijing 100091, China
2. Department of Pulmonary and Critical Care Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine

Received:2023-11-26 Published:2024-01-15 Online:2024-01-18
Contact: ^△E-mail：xielx301@126.com

宋立成, 张宇涵, 余忠阔, 解立新. 气道类器官显微注射及极性反转模型的构建[J]. 天津医药, 2024, 52(1): 4-10.

SONG Licheng, ZHANG Yuhan, YU Zhongkuo, XIE Lixin. Construction of airway organoid microinjection and polarity reversal model[J]. Tianjin Medical Journal, 2024, 52(1): 4-10.

摘要/Abstract

摘要：

目的探索通过显微注射及极性反转的方式建立高效模拟呼吸道病毒感染的新方法。方法获取8周龄雄性C57BL/6小鼠肺组织，提取呼吸道上皮细胞，建立类器官transwell培养模型。通过改进传统显微注射平台，将绿色荧光蛋白（GFP）标记的流感病毒PR8（GFP-PR8）定量注射入类器官内，观察类器官形态变化及紧密连接蛋白、微管蛋白的免疫荧光染色特点。通过悬浮培养的方法诱导极性内向反转为极性外向（AO），通过HE染色鉴定极性反转的形态学特点。对普通类器官及反转后类器官进行PR8攻毒，观察感染效率及不同浓度的病毒感染下主要通路基因的表达差异。结果普通类器官经显微注射后体积会明显增大。注射PR8后，类器官顶端区域被感染的效率明显增高且会出现明显的损伤，表现为紧密连接蛋白及微管蛋白的蛋白表达显著下调。将类器官悬浮培养后，纤毛细胞极性随时间逐渐反转向外，于第6天起反转比例趋于稳定。反转后的类器官被病毒感染的效率显著提高，细胞损伤显著。0.01感染复数（MOI）的PR8攻毒后，AO类器官出现明显的炎症通路及分化相关基因的改变；在更高浓度PR8感染后则出现与之前相反的变化趋势。结论极性反转和显微注射可以显著提高流感病毒对类器官的感染效率，这有助于类器官在呼吸道感染领域的广泛应用。

关键词: 类器官, 微量注射, 呼吸病毒感染, 极性外向, 气道类器官

Abstract:

Objective To explore novel methods for efficient respiratory viral infection of organoids by microinjection and polarity inversion techniques. Methods Lung tissue samples were obtained from 8-week-old male C57BL/6 mouse, and respiratory epithelial cells were extracted to establish a transwell organoid culture model. The green fluorescent protein (GFP) labeled influenza virus PR8 (GFP-PR8) was quantitatively injected into organoids by improving the traditional microinjection platform, and morphologic changes in organoids and the immunofluorescence staining characteristics of tight junction proteins and microtubule proteins were observed. Polarity inversion apical-out (AO) was induced by suspension culture, and the morphological characteristics of polarity inversion was determined by HE staining. Normal and inverted organoids were infected with PR8, and the infection efficiency and expression differences of key pathway genes under different virus concentrations were observed. Results Ordinary organoids showed a significant increase in volume after microinjection. Following PR8 injection, the efficiency of infection was significantly higher in the apical region of organoids, accompanied by noticeable damage, as evidenced by significant down-regulation of tight junction proteins and microtubule protein expression. After suspension culture of the organoids, the polarity of ciliated cells gradually inverted outward over time, and the proportion of AO organoids stabilized on the 6th day. The efficiency of viral infection significantly increased in the inverted organoids, accompanied by significant cellular damage. After PR8 infection at 0.01 MOI, AO organoids showed significant changes in the inflammatory pathway and differentiation-related genes, with the opposite trend observed after higher concentration of PR8 infection. Conclusion Both polarity inversion and microinjection techniques significantly enhance the efficiency of influenza virus infection in organoids, thereby facilitating organoid widespread application in the field of respiratory tract infections.

Key words: organoids, microinjections, respirovirus infections, apical-out, airway organoid

中图分类号:

R318.1

图/表 11

表1 qPCR引物序列

Tab.1 Primer sequences for qPCR

基因名称	引物序列（5′→3′）	产物大小/bp
GAPDH	上游：TGTGTCCGTCGTGGATCTGA	150
	下游：TTGCTGTTGAAGTCGCAGGAG	150
CCL2	上游：TCAGGCAGATGCAGTTAACG	117
	下游：TCTTTGGGACACCTGCTGC	117
IL-1β	上游：ACCTAGCTGTCAACGTGTGG	133
	下游：TCAAAGCAATGTGCTGGTGC	133
SCGB1A1	上游：ATGAAGATCGCCATCACAATCAC	135
	下游：GGATGCCACATAACCAGACTCT	135
SCGB3A2	上游：ACAGGGAGACGGTTGATGAG	140
	下游：GTTGGGCTTTCTGACTGCAT	140
MUC5B	上游：TCCCTAGCATGAGCGCCTTA	178
	下游：CCACGACGCAGTTGGATGTT	178
FOXJ1	上游：CCCTGACGACGTGGACTATG	114
	下游：GCCGACAGAGTGATCTTGGT	114

图1 类器官显微注射FITC-dextran后的形态学特点 A：直径<100 μm类器官显微注射后的形态变化；B：直径>100 μm类器官显微注射后的形态变化。

Fig.1 Morphological characteristics after microinjection of FITC-dextran in organoid

图2 荧光显微镜下观察显微注射后的类器官显像特点 A—C：不同体积类器官显微注射后均未见球体胀裂、荧光素渗出等现象。

Fig.2 Observation of organoid imaging characteristics under a fluorescence microscope after microinjection

图3 显微注射GFP-PR8后类器官特定蛋白表达特点（免疫荧光染色） A：普通感染GFP-PR8的类器官的病毒荧光及α-tubulin表达情况（×400）；B：显微注射GFP-PR8的类器官的病毒荧光及α-tubulin表达情况（×400）；C：显微注射PBS后类器官ZO-1及α-tubulin的表达特点（×200）；D：显微注射PR8后类器官ZO-1及α-tubulin的表达特点（×400）。

Fig.3 Expression characteristics of specific proteins in organoids after microinjection of GFP-PR8 (immunofluorescence staining)

图4 显微注射GFP-PR8后类器官特定蛋白表达特点（HE染色，×400） A：普通感染GFP-PR8的类器官结构形态特点；B：显微注射GFP-PR8的类器官结构形态特点。

Fig.4 Expression characteristics of specific proteins in organoids after microinjection of GFP-PR8 (HE staining, ×400)

图5 AI及AO类器官形态学特点比较（HE染色） A：AI类器官（×200）；B：AO类器官（×400）。

Fig.5 Morphological comparition of AI and AO organoids (HE staining)

图6 Transwell上层小室中极性反转的类器官占总体类器官的比例

Fig.6 The proportion of organoids with polarity reversal in the upper chamber of transwell to the total population of organoids

图7 类器官在不同培养条件下的形态特点（×100） A：AI类器官在基质胶内的形态特点；B：AO类器官在培养基中的形态特点。

Fig.7 Morphological characteristics of organoids under different culture conditions (×100)

图8 类器官感染GFP-PR8后的特点（×100） A：AI类器官；B：AO类器官。

Fig.8 Characteristics of organoidafter infection with GFP-PR8 (×100)

图9 PR8感染AO类器官后的形态学特点（HE染色） A：类器官内部坏死物质堆积；B：类器官结构松散，细胞排列紊乱、结构破坏。

Fig.9 Morphological characteristics of AO organoids infected by PR8 (HE staining)

图10 不同浓度病毒感染AI及AO类器官后的基因表达特点 a：极性向内；b：极性向外；n=6，＊P<0.05。

Fig.10 Gene expression characteristics in AI and AO organoids after stimulation with different virus concentrations

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气道类器官显微注射及极性反转模型的构建

Construction of airway organoid microinjection and polarity reversal model

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