耻垢分枝杆菌感染人脐带间充质干细胞模型的建立及其特征  

Establishment and characterization of a hUCMSC model of Mycobacterium smegmatis infection

作　　者：康亚莉 宁唤唤 谢燕玲 任瑞 白鹭 路延之 康健 韦垠 张琳娜[2] 柏银兰 KANG Yali;NING Huanhuan;XIE Yanling;REN Rui;BAI Lu;LU Yanzhi;KANG Jian;WEI Yin;ZHANG Linna;BAI Yinlan(Department of Microbiology and Pathogen Biology,Basic Medical Science Academy,Air Force Medical University,Xi’an 710032,Shaanxi,China;Department of Physiology and Neurobiology,School of Basic Medicine,Ningxia Medical University,Yinchuan 750001,Ningxia,China;School of Life Sciences,Yan’an University,Yan’an 716000,Shaanxi,China)

机构地区：[1]空军军医大学基础医学院微生物与病原生物学教研室,陕西西安710032 [2]宁夏医科大学基础医学院生理学和神经生物学系,宁夏银川750001 [3]延安大学生命科学学院,陕西延安716000

出　　处：《微生物学通报》2023年第11期5031-5044,共14页Microbiology China

基　　金：国家自然科学基金(82272343,81971560);国家“十三五”重大传染病专项课题(2018ZX10302302002004);陕西省重点研发计划(2022ZDLSF01-07)。

摘　　要：【背景】结核分枝杆菌(Mycobacterium tuberculosis,Mtb)休眠菌形成被认为是潜伏结核感染(latent tuberculosis infection,LTBI)的主要原因,但目前缺乏体内和体外模型进行机制研究。新近研究表明Mtb可感染间充质干细胞(mesenchymal stem cells,MSC)并以休眠状态在细胞中长期存活。然而Mtb感染细胞模型存在周期长和生物安全要求高等问题,需要探索可用的MSC感染细胞模型用于Mtb休眠机制的研究。【目的】建立快速生长型耻垢分枝杆菌(Mycobacterium smegmatis,Ms)感染人脐带间充质干细胞(human umbilical cord MSC,hUCMSC)的细胞模型并研究其特征。【方法】取分离好的hUCMSC,流式细胞术鉴定其表面标志性抗原;以Ms菌株感染hUCMSC,DiI标记细胞膜,荧光显微镜下观察细胞吞噬作用;平板法计数Ms胞内存活率;油红O染色观察细胞脂滴形成;鬼笔环肽荧光染色观察细胞骨架变化;实时荧光定量PCR(real time quantitative PCR,RT-qPCR)检测Ms感染的hUCMSC脂质代谢、静息和分化相关基因转录水平。【结果】Ms感染hUCMSC后在胞内以非复制方式存活,其感染率与感染复数呈正相关;Ms感染hUCMSC可诱导脂滴合成相关基因表达,而抑制降解相关基因转录水平,从而促进细胞内脂滴形成。Ms感染促进hUCMSC细胞骨架发生重构,RT-qPCR检测结果显示细胞增殖和分化相关基因表达增加,Ms感染促进hUCMSC向成脂、成骨细胞分化。【结论】本研究建立了可模拟Mtb休眠形成的Ms感染hUCMSC模型,可进一步用于LTBI机制的相关研究。[Background]The formation of dormant Mycobacterium tuberculosis(Mtb)is considered to be the main cause of latent tuberculosis infection(LTBI),the mechanism of which remains to be studied with in vivo and in vitro models.Mtb can infect mesenchymal stem cells(MSC)and survive in the cells in a dormant state for a long time.However,the modeling of Mtb-infected cells requires long time and high laboratory biosafety level.Therefore,it is essential to explore the available MSC for modeling Mtb infection and further deciphering the dormancy mechanism of Mtb.[Objective]To establish and characterize the human umbilical cord MSC(hUCMSC)model of Mycobacterium smegmatis(Ms)infection.[Methods]After hUCMSC were infected with Ms,flow cytometry was employed to identify the surface antigen.The cell membrane was labeled with DiI and then the phagocytosis of the hUCMSC infected with Ms was observed in a fluorescence microscope.The plate counting method was employed to measure the intracellular survival rate of Ms.The formation of lipid droplets in hUCMSC was observed by oil red O staining.Cytoskeletal changes were observed by fluorescence staining with phalloidin.The transcription levels of the genes involved in the lipid metabolism,resting,and differentiation of the hUCMSC infected with Ms were determined by RT-qPCR.[Results]Ms survived in hUCMSC in a non-replicating state,and the infection rate was positively correlated with the multiplicity of infection.Ms simulated the expression of the genes involved in lipid biosynthesis and inhibited the transcription of the genes involved in lipid degradation to promote the formation of lipid droplets in the Ms-infected hUCMSC.Ms promoted the cytoskeleton remodeling of hUCMSC.The RT-qPCR results showed up-regulated expression of the genes related to proliferation and differentiation,indicating that Ms promoted the differentiation of hUCMSC into lipoblasts and osteoblasts.[Conclusion]We successfully established a hUCMSC model of Ms infection,which could mimic the main characteristics of Mtb d

关 键 词：结核分枝杆菌 耻垢分枝杆菌 间充质干细胞 休眠菌 结核潜伏感染 

分 类 号：R378.91[医药卫生—病原生物学]