摘要
转录因子EB(TFEB)作为自噬的主要转录调节因子,通过调控细胞自噬体和溶酶体相关基因的表达影响自噬的发生。为研究TFEB在结核分枝杆菌(MTB)介导细胞自噬中的作用,本实验采用CRISPR/Cas9原理构建重组质粒pSpCas9(BB)-2A-GFP-tfeb,并测序鉴定正确后电转化小鼠单核巨噬细胞(RAW264.7细胞,简写为WT细胞),经流式细胞仪筛选GFP阳性单克隆细胞,经PCR及western blot鉴定,将鉴定正确的细胞连续传10代,每隔3代均经western blot鉴定。结果显示,经流式细胞术共筛选到25株出现绿色荧光的单克隆细胞,对这些细胞纯化后经PCR鉴定并测序结果显示,仅一株单克隆细胞敲除tfeb中PAM序列(AGG)前的第3位碱基;western blot鉴定结果显示,相比正常细胞,该细胞无57 ku的特异性条带,且每隔3代该细胞均无该57 ku的特异性条带,表明tfeb基因缺失细胞系Δtfeb RAW264.7正确构建,且其遗传稳定性较强。将MTB H37Ra株分别感染WT细胞和Δtfeb RAW264.7细胞,12 h后经western blot检测各组细胞中自噬相关蛋白LC3-II和溶酶体相关膜糖蛋白1(LAMP1)的表达水平,通过RT-qPCR检测自噬相关基因maplc3、ctsb、lamp1、tpp1的转录水平,采用激光共聚焦试验检测TFEB对H37Ra诱导细胞自噬流形成的影响。Western blot和RT-qPCR结果显示,与未感染的两种细胞相比,H37Ra菌株感染的WT细胞中LC3-II和LAMP1蛋白的表达量均极显著升高(P<0.01、P<0.001);自噬相关基因maplc3和溶酶体相关基因lamp1、ctsb和tpp1的转录水平均显著和极显著升高(P<0.05、P<0.01)。H37Ra菌株感染的Δtfeb RAW264.7细胞中上述两种蛋白的表达量均无显著差异,其上述两种蛋白的表达水平均低于WT细胞且该细胞中上述自噬体和溶酶体相关基因的转录水平均无显著差异。激光共聚焦试验结果显示,与WT细胞相比,H37Ra株感染的WT细胞中黄色荧光(成熟自噬体)和红色荧光(溶酶体)的数量均极显著增加(P<0.01),且自噬流通畅,表明WT细胞发生了自噬;H37Ra株感染的Δtfeb RAW264.7细胞与未感染的该细胞中二者的荧光数量均无显著差异,且二者荧光的数量均比WT细胞整体降低,自噬流受阻。上述结果表明,本研究构建了tfeb基因稳定缺失的细胞系Δtfeb RAW264.7,且首次证实TFEB是MTB介导巨噬细胞自噬的关键宿主因子,在巨噬细胞自噬过程中发挥关键作用,本研究为深入探究TFEB在MTB介导巨噬细胞自噬中的作用提供了细胞模型和参考依据。
As the main transcriptional regulator of autophagy,transcription factor EB(TFEB)affects the occurrence of autophagy by regulating the expression of autophagosomes and lysosome-related genes.In order to study the role of TFEB in Mycobacterium tuberculosis(MTB)-mediated autophagy,the recombinant plasmid pSpCas9(BB)-2A-GFP-tfeb was constructed using CRISPR/Cas9 system,and the sequencing identified positive recombinant plasmids were electrotransformed into mouse mononuclear macrophages(RAW264.7 cells,abreviated as WT cell),and the GFP-positive monoclonal cells were screened by flow cytometry,and confirmed by PCR amplification and western blot.The correctly identified cells were passed for 10 consecutive passages,which were identified by western blot every 3 passages.The results showed that a total of 25 monoclonal cells with green fluorescence were screened by flow cytometry,and the PCR identification and sequencing results showed that only one knocked out monoclonal cell exhibited deletion at the third base before the PAM sequence(AGG)in tfeb.The results of western blot showed that compared with normal cells,the cells did not have a specific band of 57ku,and no specific band of 57ku was observed for every 3 passages,indicating that the tfeb gene deletion cell lineΔtfebRAW264.7 was correctly constructed and the cells were genetically stable.The expression levels of autophagy-related protein LC3-II and lysosome-associated membrane glycoprotein 1(LAMP1)in each group were detected by western blot 12 hours later,and the transcription levels of autophagy-related genes maplc3,ctsb,lamp1 and tpp1 were detected by RT-qPCR.Laser confocal assay was used to detect the effect of TFEB on the formation of autophagic flux induced by H37Ra.Western blot and RT-qPCR results showed that the expression levels of LC3-II and LAMP1 proteins in WT cells infected with the H37Ra strain were significantly increased compared with those of the two uninfected cells(P<0.01,P<0.001).The transcription levels of autophagy-related gene maplc3 and lysosome-related genes lamp1,ctsb and tpp1 were significantly and extremely significantly increased(P<0.05,P<0.01).There was no significant difference in the expression levels of the above two proteins inΔtfebRAW264.7 cells infected with the H37Ra strain,and the expression levels of the above two proteins were lower than those of WT cells.There was no significant difference in transcription levels of these autophagosomes and lysosome-related genes in this cell.The results of the laser confocal assay showed that compared with WT cells,the number of yellow fluorescence(mature autophagosomes)and red fluorescence(lysosomes)in WT cells infected by the H37Ra strain was significantly increased(P<0.01),and the autophagy flowed smoothly,ensuring the occurrence of autophagy.There was no significant difference in the number of fluorescence betweenΔtfebRAW264.7 cells infected with the H37Ra strain and those of uninfected cells,and the number of fluorescence in both cells was lower than that of WT cells,indicating autophagy flow obstruction.This study provides a cell model and reference for further exploring the role of TFEB in MTB-mediated macrophage autophagy.
作者
张镇钧
冯婷婷
聂蝉蝉
段祎凡
陈春文
郭若男
高云洁
李梦雨
崔莹莹
党光辉
刘思国
ZHANG Zhen-jun;FENG Ting-ting;NIE Chan-chan;DUAN Yi-fan;CHEN Chun-wen;GUO Ruo-nan;GAO Yun-jie;LI Meng-yu;CUI Ying-ying;DANG Guang-hui;LIU Si-guo(College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China;State Key Laboratory for Animal Disease Control and Prevention,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)
出处
《中国预防兽医学报》
北大核心
2025年第6期614-622,共9页
Chinese Journal of Preventive Veterinary Medicine
基金
“十四五”国家重点研发计划(2021YFD1800403)
国家自然科学基金(32273005)
中央级公益性科研院所基本科研业务费专项(1610302024002)。
