【作者】 赵娅娅；

【导师】 潘风光；

【作者基本信息】 吉林大学 ， 食品科学， 2012， 硕士

【摘要】 本研究由农业部重大专项-抗布病与结核病关键功能基因的筛选与验证资助。当Mtb这种胞内寄生菌侵入宿主细胞后，Mtb通过一些逃逸机制逃避宿主的免疫监控作用后可成功寄生于宿主的吞噬细胞内。由于机体内存在着复杂的免疫反应，培养细胞模型所得的Mtb毒力因子表型研究数据比感染动物模型所得相关数据更准确。目前，常用细胞模型有RAW264.7与THP-1两种。本研究主要通过Solexa/Illumina高通量测序技术分析被人结核分枝杆菌（H37Rv）或牛分枝杆菌（BCG）感染3小时后的RAW264.7细胞全基因组转录表达谱变化。实验结果显示：（1）与BCG相比，RAW264.7中1,917差异表达基因中的1,135个基因被H37Rv感染后上调表达，782个基因被H37Rv下调表达；（2）上述1,917个差异表达的基因中，1822个在GenBank有记录，95个是新基因；（3）GO分析与KO分析显示，71%差异基因共参与210条代谢或信号通路；（4）与病原体识别受体和结核分枝杆菌和宿主的相互作用有关通路共28条。Real Time-PCR验证结果与高通量测序结果高度一致。上述发现的差异表达基因将为进一步研究Mtb作用的宿主细胞蛋白分子奠定基础。最新研究发现转录因子FoxO1可诱发心肌细胞内自体吞噬的产生。自体吞噬在增强宿主有效控制和杀灭入侵宿主的Mtb和提高结核疫苗效率方面起重要作用。本研究的前期实验结果发现，当细胞受到H37Rv与BCG感染RAW264.7后，RAW264.7中转录因子FoxO1表达量显著性下降。本研究利用pEGFP-N1表达载体与pCMV-tdTomato表达载体成功构建重组载体pCMV-tdTomato-FoxO1，并将其通过脂质体转染方法转入RAW264.7中。RealTime-PCR实验结果表明，被重组载体pCMV-tdTomato-FoxO1转染12h时，RAW264.7中目的基因FoxO1，FoxO3，LC3，Gabarapl1，Atg12的表达量分别为转染0h时表达量的3.6倍，1.7倍，2.9倍，2.7倍，5.2倍（p<0.05; n=3）。12h后，5个目的基因的表达量均有所降低；24h时，目的基因表达量仍高于其在对照组中的表达量，FoxO12.1倍，FoxO31.5倍，LC31.9倍，Gabarapl11.8倍，Atg123.8倍，（p<0.05; n=3）。Western blotting实验结果表明，RAW264.7被重组载体pCMV-tdTomato-FoxO1转染前12h，LC3II蛋白水平表达量逐渐提高，LC3I蛋白水平表达量逐渐降低；12h时，LC3II蛋白水平表达量达最高值；12h后，LC3II蛋白水平表达量逐渐降低。MDC染色检测自体吞噬现象实验证明，RAW264.7被重组载体pCMV-tdTomato-FoxO1转染24h后，自噬空泡的积聚显著增强。激光共聚焦观察自体吞噬相关蛋白在细胞中的分布实验证明，FoxO1过表达24h后自体吞噬标志物LAMP-1与GABARAPL1会从细胞质转移到细胞内膜上，在吞噬体周围富集；自体吞噬标志物LC3会从细胞质转移到细胞内膜上。Real Time-PCR实验结果表明，被重组载体pCMV-tdTomato-FoxO1转染12h时，RAW264.7中H₂-EAb₁与H2-Eb1的表达量分别为转染0h时表达量的4.4倍，7.5倍（p<0.05;n=3）。12h后，2个目的基因的表达量均有所降低；24h时，目的基因表达量仍高于其0h时的表达量，H2-EAb13.2倍，H₂-Eb₁5.4倍（p<0.05; n=3）。为探索RAW264.7中FoxO1过表达诱导的自体吞噬现象的发生对上调MHC II分子相关基因的表达对RAW264.7抗原呈递能力的影响，我们接着进行了抗原呈递实验。抗原呈递实验中使用的抗原为BCG与CFP10，PBS为阴性对照。实验组的抗原呈递细胞为FoxO1过表达的RAW264.7，对照组的抗原呈递细胞为FoxO1正常表达的RAW264.7。抗原呈递实验的效应细胞为用BCG、CFP10与PBS三免后的BALB/c小鼠的脾淋巴细胞或外周血单核细胞。为分析FoxO1过表达对MTB抗原的呈递效率，我们采用MTT法对淋巴细胞增殖做了测定。MTT法检测体外淋巴细胞增殖水平实验证实，FoxO1过表达可以促进体外淋巴细胞增殖。同时采用ELISA的方法对实验组与对照组抗原呈递后细胞悬液上清中IL-2、IFN-γ与TNF-α这3种细胞因子的浓度进行了测定。ELISA检测证实，FoxO1过表达可以提高RAW264.7细胞抗原处理和抗原呈递功能，提高细胞培养上清中IFN-γ、IL-2和TNF-α的含量。综上所述，转录因子FoxO1过表达可引发RAW264.7中自体吞噬现象的发生，提高RAW264.7表面MHC-II分子相关基因的表达，增强RAW264.7的抗原处理和抗原呈递效率。更多还原

【Abstract】 This research was granted by department of agriculture transgenes special purpose-screenand verification of key point function gene resist to brucelliasis and tuberculosis（2009ZX08009-163B）.Mtb is an intracellular pathogenic bacterium which can utilize various strategies to escapehost immune supervision after being ingested by phagocyte. As complex immunoreaction in vivo,phenotype of Mtb virulence factor is more accurate in vitro cell model. Using Solexa/Illuminahigh-flux sequencing technology to analyze the profile of gene expression from RAW264.7infected by H37Rv and BCG. Results showed that compared with RAW264.7infected by BCG,there were1,135up-regulated genes and782down-regulated genes in1917different expressedgenes in RAW264.7infected by H37Rv. There were1182genes found on GenBank whereas95genes had not been found in those1917different expressed genes. GO analysis and KO analysisresults showed that71%different expressed genes were involved in210Metabolism and signalpathways.28pathways were involved with pathogen recognize receptor and interaction betweenMtb and host. All the results of Solexa/Illumina high-flux sequencing were validated by real timePCR, and real time PCR results were coincidence with that of Solexa/Illumina high-fluxsequencing.Solexa/Illumina high-flux sequencing results showed that transcription factor FoxO1hadbeen affected by H37Rv and BCG significantly. Arunima Sengupta et al. found transcription factorFoxO can induce autophagy in myocardial cell. Importantly, autophagy can enhance the ability ofhost to kill and control Mtb that invaded host and Mtb vaccine. Using pEGFP-N1andpCMV-tdTomato built successfully pCMV-tdTomato-FoxO1which was transfected intoRAW264.7by using TransLipid Transfection Reagent. FoxO1, FoxO3, LC3, Gabarapl1and Atg12mRNA levels is at the summit on12-hour liposome transfection, FoxO13.6fold, FoxO31.7fold,LC32.9fold, Gabarapl12.7fold and Atg125.2fold （p<0.05; n=3）. After the time of12-hourliposome transfection, the mRNA levels of those studied gene began to decrease. It is interestingto note that the mRNA levels of those studied gene had been higher than the beginning mRNAlevel of them, FoxO12.1fold, FoxO31.5fold, LC31.9fold, Gabarapl11.8fold and Atg123.8fold （p<0.05; n=3）. Western blotting results showed, at the first12-hour liposome transfection,LC3II protein isoform was increased as LC3I protein isoform was decreased. LC3II protein isoform was at the peak at the time of12-hour liposome transfection. Following48h, LC3IIprotein isoform was gradually decreased.MDC dyeing results showed FoxO1over expression canenhance gathering vacuolar. Immunofluorescence results showed that FoxO1over expression canenhance LAMP-1, GABARAPL1and LC3transport from cytoplasm to Intracellular Membranes.Real time PCR results showed that, at the first6-hour liposome transfection, the mRNAlevels of studied gene （H₂-EAb₁, H₂-Eb₁） were all a few lower than the beginning mRNA level ofthem. Expression of H2-EAb1, H2-Eb1mRNA levels is at the summit on12-hour liposometransfection, H₂-EAb₁4.4fold, H2-Eb17.5fold （p<0.05; n=3）. After the time of12-hour liposometransfection, the mRNA levels of those studied gene began to decrease. The mRNA levels of thosestudied gene had been higher than the beginning mRNA level of them, H2-EAb13.2fold, H2-Eb15.4fold （p<0.05; n=3）. In order to study the effect of transcription factor FoxO1on autophagy andefficiency of antigen presentation in RAW264.7, antigen presentation analysis were done withFoxO1over expressed RAW264.7as antigen presentation cell, BCG and CFP10as antigen. Afterantigen presentation, MTT results showed that FoxO1over expression can increase perilymphaticcell proliferation in vitro. ELISA results showed that secrete level of IL-2, IFN-γ and TNF-α incell suspension after antigen presentation was increased significantly.Above all, transcription Factor FoxO1has can induce the the autophagy, increase theexpression of MHC-II related gene and enhance the efficiency of antigen presentation inRAW264.7.更多还原