环形泰勒虫介导的宿主细胞代谢改变及其重编程分子机制研究

Bovine B lymphocytes and macrophages can be transformed when infected by Theileria annulata, and the parasitized cells acquire continuous proliferation ability as tumor cells cultured in vitro. T. annulata and T. parva are the only known eukaryotes that can transform mammalian cells and this kind of transformation induced by T. annulata is entirely reversible. This illustrates that Theileria annulata mediated metabolic reprogramming in host cells, but the mechanisms underlying this transformation are not well understood. The objectives of the present project are to study the metabolomics of PBMCs of uninfected bovine, T. annulata transformed cells before and after with BW720c treatment by an integrated approach using Gas chromatography tandem mass spectrometry(GC-MS) and multivariate data processing technique. Differential metabolites will be mapped in the KEGG database to search for the differential metabolic pathway and key molecules involved in metabolic pathways. Moreover, the molecular mechanisms of metabolic reprogramming in the transformed cells will be investigated by a series of technique methods, such as gene overexpression, RNA interference, Real-time PCR and Western blotting. Our studies will establish a foundation for further better understanding transformation mechanisms of this parasite, and for better understanding the molecular mechanisms of interactions between T. annulata and host cells.

环形泰勒虫可使感染的牛B淋巴细胞和巨噬细胞发生转化，体外培养时获得类似肿瘤样细胞的无限增殖能力；它与同属的小泰勒虫是截至目前已发现的唯一一类可使哺乳动物细胞发生永生性转化的真核生物，而且这类细胞的转化是可逆的；说明环形泰勒虫可介导宿主细胞发生代谢重编程，但其分子机制尚不明确。本项目以非感染牛外周血单个核细胞和抗泰勒虫药物布帕伐醌（BW720c）作用前后的环形泰勒虫转化细胞为研究对象，通过气相色谱-质谱(GC-MS) 技术及多变量数据处理代谢组学方法，分析差异代谢物并在KEGG数据库中映射，寻找差异代谢通路以及影响该代谢通路的关键分子；进而利用过表达、RNA干扰、Real-time PCR和Western blotting等技术研究宿主细胞代谢重编程的分子机制，为系统研究环形泰勒虫与宿主细胞相互作用，揭示其转化机制奠定基础。

本研究采用细胞培养、代谢组学、转录组学、酵母双杂、Real-time PCR和Western Blotting 等技术研究了环形泰勒虫转化宿主细胞的代谢谱及转录谱特点，阐释了环形泰勒虫介导宿主细胞代谢重编程的分子机制，并获得了以下结果：.1．代谢谱研究：在BW720c 作用的环形泰勒虫转化细胞中，负离子模式下检测到1425个代谢物，在正离子模式下检测到1298个代谢物。药物处理24h、48h、72h后，负离子模式下有56、162、243个差异代谢物，正离子模式下有35、121、177个差异代谢物， 这些差异代谢物主要集中在各种必需氨基酸上。.2．能量代谢谱研究：对转化细胞的药物试验发现，相对于对照组，筛选到21种组间差异的代谢物，药物处理组中有８个代谢物上调，13个代谢物下调。经富集分析发现这些代谢物主要是与能量代谢相关的代谢物，如谷氨酰胺、Ｌ－乳酸、苹果酸、丙酮酸、异柠檬酸等。.3．转录组学研究：筛选出差异表达基因一共3935个，其中共同表达的基因为416个，有196个基因显著上调，171个基因显著下调；确定环形泰勒虫介导宿主细胞转化的过程中的关键信号通路主要包括Central carbon metabolism in cancer , Pathways in cancer, HIF-1 signaling pathway。 .4．宿主互作虫体蛋白筛选验证：TFG基因在环形泰勒虫转化细胞中高表达。通过酵母双杂交筛选到与其互作虫体蛋白9个，排出7个假阳性结果，最终筛选出两个环形泰勒虫蛋白TA21130和TA11965。并对虫体基因序列进行了初步分析。.通过本项目实施，确定了BW720c 作用前后环形泰勒虫转化细胞的差异代谢物和差异代谢途径，明确了环形泰勒虫对宿主细胞代谢的影响，初步揭示了环形泰勒虫引起宿主细胞发生代谢重编程的分子机制。