低氧微环境下转录因子HIF-1α活化导致甜菜夜蛾原代细胞转化机制研究

Cell line is an important experimental material for verifying scientific hypothesis in vitro. Compared with establishing the mammalian cell lines, insect cell lines is weak in establishing basic technology foundation, time-consuming, harder to get the second passage after the first passage, and has lower success rate in establishing cell lines. So, what causes the huge difference between the two? Previously, We have observed that it is more prone, in the primary culture progress of Beet Armyworm, to get a large number of cell clusters increasing orders of magnitude when the cell culture medium is not replaced frequently and this phenomenon will not appear when adding conditioned medium. The preliminary study shows that the differentially expressed genes, in the progress of cell transformation, except the retardant in the cell cycle of G2/M, participate in the HIF-1 regulating signal pathways of ubiquitin-proteasome and the signal pathways are consisted with the one that is changing in the mammal tumor genesis induced by hypoxic microenvironment. So we speculated that the activation of transcription factors HIF-1 is the key reason leading to the cell transformation of the beet Armyworm primary cells. The results of this study will modify, in the traditional process of establishing insect cell lines, the neglect of cell using of O2 in carbonate buffer system culture medium, and will provide theoretical basis and new ideas for building insect cell lines to promote the development of insect science.

细胞系是体外水平验证科学假设的重要实验材料。与哺乳动物建立细胞系相比，昆虫细胞系的建立技术基础更薄弱，耗时更长，成功率更低。那么，造成两者巨大差异的原因是什么？我们前期通过观察发现：甜菜夜蛾原代培养过程中不经常替换培养液更容易出现大量多簇细胞呈数量级增殖，且加入条件培养液并不会出现类似的现象。前期研究表明，细胞发生转化的过程中，除了细胞周期发生G2/M阻滞外，差异表达的基因参与了调节低氧诱导因子HIF-1活性的泛素-蛋白酶体信号等通路，并与哺乳动物肿瘤发生时产生变化的信号通路具有一致性。因此我们推测，低氧微环境下，转录因子HIF-1α的活化是导致甜菜夜蛾原代细胞转化的关键原因。我们将从低氧微环境模型构建、SeHIF-1α的上下游调控及功能论证其与原代细胞转化的因果关系。本研究成果将修正传统昆虫细胞系的建立过程中，细胞在CO2缓冲体系的培养液中对氧气利用方面的忽视，为昆虫建系策略提供理论基础

细胞系是体外水平验证科学假设的重要实验材料，快速有效地建立来源于不同物种和不同组织的昆虫细胞系，对于昆虫学科的发展具有重要意义。在昆虫细胞系建立过程中，原代细胞转化是其最关键步骤，其技术方法尚不成熟，其机制有待明确。本项目通过分析体内体外微环境差异，建立了甜菜夜蛾原代细胞永生化的低氧诱导模型；并发现此方法具有一定的普适性，建立了27株来自4个鳞翅目昆虫种类的细胞系。本研究同时通过ROS，线粒体膜电位，伪足，Atg8及凋亡相关基因表达分析，明确了细胞由体内到体外后发生了自噬，而不是凋亡。然而经过低氧诱导后，细胞总抗氧化能力增强，细胞周期S期细胞所占比例增加，转录组数据分析发现在体外培养中逐步淘汰因外界环境变化而改变的细胞，留下仍然保持组织体内细胞状态的细胞，进而取得优势最后建立成系。最后，研究了Sehif-1α基因的部分功能，发现HIF通路在低氧诱导甜菜夜蛾原代细胞转化中必不可少。本研究成果将为昆虫细胞系的建立策略提供理论基础和新的思路，从而推动昆虫科学的发展。