新型融合蛋白rONC-TFn抗肿瘤的靶向作用及其机制研究

Onconase (ONC) as a novel anti-tumor drug has a significant killing effect on a variety of tumor cells. Drug delivery system mediated by transferrin (TF) and TF receptor, which can significantly increase the amount of drug uptake in the tumor cells, enhance the initiative target efficiency of drugs and reduce its toxic side effects. It has been widely used in drug delivery and clinical trials. In our previous studies, the fusion protein (rONC-TFn) was expressed in Escherichia coli by linking ONC with the N-terminal domain protein of TF and prepared in highly purified form. Our results showed that the toxicities of rONC-TFn on stationary phase and logarithmic phase of tumor cells were significant higher than those of rONC. In the present study, we will in-depth investigate the targeted anti-tumor effects of rONC-TFn in vitro and in vivo, and analyze the expression of the signal transduction pathway related proteins in the process of killing tumor cells. Our studies will reveal the anti-tumor effects of rONC-TFn and further clarify its molecular mechanism, which will offer a new theory for tumor treatment and set a foundation for the further development of highly effective and targeted anti-tumor drugs.

豹蛙抗瘤酶（ONC）是一种新型的抗肿瘤药物，对多种肿瘤细胞具有显著的杀伤作用。转铁蛋白（TF）和转铁蛋白受体介导的给药系统可以显著提高肿瘤细胞对药物的摄取量、增强药物主动靶向性和降低其毒副作用，在药物转运和临床上得到越来越多的应用。申请人前期将ONC与TF的N端结构域蛋白进行融合表达，并成功制备出纯度较高的融合蛋白（rONC-TFn），前期结果表明rONC-TFn对静止期和对数期肿瘤细胞的毒性都显著高于rONC。本项目拟在体内和体外水平上深入研究rONC-TFn靶向杀伤肿瘤的效果，细胞水平上分析杀伤肿瘤细胞过程中相关信号通路蛋白的变化情况。本研究将揭示rONC-TFn抗肿瘤的靶向作用，进而明确rONC-TFn杀伤肿瘤细胞的分子机制，为开发高效的、具有靶向性的抗肿瘤药物奠定基础。

为了进一步阐明rONC-TFn抗肿瘤的靶向作用，本研究首先明确了rONC-TFn杀伤不同生长阶段肿瘤细胞的效果和抗肿瘤的靶向性，并建立肝癌移植瘤小鼠模型，深入研究rONC-TFn作用于肿瘤细胞的靶向性及对肿瘤相关信号通路的影响。. 本项目进行了三方面的研究：.1. 比较了rONC-TFn与rONC对多种肿瘤细胞的杀伤作用效果，检测TF对rONC-TFn结合肿瘤细胞的竞争抑制作用，研究rONC-TFn杀伤肿瘤细胞的靶向性。.2. 建立肝癌移植瘤小鼠模型，并分别对模型小鼠进行rONC-TFn和rONC的给药处理，对实验组小鼠的各项指标进行检测，在体内水平上研究rONC-TFn杀伤肿瘤细胞的靶向性。.3. 研究rONC-TFn对肿瘤相关信号通路p38MAPK/JNK的影响。. 结果显示：.1. rONC-TFn对正常细胞没有杀伤作用，而对肿瘤细胞的杀伤作用显著高于野生型rONC，并且肿瘤细胞对rONC-TFn的摄取率显著高于rONC。TF对rONC-TFn结合肿瘤细胞方面存在竞争抑制作用，说明rONC-TFn杀伤肿瘤细胞具有靶向性。.2. 与rONC相比，rONC-TFn能更好的抑制模型小鼠肝癌移植瘤大小的增长。.3. 研究发现rONC-TFn可通过上调p38MAPK和JNK磷酸化水平来杀伤肿瘤细胞。. 此外，在项目执行过程中为了兼顾研究成果的产业化应用，还研究了本项目中纯化制备的rONC蛋白在改善肺纤维化及抗猪瘟病毒（CSFV）两方面的应用效果。结果发现：.1. rONC可以抑制TGF-β1诱导A549细胞EMT（上皮间质转化）及BLM（博莱霉素）诱导小鼠肺纤维化，说明rONC具有治疗肺纤维化的潜能。rONC可能通过下调miR-155和miR-21的表达来改善肺纤维化水平，这一研究为rONC用于临床治疗肺纤维化奠定了理论基础。.2. rONC抗病毒研究方面，数据显示rONC可以显著抑制CSFV mRNA水平的表达，且具有剂量和时间依赖性；说明了rONC能够降解猪瘟病毒，本研究为rONC成为抗病毒的候选药物提供理论依据，也为其在兽药领域的应用奠定基础。