荞麦Potato I 型蛋白酶抑制剂诱导Hep G2细胞线粒体自噬的分子机制

Potato I型蛋白酶抑制剂是植物中广泛存在的一类诱导型抑制剂，在植物抵抗病虫害及应对干旱，水涝，盐胁迫等极端条件中发挥重要的作用。近几年，蛋白酶抑制剂作为抗肿瘤药物也受到国内外研究者的特别关注。我们前期的研究获得一种分子量小，活性稳定的荞麦Potato I 型抑制剂（rBTI）。功能分析揭示rBTI可作用于不同肿瘤细胞，能通过诱导肿瘤细胞自噬抑制肿瘤细胞增殖，但其诱导肿瘤细胞自噬的分子机制仍不清楚。该项目采用Western blot, 流式细胞术，透射电子显微镜等手段，研究rBTI 对Hep G2 细胞线粒体的损伤，诱导细胞内ROS升高，导致细胞自噬的分子机制，检测rBTI诱导Hep G2细胞线粒体受损后ATP生成量的变化。通过本项研究初步阐明rBTI诱导Hep G2细胞自噬的分子机制以及对肿瘤细胞能量代谢的影响，为蛋白酶抑制剂基因工程抗肿瘤药物的深入研究及临床应用研究奠定基础。

Potato I type protease inhibitor is a widespread inducible inhibitors, and play an important role in plant resistance to various external stimuli including wounding, insect feeding and microbial infections.Protease inhibitors as anticancer drugs have been an international research hotpot.Our group has obtained Potato I inhibitor (rBTI),which is stable,high inhibition efficiency of tumor.Experiment data indicates that rBTI could induce Hep G2 cells autophagy and inhibit tumor cell proliferation, but the molecular mechanisms is unclear. In addition, a lot of reactive oxygen species (ROS) generated in Hep G2 cells after treating with rBTI. Based on these findings, we speculate that mitochondrial dysfunction is involved in regulating ROS generation, mitophagy and ATP production. In this project, we are going to multilevelly investigate the relationship between mitochondrial dysfunction, ROS generation and mitophagy by using different means, including western blot, flow cytometry, transmission electron microscopy, and so on. Futher, we will identify the molecular mechanisms of mitophagy induced by BTI. At the same time, we will explore the effect of mitophagy induced by BTI on the cells energy metabolism by some methods of biochemistry, physilogy and molecular biology. This project will provide the theoretical basis for the safe and effective utility for the clinical application of BTI, has important theoretical significances and application value. The research of molecular mechanisms of mitophagy induced by potato I type inhibitor has not yet been investigated at home and abroad.