神经酰胺信号通路在吴茱萸碱诱导胃癌细胞凋亡中的作用及机理研究

Ceramides, a family of lipid molecules, have been known as a second messenger involved in regulating the signaling pathway of endogenous and exogenous apoptosis in cell. However, the mechanism of ceramide pathway involved in evodiamine-based induction of gastric cancer cells apoptosis has not been completely demonstrated. Our previous experiments have shown that the process of evodiamine induction of gastric cancer cells apoptosis induced the increase of ceramide generation and the up-regulation of acid sphingomyelinase expression, indicating that ceramide signal transduction may be concerned with evodiamine-based induction of tumor cell apoptosis and play an important role in the regulation process, for which the exact mechanism is not clear yet. With the cell lines and nude mouse model of gastric cancer as the subjects, this study started from the effect of evodiamine on ceramide generation and apoptosis signaling pathway. Some technologies such as chemical intervention method and RNA interference technology will be used to observe the relationship between ceramide generation and evodiamine-induced apoptosis, and the effect on the expression of key proteins in the downstream apoptosis signaling pathway, such as JNKs, endoplasmic reticulum,PI3K-Akt-mTOR, mitochondria pathway et al. Besides, LC-MS technology was employed to analyze the regulation and control relationship between the content of ceramide components and its related metabolites and the apoptosis pathway. From molecular，cellular and systemic treatment three levels, to observe the in vitro and in vivo anticancer effects of evodiamine. Therefore, we will not only manifest the effect of ceramide on evodiamine-based induction of gastric cancer cells apoptosis and the regulating mechanism thereof, but also demonstrate molecular and cellular experimental evidence for developing evodiamine into a new kind of anticancer drug.

神经酰胺是细胞内第二信使效应分子，广泛参与了凋亡信号通路的调节，但在吴茱萸碱诱导胃癌细胞凋亡中的作用及信号转导机制尚不完全清楚。我们前期发现，吴茱萸碱在诱导胃癌细胞凋亡过程中，涉及神经酰胺的生成增加和酸性鞘磷脂酶的表达上调，提示神经酰胺信号转导可能参与了吴茱萸碱诱导肿瘤细胞的凋亡过程并存在调控的关系。本研究通过胃癌细胞株模型和构建裸鼠胃癌模型，以吴茱萸碱作用神经酰胺的生成机制与凋亡信号通路的关联为切入点，采用化学物干预方法、RNA干扰等技术，研究生成的神经酰胺与凋亡的关系，以及对下游调节凋亡关键信号通路如JNKs、内质网、PI3K-Akt-mTOR和线粒体信号通路等的影响；用LC-MS技术分析神经酰胺组分及相关代谢物含量的变化与凋亡通路的调控关系。从分子、细胞及整体层面，探讨神经酰胺信号通路在吴茱萸碱诱导胃癌细胞凋亡中的作用及其调控机制，也为吴茱萸碱开发成抗癌新药提供细胞与分子生物学依据。

神经酰胺是细胞内第二信使效应分子，广泛参与了凋亡信号通路的调节，但在吴茱萸碱诱导胃癌细胞凋亡中的作用及信号转导机制尚不完全清楚。本研究通过吴茱萸碱处理胃癌SGC-7901、BGC-823细胞，研究神经酰胺信号通路在吴茱萸碱诱导胃癌SGC-7901、BGC-823细胞凋亡中的作用及机制。本研究利用Real time PCR、Western Blot、MTT/CCK8分析、彗星实验、流式细胞术、HPLC以及透射电镜等技术，获得系列有意义的结果：确定了吴茱萸碱抑制胃癌细胞增殖、诱导胃癌细胞凋亡的作用；阐明了吴茱萸碱通过诱导催化产生神经酰胺的鞘磷脂酶aSMase和nSMase基因表达，促进神经酰胺生成进而诱导胃癌细胞凋亡的机制；揭示了神经酰胺介导吴茱萸碱的诱导胃癌细胞凋亡的初步机制，即通过活化Caspase、PTEN/mTOR、PI3K/AKT、Bcl2/Bax家族及内质网途径等凋亡信号通路，实现诱导胃癌SGC-7901、BGC-823细胞凋亡的作用，其过程涉及Caspase依赖通路和非Caspase依赖通路；提示吴茱萸碱对胃癌SGC-7901、BGC-823细胞周期体现不同的周期阻滞作用；以及吴茱萸碱可能具有胃癌或肿瘤的靶向特性，为吴茱萸碱用于胃癌治疗的分子机制研究及治疗靶点提供理论和实践的依据。