Lgr5-Wnt/β-catenin信号通路对胃癌干样细胞恶性生物学行为的影响及其分子调控机制研究

Recent studies show that Lgr5 (a new stem cell marker) and Wnt/β-catenin make up an important signal pathway and is involved in activity regulation of tumor stem cells. However, the relationship between this pathway and gastric cancer stem cells has not yet been investigated. Our preliminary experiments showed that the expression of key protein in this signal pathway was significantly higher in gastric cancer stem-like cells than that in ordinary gastric cancer cells. Lgr5 expression was positively associated with malignant degree and microvessel density. Therefore, we hypothesize: Lgr5-Wnt/β-catenin signal pathway is involved the regulations of gastric cancer stem activity, and maintains the malignant biological behavior of gastric cancer stem-like cell by regulating the expression of EphA3/ephrinA5. In this study, we will use the RNAi technology to block this pathway to observe the changes of the stem-like cell characteristics, as well as EphA3/ephrinA5 gene, and the use of the method of MTT, annexinV detection, flow cytometry, MircroPET / CT and other analysis of gastric cancer stem-like cell proliferation, apoptosis, and the changes of angiogenesis and oxygen metabolism and other biological change behavior. The subject carry out not only provide a new target for the future research of gastric cancer stem cell, but also lay the foundation for further biological treatment of gastric cancer.

近年来研究表明lgr5作为新的干细胞标记物，它与Wnt/β-catenin共同组成了重要的信号通路，调控了肿瘤干细胞生物学活性。然而，有关该通路与胃癌干细胞方面的研究，却鲜有报道。前期研究表明该通路关键蛋白在胃癌干样细胞中的表达高于普通胃癌细胞，Lgr5与胃癌恶性程度及微血管密度呈正相关。因此，我们提出假设：Lgr5-Wnt/β-catenin通路是否参与胃癌干样细胞的活性调控，并通过调节EphA3/ephrinA5的表达维持了胃癌干样细胞的恶性生物学行为。本实验拟通过RNAi技术阻断该通路，观察干扰后干细胞生物学特性以及EphA3/ephrinA5等靶基因表达变化，并利用MTT、annexinV标记、流式细胞仪、MircroPET/CT等方法综合分析胃癌干样细胞的增殖、凋亡以及血管生成、氧代谢等生物学行为改变情况。借此寻找一个全新的胃癌干细胞研究靶点，为今后开展胃癌生物治疗奠定基础。

近年来的研究表明，Lgr5与Wnt/β-catenin共同组成了重要的干细胞相关信号通路，然而有关该信号通路与胃癌干细胞方面的研究却罕有报道。本研究中，我们首先对胃癌干样细胞提纯研究，随机选取四种人胃癌细胞系（AGS、BGC-823、MKN-28、MKN-45）进行悬浮球培养及CD44+CD54+流式细胞分选，通过悬浮球形成、平板克隆、增殖检测、抗化疗药物及裸鼠成瘤等实验对比实验组细胞和对照组细胞的差异。随后通过慢病毒干扰稳定转染的方式感染胃癌干样细胞，研究Lgr5的改变对胃癌干样细胞的干细胞特性的影响。结果显示CD44+CD54+悬浮球细胞具有更强的干细胞特性，是富集度更高的胃癌干样细胞。功能实验显示，Lgr5干扰后，胃癌干样细胞的悬浮球形成能力、平板克隆能力、增殖能力、抗化疗耐药能力及裸鼠成瘤能力均较感染前明显下降。同时检测β-catenin,Ki67, EphA3和Ephrin A5等mRNA和蛋白表达，发现其同Lgr5的变化具有一致性，这些均说明Lgr5-Wnt/β-catenin信号通路在维持胃癌干样细胞的生物学特性方面起到了重要作用。