PTEN-PI3K-Akt-mTOR-自噬与凋亡交互介导胃粘膜萎缩机制及安胃汤的拮抗效应

"The Chronic Gastritis → gastric atrophy → intestinal metaplasia → dysplasia → gastric cancer" is the accepted mode of intestinal type gastric cancer. Apoptosis and cell proliferation imbalance is its main mechanism.But it was unknown to the mechanism of "The Chronic Gastritis → gastric atrophy → intestinal metaplasia".So Our hypothesis is: autophagy and apoptosis jointly mediated pathological process of gastric atrophy, and which was really induced intestinal metaplasia and dysplasia ,after autophagy and apoptosis runaway cell proliferation . Beclin1 from the PI3K-Akt-mTOR pathway mediating autophagy, such as Bcl-2 mediating apoptosis which activates downstream of their cross-talk, Our subject will copy chronic atrophic gastritis rat model and in vitro cell culture, use modern biology techniques Western Blot, ELISA, immunohistochemistry method detect the expression of PTEN, PI3K, Akt, mTOR, Beclin1, Bcl-2 and other proteins change, transmission electron microscopy observe the ultrastructural changes of gastric mucosa, ad flow cytometry detect apoptosis,Coimmunoprecipitation detect Beclin1 in Bcl-2 protein, so as to explore the interaction between the them.We will explore the mechanism of autophagy and apoptosis cross-talk which mediates atrophic gastritis gastric mucosal atrophy and Investigate antagonistic effects of AnWeiTang, so we provide an objective basis for traditional Chinese medicine in prevention of chronic atrophic gastritis.

"慢性胃炎→胃黏膜萎缩→肠化→异型增生→胃癌"是肠型胃癌形成的公认模式。细胞凋亡与细胞增殖失衡是其主要机制。但对"慢性胃炎→胃黏膜萎缩→肠化"过程病变机制阐释不明。我们假说：细胞自噬与凋亡共同介导了"胃黏膜萎缩"的病理过程，在细胞自噬与凋亡失控后细胞过度增殖真正诱导了肠化及异型增生。本课题从PI3K-Akt-mTOR通路激活下游Beclin1，Bcl-2等介导细胞自噬与凋亡交互作用(cross-talk)的角度，复制慢性萎缩性胃炎大鼠模型，并体外细胞培养，运用现代生物学技术Western Blot，ELISA，免疫组化法检测PTEN、PI3K，Akt，mTOR，Beclin1，Bcl-2等蛋白表达变化，透射电镜观察胃粘膜超微结构改变，流式细胞仪检测细胞凋亡，探讨细胞自噬与凋亡交联介导萎缩性胃炎的胃粘膜萎缩机制及临床疗效确切的"安胃汤"对其的拮抗效应，为中药防治慢性萎缩性胃炎提供客观依据。

项目背景：胃黏膜萎缩被公认为是胃癌的起始阶段。阻断萎缩性胃炎的发生发展对于预防胃癌有重要意义。细胞凋亡与细胞增殖失衡是胃癌主要机制。我们假说：细胞自噬与凋亡共同介导了“胃黏膜萎缩”的病理过程。主要研究内容：本课题从 PI3K-Akt-mTOR 通路激活下游 Beclin1，Bcl-2 等介导细胞自噬与凋亡交互作用(cross-talk)的角度，复制慢性萎缩性胃炎大鼠模型，并用临床疗效确切的中药服方安胃汤干预萎缩性胃炎大鼠，并体外细胞培养，运用现代生物学技术 Western Blot，ELISA，免疫组化法检测 PTEN、PI3K，Akt，mTOR，Beclin1，Bcl-2 等蛋白表达变化，透射电镜观察胃癌AGS细胞超微结构改变，流式细胞仪检测细胞凋亡。重要结果：(1)胃黏膜组织HE染色表明安胃汤对萎缩性胃炎有逆转作用。胃复春组、安胃汤低剂量组、安胃汤高剂量组治疗CAG的有效率分别50%、58.33%、83.33%，各组比较有统计学意义（P<0.05）；(2)胃黏膜组织Q-PCR和大鼠血清ELISA结果表明，PI3K，AKT，mTOR和Bcl-2蛋白在慢性胃炎组织中表达量明显升高，用药组明显降低；PTEN和Beclin-1的表达正好相反；从胃黏膜免疫组化结果可以看出，PTEN、PI3K，AKT，mTOR，Beclin-1和Bcl-2蛋白表达变化趋势和Q-PCR和ELISA结果基本一致。各组比较均有统计学意义（P<0.05）。(3)透射电镜结果安胃汤可减少胃癌细胞自噬。(4)Western Blot 检测结果表明：PI3K，mTOR，Bcl-2，AKT蛋白在AGS空白细胞中表达较高，3MA组除外，经过药物治疗后，均有一定程度的降低，而PTEN和Beclin-1蛋白表达则相反。其中雷帕霉素组效果最好。差异有统计学意义（P<0.05）。(5)Q-PCR结果和ELISA，Western Blot结果变化趋势基本一致。(6)免疫共沉淀结果发现，安胃汤可以通过促进Beclin-1和BCL-2抑制细胞自噬。科学意义：以上实验结果表明安胃汤可通过拮抗PI3K-Akt-mTOR 通路调节 Beclin1和Bcl-2的相互作用，调节细胞的自噬和凋亡相互作用，从而逆转萎缩性胃炎的发展。