PPI抑制胃癌细胞作用的系统生物学研究

发布时间：2018-07-03 10:10

本文选题：奥美拉唑 + 质子泵抑制剂(PPI)　；参考：《皖南医学院》2017年硕士论文

【摘要】：目的:探索H+-K+-ATP酶抑制剂(proton pump inhibitor,PPI)对胃癌细胞生物学功能方面以及对胃癌细胞代谢的影响,揭示奥美拉唑抑制肿瘤的机制。方法:本实验采用Cell Counting Kit-8(CCK-8)及细胞克隆形成实验来检测胃癌细胞的增殖能力;运用Transwell细胞迁移及侵袭实验和划痕实验来检测PPI对胃癌细胞迁移和侵袭的影响;采用Western blot的方法检测PPI对胃癌细胞SGC-7901及MGC-803凋亡相关蛋白Bax(促凋亡蛋白)、Bcl-2(抗凋亡蛋白)及上皮间质转化(EMT)相关蛋白Vimentin、E-cadherin、β-catenin的影响。通过裸鼠成瘤实验验证PPI对胃癌的影响。采用流式细胞仪检测PPI对胃癌细胞的周期(碘化丙啶PI单染法)及凋亡(Annexin V-FITC/PI双染法)的影响。采用Fluo-3 AM探针染色,通过共聚焦显微镜荧光定量来检测PPI引起肿瘤细胞SGC-7901及MGC-803细胞内钙离子的变化情况。细胞内外的小分子的代谢物质(分子量小于10000)可以用代谢组学的方法进行鉴定和分析,实验中,我们采用基于核磁共振的代谢组学方法(1H-NMR-based metabonomics)对细胞内代谢物进行定量分析,研究PPI作用于胃癌细胞所引起的一系列胞内胞外代谢物微环境的改变,寻找PPI对细胞代谢相关通路的影响。结果:PPI影响胃癌细胞的生物学功能,CCK-8及细胞克隆形成实验说明PPI可以显著抑制细胞的增殖,Transwell细胞迁移及侵袭实验和划痕实验说明PPI可以抑制细胞的迁移和侵袭。通过裸鼠成瘤实验体外验证了PPI对胃癌细胞具有抑制的作用,结果具有统计学意义(p0.05)。Western-blot结果显示PPI对胃癌细胞SGC-7901及MGC-803抗凋亡蛋白Bcl-2进行下调而上调促凋亡蛋白Bax;实验也发现PPI可以影响EMT相关蛋白的表达,即上调E-cadherin、下调Vimentin、β-catenin蛋白的表达。代谢组学分析显示在MGC-803胃癌细胞系中,与对照组相比,PPI组细胞对缬氨酸(Valine)、亮氨酸(Leucine),异亮氨酸(Isoleucine),谷氨酰胺(Glutamine),丙酮酸(Pyruvate)及葡萄糖(Glucose)的利用明显减少。细胞内乙醇(Ethanol),乳酸(Lactate)大量堆积;细胞代谢物肌醇(myo-Inositol),酪氨酸(Tyrosine)及苯丙氨酸(Phenylalanine)等代谢物合成减少。结论:PPI可以抑制胃癌细胞的增殖、迁移和侵袭;PPI通过增加细胞钙离子内流、上调促凋亡蛋白Bax蛋白、下调抗凋亡Bcl-2蛋白及阻滞细胞周期来引起胃癌细胞凋亡;PPI通过抑制Wnt/β-catenin信号通路抑制胃癌细胞的侵袭和迁移。同时,PPI可以影响胃癌细胞的氨基酸、葡萄糖的利用,影响细胞代谢产物代谢抑制胃癌细胞的生长。PPI发挥抗肿瘤作用是多因素共同作用的结果。
[Abstract]:Aim: to investigate the effects of H K ATPase inhibitor (proton pump inhibitor PPI on the biological function and metabolism of gastric cancer cells, and to explore the mechanism of omeprazole inhibiting tumor. Methods: cell Counting Kit-8 (CCK-8) and cell clone formation assay were used to detect the proliferation of gastric cancer cells, and the effects of PPI on the migration and invasion of gastric cancer cells were detected by Transwell cell migration and invasion assay and scratch test. Western blot was used to detect the effect of PPI on apoptosis related protein Bax (pro-apoptotic protein) Bcl-2 (anti-apoptotic protein) and Vimentinin-E-cadherin (尾 -catenin) in gastric cancer cell line SGC-7901 and MGC-803. The effect of PPI on gastric cancer was tested by tumorigenesis in nude mice. Flow cytometry was used to detect the effect of PPI on cell cycle (Pi single staining) and apoptosis (Annexin V-FITC / Pi double staining) in gastric cancer cells. The changes of calcium in SGC-7901 and MGC-803 cells induced by PPI were detected by Fluo-3AM probe staining and confocal microscopy. The metabolites of small molecules (molecular weight less than 10000) in and out of cells can be identified and analyzed by metabonomics. In the experiment, we used 1H-NMR-based metabonomics to quantitatively analyze the metabolites in cells. To study the microenvironment changes of extracellular metabolites induced by PPI in gastric cancer cells, and to find out the effect of PPI on the pathway related to cell metabolism. Results the biological function and cell clone formation of gastric cancer cells were affected by: PPI. The results showed that PPI could significantly inhibit cell proliferation, migration and invasion of Transwell cells, and scratch test, which indicated that PPI could inhibit the migration and invasion of gastric cancer cells. The inhibitory effect of PPI on gastric cancer cells was confirmed by tumorigenesis in nude mice in vitro. Results there were significant (p0.05) .Western-blot results showed that PPI down-regulated apoptosis protein Bcl-2 in SGC-7901 and MGC-803 cells and up-regulated the expression of apoptosis-promoting protein Bax.The results also showed that PPI could affect the expression of EMT related protein, that is, up-regulation of E-cadherin and down-regulation of Vimentin, 尾 -catenin protein. In MGC-803 gastric cancer cell line, the utilization of Valine, Leucine, Isoleucine, Glutamine, Pyruvate and glucose were significantly decreased in MGC-803 gastric cancer cell line compared with the control group, the utilization of valine (Valine), leucine (Leucine), Isoleucine (Isoleucine), glutamine (Glutamine), pyruvate (Pyruvate) and glucose (glucose) were significantly decreased. Intracellular ethanol (Ethanol), lactic acid (Lactate) accumulation and cell metabolites myo-Inositol (Inositol), tyrosine (Tyrosine) and Phenylalanine (Phenylalanine) were reduced. ConclusionPPI can inhibit the proliferation of gastric cancer cells. Migration and invasion of PPI can up-regulate the apoptotic protein Bax by increasing calcium influx. PPI inhibited the invasion and migration of gastric cancer cells by inhibiting Wnt- 尾 -catenin signaling pathway. At the same time, PPI can affect the utilization of amino acids and glucose in gastric cancer cells, and affect the metabolism of cell metabolites to inhibit the growth of gastric cancer cells.
【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

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