本文選題：奧美拉唑 + 質(zhì)子泵抑制劑(PPI)�。� 參考：《皖南醫(yī)學(xué)院》2017年碩士論文

【摘要】：目的:探索H+-K+-ATP酶抑制劑(proton pump inhibitor,PPI)對(duì)胃癌細(xì)胞生物學(xué)功能方面以及對(duì)胃癌細(xì)胞代謝的影響,揭示奧美拉唑抑制腫瘤的機(jī)制。方法:本實(shí)驗(yàn)采用Cell Counting Kit-8(CCK-8)及細(xì)胞克隆形成實(shí)驗(yàn)來(lái)檢測(cè)胃癌細(xì)胞的增殖能力;運(yùn)用Transwell細(xì)胞遷移及侵襲實(shí)驗(yàn)和劃痕實(shí)驗(yàn)來(lái)檢測(cè)PPI對(duì)胃癌細(xì)胞遷移和侵襲的影響;采用Western blot的方法檢測(cè)PPI對(duì)胃癌細(xì)胞SGC-7901及MGC-803凋亡相關(guān)蛋白Bax(促凋亡蛋白)、Bcl-2(抗凋亡蛋白)及上皮間質(zhì)轉(zhuǎn)化(EMT)相關(guān)蛋白Vimentin、E-cadherin、β-catenin的影響。通過(guò)裸鼠成瘤實(shí)驗(yàn)驗(yàn)證PPI對(duì)胃癌的影響。采用流式細(xì)胞儀檢測(cè)PPI對(duì)胃癌細(xì)胞的周期(碘化丙啶PI單染法)及凋亡(Annexin V-FITC/PI雙染法)的影響。采用Fluo-3 AM探針染色,通過(guò)共聚焦顯微鏡熒光定量來(lái)檢測(cè)PPI引起腫瘤細(xì)胞SGC-7901及MGC-803細(xì)胞內(nèi)鈣離子的變化情況。細(xì)胞內(nèi)外的小分子的代謝物質(zhì)(分子量小于10000)可以用代謝組學(xué)的方法進(jìn)行鑒定和分析,實(shí)驗(yàn)中,我們采用基于核磁共振的代謝組學(xué)方法(1H-NMR-based metabonomics)對(duì)細(xì)胞內(nèi)代謝物進(jìn)行定量分析,研究PPI作用于胃癌細(xì)胞所引起的一系列胞內(nèi)胞外代謝物微環(huán)境的改變,尋找PPI對(duì)細(xì)胞代謝相關(guān)通路的影響。結(jié)果:PPI影響胃癌細(xì)胞的生物學(xué)功能,CCK-8及細(xì)胞克隆形成實(shí)驗(yàn)說(shuō)明PPI可以顯著抑制細(xì)胞的增殖,Transwell細(xì)胞遷移及侵襲實(shí)驗(yàn)和劃痕實(shí)驗(yàn)說(shuō)明PPI可以抑制細(xì)胞的遷移和侵襲。通過(guò)裸鼠成瘤實(shí)驗(yàn)體外驗(yàn)證了PPI對(duì)胃癌細(xì)胞具有抑制的作用,結(jié)果具有統(tǒng)計(jì)學(xué)意義(p0.05)。Western-blot結(jié)果顯示PPI對(duì)胃癌細(xì)胞SGC-7901及MGC-803抗凋亡蛋白Bcl-2進(jìn)行下調(diào)而上調(diào)促凋亡蛋白Bax;實(shí)驗(yàn)也發(fā)現(xiàn)PPI可以影響EMT相關(guān)蛋白的表達(dá),即上調(diào)E-cadherin、下調(diào)Vimentin、β-catenin蛋白的表達(dá)。代謝組學(xué)分析顯示在MGC-803胃癌細(xì)胞系中,與對(duì)照組相比,PPI組細(xì)胞對(duì)纈氨酸(Valine)、亮氨酸(Leucine),異亮氨酸(Isoleucine),谷氨酰胺(Glutamine),丙酮酸(Pyruvate)及葡萄糖(Glucose)的利用明顯減少。細(xì)胞內(nèi)乙醇(Ethanol),乳酸(Lactate)大量堆積;細(xì)胞代謝物肌醇(myo-Inositol),酪氨酸(Tyrosine)及苯丙氨酸(Phenylalanine)等代謝物合成減少。結(jié)論:PPI可以抑制胃癌細(xì)胞的增殖、遷移和侵襲;PPI通過(guò)增加細(xì)胞鈣離子內(nèi)流、上調(diào)促凋亡蛋白Bax蛋白、下調(diào)抗凋亡Bcl-2蛋白及阻滯細(xì)胞周期來(lái)引起胃癌細(xì)胞凋亡;PPI通過(guò)抑制Wnt/β-catenin信號(hào)通路抑制胃癌細(xì)胞的侵襲和遷移。同時(shí),PPI可以影響胃癌細(xì)胞的氨基酸、葡萄糖的利用,影響細(xì)胞代謝產(chǎn)物代謝抑制胃癌細(xì)胞的生長(zhǎng)。PPI發(fā)揮抗腫瘤作用是多因素共同作用的結(jié)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.
【學(xué)位授予單位】：皖南醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.2

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本文編號(hào)：2093291