【摘要】：PFK15是糖酵解通路關(guān)鍵調(diào)節(jié)酶PFKFB3的特異性小分子抑制劑。PFKFB3通過調(diào)節(jié)F2,6P2的生成量而影響糖酵解過程中限速酶PFK-1的活性進(jìn)而控制糖酵解通量。本文旨在考察PFK15在體內(nèi)外模型中對(duì)胃癌的抗腫瘤作用和潛在的作用機(jī)制以及與sorafenib聯(lián)合用藥對(duì)腫瘤血管生成的影響。本文應(yīng)用蛋白免疫印跡方法驗(yàn)證PFKFB3在胃癌細(xì)胞移植瘤、血管內(nèi)皮細(xì)胞和正常組織中的蛋白表達(dá)水平以確定PFKFB3蛋白表達(dá)的組織特異性。考察PFK15對(duì)胃癌細(xì)胞和內(nèi)皮細(xì)胞糖酵解通量中葡萄糖攝取和F2,6P2產(chǎn)物的影響以及F2,6P2對(duì)PFK15誘導(dǎo)的細(xì)胞增殖能力的影響以確定PFK15對(duì)PFKFB3的"on-target"效應(yīng)。應(yīng)用細(xì)胞活力分析、細(xì)胞凋亡分析、細(xì)胞周期分析、劃痕實(shí)驗(yàn)、Transwell小孔穿梭實(shí)驗(yàn)、細(xì)胞骨架檢測(cè)實(shí)驗(yàn)分別考察PFK15對(duì)胃癌細(xì)胞的增殖、凋亡、周期、遷移、侵襲和骨架的影響。應(yīng)用內(nèi)皮細(xì)胞管狀結(jié)構(gòu)形成實(shí)驗(yàn)、體外3D內(nèi)皮細(xì)胞球狀體出芽實(shí)驗(yàn)和小鼠Matrigel Plug實(shí)驗(yàn)考察PFK15對(duì)血管新生的影響;應(yīng)用體內(nèi)裸小鼠移植瘤模型分析PFK15對(duì)胃癌的體內(nèi)抗腫瘤活性和抗血管生成作用;應(yīng)用蛋白免疫印跡分析和免疫組化分析檢測(cè)PFK15對(duì)腫瘤細(xì)胞增殖、凋亡、周期和血管新生相關(guān)蛋白和信號(hào)通路的影響。另外,本文還在體內(nèi)和體外模型中考察了 PFK15與sorafenib聯(lián)用對(duì)腫瘤血管新生的影響。結(jié)果表明,相比于其它正常組織,PFKFB3在胃癌移植瘤和內(nèi)皮細(xì)胞中是過量表達(dá)的。PFK15在實(shí)驗(yàn)劑量范圍內(nèi)可劑量依賴性的降低胃癌細(xì)胞(MKN45和AGS)和內(nèi)皮細(xì)胞(EA.hy926)中葡萄糖的攝取和F2,6P2的產(chǎn)生,從而降低糖酵解通量;而F2,6P2加入后可以消除PFK15誘導(dǎo)的細(xì)胞增殖抑制作用,說明PFKFB3為PFK15的特異性靶點(diǎn)。PFK15在實(shí)驗(yàn)劑量范圍內(nèi)劑量依賴性的抑制胃癌細(xì)胞MKN45和AGS的增殖,PFK15引起的G0/G1期細(xì)胞周期阻滯與阻斷Cyclin-CDKs/Rb/E2F信號(hào)通路有關(guān),引起的細(xì)胞凋亡與線粒體途徑誘導(dǎo)的細(xì)胞凋亡有關(guān),引起的對(duì)細(xì)胞的遷移和侵襲的影響與改變細(xì)胞內(nèi)F-actin數(shù)量和排列、改變細(xì)胞相關(guān)黏附分子的蛋白表達(dá)有關(guān)。PFK15可顯著抑制內(nèi)皮細(xì)胞EA.hy926的增殖、管狀結(jié)構(gòu)的形成、球狀體的出芽和小鼠植入的基質(zhì)膠塊中血管的生成。在MKN45和AGS裸小鼠移植瘤模型中,PFK15都能顯著抑制移植瘤的腫瘤體積和瘤重的增長(zhǎng),PFK15還能顯著降低移植瘤中CD31陽性的染色面積。另外,PFK15與sorafenib聯(lián)用顯著抑制內(nèi)皮細(xì)胞EA.hy926的增殖、管狀結(jié)構(gòu)的形成、球狀體的出芽、Matrigel Plug和移植瘤中血管的發(fā)生,并且聯(lián)用組對(duì)血管新生的抑制作用大于任何單獨(dú)用藥組。綜上所述,本研究初步闡明了 PFK15抗胃癌活性的作用機(jī)制以及PFK15與sorafenib聯(lián)用的抗血管新生作用,為糖酵解抑制劑的進(jìn)一步開發(fā)提供理論基礎(chǔ)。
[Abstract]:PFK15 is a specific small molecule inhibitor of PFKFB3, which is the key regulator of glycolysis pathway. PFKFB3 affects the activity of rate-limiting enzyme PFK-1 during glycolysis by regulating the production of F2and 6P2, and then controls the glycolysis flux. The purpose of this study was to investigate the antitumor effect and potential mechanism of PFK15 on gastric cancer in vitro and in vivo, as well as the effect of sorafenib on tumor angiogenesis. In order to determine the tissue specificity of PFKFB3 protein expression in gastric cancer cell transplantation tumor, vascular endothelial cells and normal tissues, the expression level of PFKFB3 protein was confirmed by Western imprinting. The effects of PFK15 on glucose uptake and F26P2 products in glycolysis fluxes of gastric cancer cells and endothelial cells and the effects of F2and 6P2 on the proliferation induced by PFK15 were investigated to determine the "on-target" effect of PFK15 on PFKFB3. The effects of PFK15 on proliferation, apoptosis, cycle, migration, invasion and skeleton of gastric cancer cells were investigated by cell vitality analysis, apoptosis analysis, cell cycle analysis, scratch test, Transwell pore shuttle test and cytoskeleton detection. The effects of PFK15 on angiogenesis were investigated by endothelial cell tubular structure test, 3D endothelial cell bulbous sprouting test and mouse Matrigel Plug test in vitro, and the antitumor activity and anti-angiogenic effect of PFK15 on gastric cancer in vivo were analyzed by transplanting tumor model in nude mice. The effects of PFK15 on proliferation, apoptosis, cycle and neovascularization related proteins and signaling pathways of tumor cells were detected by Western imprinting and immunohistochemical analysis. In addition, the effects of PFK15 combined with sorafenib on tumor angiogenesis were also investigated in vivo and in vitro. The results showed that compared with other normal tissues, PFKFB3 was overexpressed in gastric cancer xenografts and endothelial cells. PFK15 could decrease glucose uptake and F26P2 production in gastric cancer cells (MKN45 and AGS) and endothelial cells (EA.hy926) in a dose-dependent manner, thus reducing glycolysis flux. The addition of F2and 6P2 could eliminate the inhibitory effect of PFK15 on cell proliferation, indicating that PFKFB3 was a specific target of PFK15. PFK15 inhibited the proliferation of MKN45 and AGS in a dose-dependent manner. The cell cycle arrest in G0/G1 phase induced by PFK15 was related to the blocking of Cyclin-CDKs/Rb/E2F signaling pathway, and the apoptosis was related to apoptosis induced by mtDNA pathway. The effect of PFK15 on cell migration and invasion was related to changing the number and arrangement of F-actin in cells and changing the protein expression of cell-associated adhesion molecules. PFK15 could significantly inhibit the proliferation of endothelial cells, the formation of tubular structure, the budding of bulbous bodies and the formation of blood vessels in matrix glue blocks implanted in mice. In both MKN45 and AGS nude mice, PFK15 could significantly inhibit the increase of tumor volume and tumor weight, and PFK15 could also significantly reduce the positive staining area of CD31 in nude mice. In addition, the combination of PFK15 and sorafenib significantly inhibited the proliferation of endothelial cell EA.hy926, the formation of tubular structure, the budding, Matrigel Plug of bulbous body and the occurrence of blood vessels in the transplantation tumor, and the inhibitory effect of the combined group on vascular regeneration was greater than that of any single treatment group. In summary, this study preliminarily clarified the mechanism of anti-gastric cancer activity of PFK15 and the antiangiogenic effect of PFK15 combined with sorafenib, which provided a theoretical basis for the further development of glycolysis inhibitors.
【學(xué)位授予單位】：沈陽藥科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.2

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1 朱偉;PFK15對(duì)人胃癌的抗腫瘤活性及潛在的機(jī)制研究[D];沈陽藥科大學(xué);2016年

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本文編號(hào)：2499666