【摘要】：銅是人體必須的微量元素,其希夫堿的配合物具有抗氧化、抗菌、抗腫瘤等多種作用,尤其是希夫堿銅配合物的抗腫瘤作用受到廣泛的關(guān)注,但相關(guān)機(jī)制報(bào)道較少。本論文中介紹了一種新合成的希夫堿銅配合物,并以此為研究對(duì)象,從細(xì)胞凋亡和自噬兩方面,在細(xì)胞水平上探討新型希夫堿銅配合物抑制胃癌SGC-7901、BGC-823的增殖作用,同時(shí)通過(guò)檢測(cè)ROS,分析了ROS在新型希夫堿銅配合物促進(jìn)這兩種胃癌細(xì)胞凋亡過(guò)程中發(fā)揮的作用。研究?jī)?nèi)容及其相關(guān)檢查如下:(1)首先將水楊醛和水楊酰肼溶于乙醇水浴加熱回流后得到希夫堿的配體,再將希夫堿配體溶于乙醇,加入硫酸銅后水浴加熱,最后經(jīng)冷卻過(guò)濾得到新型希夫堿銅配合物。(2)通過(guò)MTS、流式細(xì)胞檢測(cè)發(fā)現(xiàn)新型希夫堿銅配合物對(duì)胃癌細(xì)胞SGC-7901、BGC-823具有增殖抑制及促進(jìn)其凋亡的作用,具有濃度依賴性及時(shí)間依賴性,并發(fā)現(xiàn)新型希夫堿銅配合物能使細(xì)胞停滯在G1期。利用電鏡觀察細(xì)胞凋亡后細(xì)胞形態(tài)及細(xì)胞器的變化。采用裸鼠成瘤實(shí)驗(yàn)證明新型希夫堿銅配合物能在體內(nèi)誘導(dǎo)瘤體變小。(3)探討新型希夫堿銅配合物誘導(dǎo)胃癌細(xì)胞凋亡的機(jī)制。通過(guò)AO-EB、Hoechst33258、DAPI染色觀察到新型希夫堿銅配合物能夠使胃癌細(xì)胞SGC-7901、BGC-823凋亡,經(jīng)過(guò)Western Blot可以明確新型希夫堿銅配合物可以激活死亡受體通路增加其相關(guān)配體的表達(dá),進(jìn)而激活Caspase-8使凋亡誘導(dǎo)蛋白Bid形成活化的t-Bid。同時(shí)新型希夫堿銅配合物能夠降低線粒體膜電勢(shì)進(jìn)而通過(guò)線粒體凋亡途徑誘導(dǎo)細(xì)胞凋亡。此外NF-κB也參與了胃癌細(xì)胞的凋亡過(guò)程。但是通過(guò)克隆形成實(shí)驗(yàn)發(fā)現(xiàn)NF-κB的抑制劑并不能完全阻止新型希夫堿銅配合物對(duì)胃癌細(xì)胞的凋亡誘導(dǎo)作用,那么一定存在其他導(dǎo)致細(xì)胞凋亡的途徑。利用AO、MDC染色發(fā)現(xiàn)新型希夫堿銅配合物作用于胃癌細(xì)胞SGC-7901、BGC-823時(shí)存在細(xì)胞自噬。Western發(fā)現(xiàn)自噬的特征性蛋白IC3和Beclin-1高表達(dá)。同時(shí)利用熒光染色發(fā)現(xiàn)新型希夫堿銅配合物能夠顯著增加細(xì)胞內(nèi)ROS水平,而當(dāng)新型希夫堿銅配合物與ROS的抑制劑NAC共同作用于胃癌細(xì)胞是發(fā)現(xiàn)NF-κB及自噬的特征蛋白表達(dá)均明顯減少。故ROS為NF-κB和自噬的上游刺激因子,影響新型希夫堿銅配合物對(duì)胃癌細(xì)胞SGC-7901和BGC-823的凋亡和自噬。
[Abstract]:Copper is a necessary trace element in human body. Its Schiff base complexes have many functions, such as antioxidation, antimicrobial, anti-tumor and so on. Especially, the antitumor effects of Schiff base copper complexes have been paid more attention to, but the related mechanisms are seldom reported. In this paper, a new Schiff base copper complex was introduced. From the aspects of cell apoptosis and autophagy, the inhibitory effect of new Schiff base copper complex on the proliferation of gastric cancer SGC-7901BGC-823 was studied at the cell level. At the same time, the role of ROS in the process of Schiff base copper complex promoting apoptosis of gastric cancer cells was analyzed. The contents of the study were as follows: (1) Schiff base ligands were obtained by refluxing salicylaldehyde and salicylic hydrazine in ethanol water bath, then Schiff base ligands were dissolved in ethanol and then heated in water bath with copper sulfate. Finally, a new type of Schiff base copper complex was obtained by cooling filtration. (2) by MTSand flow cytometry, it was found that the new Schiff base copper complex could inhibit the proliferation and promote the apoptosis of gastric cancer cell line SGC-7901 and BGC-823 in a concentration-and time-dependent manner. It was also found that the new Schiff base copper complex could arrest cells in G 1 phase. The changes of cell morphology and organelle after apoptosis were observed by electron microscope. The results showed that the new Schiff base copper complex could induce the tumor to become smaller in vivo. (3) to explore the mechanism of the new Schiff base copper complex inducing apoptosis of gastric cancer cells. By AO-EBN Hoechst33258 DAPI staining, it was observed that the new Schiff base copper complex could induce apoptosis in gastric cancer cell line SGC-7901BGC-823. The Western Blot showed that the new Schiff base copper complex could activate the death receptor pathway to increase the expression of its associated ligand. Activation of Caspase-8 resulted in the formation of an activated t-Bidi of apoptosis-inducing protein Bid. At the same time, the new Schiff base copper complex can reduce the mitochondrial membrane potential and induce apoptosis through the mitochondrial apoptosis pathway. In addition, NF- 魏 B is involved in the apoptosis of gastric cancer cells. However, it was found that the inhibitor of NF- 魏 B could not completely prevent the apoptosis of gastric cancer cells induced by new Schiff base copper complex, so there must be other pathways leading to apoptosis. A novel Schiff base copper complex was found to be highly expressed in SGC-7901 cell line BGC-823 by AOA MDC staining. The characteristic protein IC3 and Beclin-1 of autophagy were found in SGC-7901 BGC-823 cells. At the same time, fluorescence staining showed that the new Schiff base copper complex could significantly increase the intracellular ROS level. The expression of NF- 魏 B and autophagy in gastric cancer cells was significantly decreased when the new Schiff base copper complex and NAC, an inhibitor of ROS, co-acted on gastric cancer cells. Therefore, ROS is the upstream stimulating factor of NF- 魏 B and autophagy, which affects the apoptosis and autophagy of SGC-7901 and BGC-823 in gastric cancer cells induced by novel Schiff base copper complexes.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R914;R96

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