新型希夫碱铜配合物的合成及其抑制胃癌的研究

发布时间：2018-08-14 11:05

【摘要】：铜是人体必须的微量元素,其希夫碱的配合物具有抗氧化、抗菌、抗肿瘤等多种作用,尤其是希夫碱铜配合物的抗肿瘤作用受到广泛的关注,但相关机制报道较少。本论文中介绍了一种新合成的希夫碱铜配合物,并以此为研究对象,从细胞凋亡和自噬两方面,在细胞水平上探讨新型希夫碱铜配合物抑制胃癌SGC-7901、BGC-823的增殖作用,同时通过检测ROS,分析了ROS在新型希夫碱铜配合物促进这两种胃癌细胞凋亡过程中发挥的作用。研究内容及其相关检查如下:(1)首先将水杨醛和水杨酰肼溶于乙醇水浴加热回流后得到希夫碱的配体,再将希夫碱配体溶于乙醇,加入硫酸铜后水浴加热,最后经冷却过滤得到新型希夫碱铜配合物。(2)通过MTS、流式细胞检测发现新型希夫碱铜配合物对胃癌细胞SGC-7901、BGC-823具有增殖抑制及促进其凋亡的作用,具有浓度依赖性及时间依赖性,并发现新型希夫碱铜配合物能使细胞停滞在G1期。利用电镜观察细胞凋亡后细胞形态及细胞器的变化。采用裸鼠成瘤实验证明新型希夫碱铜配合物能在体内诱导瘤体变小。(3)探讨新型希夫碱铜配合物诱导胃癌细胞凋亡的机制。通过AO-EB、Hoechst33258、DAPI染色观察到新型希夫碱铜配合物能够使胃癌细胞SGC-7901、BGC-823凋亡,经过Western Blot可以明确新型希夫碱铜配合物可以激活死亡受体通路增加其相关配体的表达,进而激活Caspase-8使凋亡诱导蛋白Bid形成活化的t-Bid。同时新型希夫碱铜配合物能够降低线粒体膜电势进而通过线粒体凋亡途径诱导细胞凋亡。此外NF-κB也参与了胃癌细胞的凋亡过程。但是通过克隆形成实验发现NF-κB的抑制剂并不能完全阻止新型希夫碱铜配合物对胃癌细胞的凋亡诱导作用,那么一定存在其他导致细胞凋亡的途径。利用AO、MDC染色发现新型希夫碱铜配合物作用于胃癌细胞SGC-7901、BGC-823时存在细胞自噬。Western发现自噬的特征性蛋白IC3和Beclin-1高表达。同时利用荧光染色发现新型希夫碱铜配合物能够显著增加细胞内ROS水平,而当新型希夫碱铜配合物与ROS的抑制剂NAC共同作用于胃癌细胞是发现NF-κB及自噬的特征蛋白表达均明显减少。故ROS为NF-κB和自噬的上游刺激因子,影响新型希夫碱铜配合物对胃癌细胞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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R914;R96

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