新型卟啉化合物体外抗肿瘤作用筛选及机理研究

发布时间：2018-06-18 05:47

本文选题：卟啉 + 光动力疗法　；参考：《遵义医学院》2014年硕士论文

【摘要】：目的：观察新合成的金属卟啉化合物对肝癌细胞(HepG2)、胃癌细胞(SGC-7901)、结肠癌细胞(sw480)、高分化鼻咽癌细胞(CNE-1)、低分化鼻咽癌细胞(CNE-2)、小鼠单核巨噬细胞(RAW264.7)、肺癌细胞(A549)以及乳腺癌细胞(MCF-7)的抗肿瘤作用，并筛选疗效较好的药物进一步研究卟啉化合物抗肿瘤作用机理。方法：1.药物抗肿瘤活性筛选：SRB法和MTT法测定药物对肿瘤细胞增殖的影响；邻二氮菲-Fe2+氧化法和邻苯三酚自氧化法测定药物对肿瘤细胞清除羟自由基和超氧阴离子自由基能力的影响；Griess法测定肿瘤细胞中NO自由基含量的变化；Giemsa染色法观察肿瘤细胞形态学的变化。2.药物抗肿瘤机理研究：罗丹明123测定线粒体膜电位变化；流式细胞仪检测细胞凋亡；DNA完整性检测；实时荧光定量PCR法检测肿瘤细胞C-myc基因的mRNA表达量的变化；ELISA法测定肿瘤细胞端粒酶活性的变化。结果：八种金属卟啉化合物对肿瘤细胞的生长均具有一定的抑制作用，且随着药物浓度的升高，，细胞的生长抑制率基本呈上升的趋势；细胞对羟自由基和超氧阴离子自由基的清除能力下降；细胞上清液中NO自由基的含量升高；Giemsa染色后观察到凋亡细胞数目明显增加且药物对HepG2细胞作用最显著。上述效果最好的两种药物是Rup-03和Rup-04，Rup-03对五种肿瘤细胞(HepG2、CNE-1、CNE-2、sw480以及RAW264.7细胞)的杀伤作用较好，Rup-04对SGC-7901细胞的杀伤作用较好。随着Rup-03浓度的增加(10-9～10-6mol/L)，线粒体膜电位荧光强度减弱；流式细胞仪显示HepG2细胞凋亡率逐渐增加；琼脂糖凝胶电泳图显示DNA发生了降解；C-myc基因的mRNA相对表达量减少且端粒酶活性降低。这些结果均表明，细胞凋亡程度随着药物浓度的增加而加重。结论：八种新型金属卟啉化合物对肿瘤细胞均具有一定的杀伤作用，且杀伤作用与浓度具有一定相关性，总体效果最好的药物为Rup-03，适宜浓度为10-9～10-6mol/L。该新型卟啉化合物的抗肿瘤作用机理可能是经光照后生成自由基，进而抑制肿瘤细胞C-myc基因的表达以及端粒酶活性并诱导细胞发生凋亡。
[Abstract]:Objective: to observe the effects of newly synthesized metalloporphyrin compounds on hepatocellular carcinoma cell line HepG2, gastric cancer cell line SGC-7901, colon cancer cell line SW480, well-differentiated nasopharyngeal carcinoma cell line CNE-1, poorly differentiated nasopharyngeal carcinoma cell line CNE-2, mouse mononuclear macrophage RAW264.7, lung cancer cell A549) and breast cancer. The antitumor effect of MCF-7), The mechanism of anti-tumor action of porphyrin compounds was further studied. Method 1: 1. The effects of antitumor drugs on the proliferation of tumor cells were determined by the method of antitumor activity: the effects of phenanthrene Fe2 oxidation and pyrogallol autooxidation on the ability of scavenging hydroxyl radical and superoxide anion free radical in tumor cells were measured. The changes of no Free radical content in tumor cells by Griess method; the morphological changes of tumor cells were observed by Giemsa staining. The mechanism of drug anti-tumor: Rhodamine 123 was used to detect mitochondrial membrane potential, flow cytometry was used to detect apoptosis and DNA integrity, real-time fluorescence quantitative PCR was used to detect the mRNA expression of C-myc gene in tumor cells. Telomerase activity of tumor cells was determined by Elisa. Results: all of the eight metalloporphyrin compounds could inhibit the growth of tumor cells, and the growth inhibition rate of tumor cells increased with the increase of drug concentration. The activity of scavenging hydroxyl radical and superoxide anion radical was decreased, and the content of no free radical in supernatant increased after Giemsa staining, and the number of apoptotic cells was obviously increased, and the effect of drugs on HepG2 cells was most significant. The two most effective drugs were Rup-03 and Rup-04 Rup-03. Rup-04 had better killing effect on SGC-7901 cells than Rup-04 on five kinds of tumor cells (HepG2CNE-1 CNE-2Osw480 and RAW264.7). With the increase of Rup-03 concentration, the fluorescence intensity of mitochondrial membrane potential decreased, the apoptosis rate of HepG2 cells increased by flow cytometry, the DNA degradation occurred by agarose gel electrophoresis. The relative expression of C myc mRNA and telomerase activity were decreased. These results indicate that the degree of apoptosis increases with the increase of drug concentration. Conclusion: all the eight new metalloporphyrin compounds have a certain killing effect on tumor cells, and the killing effect is related to the concentration of Rup-03. the best drug is Rup-03, and the optimum concentration is 10-9 ~ 10-6 mol 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) ~ (-1) ~ (-1) ~ (-1) ~ (-1). The anti-tumor mechanism of the new porphyrin compound may be the formation of free radicals after irradiation, which can inhibit the expression of C-myc gene, telomerase activity and induce apoptosis of tumor cells.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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