【作者】 杨颖；

【导师】 邹伟；

【作者基本信息】 辽宁师范大学 ， 细胞生物学， 2009， 硕士

【摘要】 5-氟尿嘧啶(5-Fluorouracil,5-Fu)属于抗肿瘤代谢药物,由于其具有快速、高效和低毒副作用的特征,目前已成为临床治疗恶性肿瘤如胃肠道癌、肝癌、乳腺癌等首选的化疗药物。但同时也发现,在不同的癌症治疗过程中,使用该药物表现出来的药效不同,而且难以逃脱目前众多抗癌药物本身带来的毒性大等缺点,使其在应用上具有一定的局限性。随着现代医药生物技术的发展,为提高5-氟尿嘧啶的药效,采用5-氟尿嘧啶与其它药物联合使用,极大地提高了药物的效用。但是,关于5-氟尿嘧啶与其它药物联合使用的种类、剂量-药效关系以及细胞机制尚不完全清楚。研究发现,还原型叶酸-甲酰四氢叶酸钙(CF)作为核酸合成的重要辅酶对5-Fu有增效的作用,其优点是毒副作用较轻。因此,本文用5-氟尿嘧啶与甲酰四氢叶酸钙联合使用处理人乳腺癌细胞MCF-7,通过MTT法检测药物毒性及其对细胞增殖的影响;利用琼脂糖凝胶电泳检测DNA梯状带、荧光染色观察细胞核的形态变化、流式细胞检测分析凋亡细胞的周期变化;凋亡相关蛋白Bcl-2、Bax等的动态变化,比较了联合用药和单独用药对细胞凋亡的影响,探讨了联合用药可能的机制,旨在为临床提高恶性肿瘤化疗的疗效,减少药物的毒副作用提供一定的理论和实验依据。结果如下:(1)5-氟尿嘧啶单独作用于乳腺癌细胞株MCF-7产生的抑制效应随时间的延长而增加;不同浓度的5-氟尿嘧啶与不同浓度的甲酰四氢叶酸钙作用于MCF-7细胞,5-氟尿嘧啶分别与2.5μg/ml、5μg/ml的甲酰四氢叶酸钙联合作用的抑制效应随甲酰四氢叶酸钙剂量的增加而增强。相同时间内,单独用5-氟尿嘧啶作用于MCF-7细胞较之与叶酸联合作用的抑制效应差。5-氟尿嘧啶联合使用较单独使用效果要好,且与甲酰四氢叶酸钙的联合抑制作用最佳;(2)细胞形态学检测发现,凋亡细胞体积变圆,胞浆浓缩,核DNA固缩深染,贴壁能力很差,有的已经脱落至培养液中。早期凋亡细胞的细胞膜正常,呈圆珠状小体,晚期凋亡细胞被染成红色,其核形态与早期凋亡细胞相近。坏死细胞体积明显变大,呈不均匀的橙红色荧光,也有的已经变成颗粒状无定形状的碎片。随着5-氟尿嘧啶浓度的增加,凋亡细胞的比例呈剂量依赖型;琼脂糖凝胶电泳时,凝胶成像系统下可观察到凋亡小体。流式细胞仪检测过程中,可以明显看到凋亡峰,并且中期细胞增多。早期凋亡的细胞数量增多,差异极显著(P < 0.01)。晚期凋亡细胞数量增多,差异显著(P < 0.05)。(3)5-氟尿嘧啶与甲酰四氢叶酸钙作用于MCF-7细胞后,分别加入相对应的一抗、二抗,流式细胞仪检测凋亡相关蛋白Bcl-2、Bax和p53的表达,得到了Bcl-2表达降低,Bax表达升高的结果。结论:(1)5-氟尿嘧啶不论是单独作用还是联合作用,都对乳腺癌MCF-7细胞产生抑制作用,其作用效果有差异,5-氟尿嘧啶联合使用较单独使用效果要好,且与甲酰四氢叶酸钙的联合抑制作用最佳。5-Fu联合CF诱导癌细胞凋亡有明显的时间、剂量依赖性。(2)5-Fu具有明显的诱导人体外癌细胞凋亡的作用,随着5-氟尿嘧啶剂量的增加,5-氟尿嘧啶联合甲酰四氢叶酸钙导致MCF-7细胞早期凋亡率明显增加,晚期凋亡细胞也有递增之势。(3)5-氟尿嘧啶联合甲酰四氢叶酸钙诱导MCF-7细胞凋亡时Bcl-2降低、Bax增加,同时Bcl-2/ Bax的比率亦随时间延长及剂量的增加而逐渐下降。5-氟尿嘧啶联合甲酰四氢叶酸钙诱导MCF-7细胞凋亡与Bcl-2降低、Bax增加有关。更多还原

【Abstract】 5-Fluorouracil (Fluorouracil, 5-Fu) is a anti-tumor drug, which is in possession of speediness, high efficiency and low toxicity, and has been the first choose drug in malignant cancer therapies, e.g mammary cancer, gastro-intestinal tract cancer, liver cancer, and so on. At the mean time, it has been found that the effects of 5- Fluorouracil in different clinical therapies were different because of its toxicity, so that it is somewhat restricted in its applications of clinic. Along with the modern medicine biological technology development, 5-Fluorouracil associated with other medicine was used to enhance the drug efficacy successfully. However, some kinds, dose-effect and the mechanisms of 5- Fluorouracil associated with other drugs, are still unknown clearly.Some studies shows that reductive Leucovorin (CF) with low toxicity as assistant enzyme has an important role in DNA corporation and could enhance the effect of 5- Fluorouracil Presently, along with the develop of technology, some people use to carry on certain process to 5-Fluorouracil, or use other medicine together, seek the low poisonous highly effective way, overcome 5-Fluorouracil anticancer treatment medicine’s some shortcomings, enable it to obtain a more widespread application in the clinical aspect. Thus, here we adopt the way of MTT, and so on, take 5-Fluorouracil with Calcium Leucovorin(CF) on MCF-7 cell line, which is used for different effect of the dose of 5-Fluorouracil Calcium Leucovorin, also to detect the best time and the dose of the drugs with MCF-7 cell line. After take doses of 5-Fluorouracil and Calcium Leucovorin, we observe the cell apoptosis with morphological ways, AO/EB staining, DNA ladder and also we use Cyto-Flows to see the change of phases with MCF-7 cell lines. Also, we detect the expression relative protein of Bcl-2, Bax and so on, discuss possible mechanisms, in order to offer some theories and gist of upgrade the effec of application in clinic, lower the toxicity.Our results showed that:(1)This topic that mechanism of restraining cancer in view of 5-Fluorouracil, find the best dose to MCF-7 cell line to with Fluorouracil and also the best response time; Different doses are used to see the different specificity and the best time and dose. There are differences according to the different cell logarithm period, which will cause the cell growth condition at the logarithmic growth time, to take each experiment smoothly. MTT method to find the best medication dosage as well as best medication time, with different dose of 5-Fluorouracil with Calcium Leucovorin (2.5μg/ml、5μg/ml). 5-Fluorouracil, which is used single, is not better than that both used 5-Fluorouracil and Calcium Leucovorin.(2)Using morphology examination and using fluorescence staining technique under the microscope, cells of apoptosis are getting round and round, the color of DNA become dark and some fell off. We see the changes of phases to the cells which are dosed with 5-Fluorouracil and Calcium Leucovorin, with cyto-flows, different period of apoptosis could be detected.In the early phase of apoptosis cells, we could see some small bodies in cells, late phase of apoptosis cells become red, shapes are similar to the early ones.The death cells are all red without intact shapes.(3)After dosing with 5-Fluorouracil and Calcium Leucovorin with some time, collecting cells, using relative antibodies, we detect the expression of apoptosis relative proteins bcl-2, Bax, and p53. The result is that the expression of Bcl-2 is falling, the expression of Bax is rising.Conclusion:(1) 5-Fluorouracil would get more effect on cancer cells if it is used together with Calcium Leucovorin. To some sides, there are some dose depending between the medicine and the ratio of death of MCF-7 and other cancer cells.(2) The cells rise with the doses of 5-Fluorouracil and Calcium Leucovorin, and both the phases of early and the late cells rise, too.(3) Apoptosis of cancer cells are related to both Bcl-2 and Bax, which the ratio of Bcl-2 and Bax are also taken some rising effect.更多还原