新型希夫碱锌配合物的合成及其抗骨肿瘤活性的研究

发布时间：2019-06-04 00:41

【摘要】：研究背景骨肿瘤的发病率并不高,在常见肿瘤中仅占2%左右,但以我国高达13亿人口的统计基数而论,骨肿瘤的患病人群仍已达到一个不容忽视的规模。同时,一些高发肿瘤如肺癌、乳腺癌、前列腺癌等容易并发肿瘤的骨转移,因此实际上总的骨肿瘤患病人数仍然相当庞大。目前,对骨肿瘤的治疗已经从单纯的外科手术逐步向综合治疗的方向发展,尤其是新型化疗药物的出现,将骨肿瘤的治疗带入了一个新的时代。早在上世纪60年代,人们就已经发现希夫碱具有抗肿瘤、抗菌、催化活性等特性,近年来有学者研制出希夫碱的铂、铜等金属配合物,并显示出强大的抗肿瘤作用。本研究中使用一种新型的希夫碱锌配合物并研究其对骨肉瘤以及常见骨转移瘤-乳腺癌的抑制作用及其抗肿的作用机制,为骨肿瘤的治疗提供一种新的可能方案。目的制备一种全新的希夫碱锌配合物并研究其对MG-63人骨肉瘤细胞及MCF-7人乳腺癌细胞的杀伤作用并对其可能机制进行研究。方法(1)新型希夫碱锌配合物的合成。(2)CCK-8法测定新型希夫碱锌配合物对体外培养的上述两种细胞的增值抑制作用;采用Annexin V-PI双染法分析新型希夫碱锌配合物诱导两种细胞的凋亡率以及对细胞周期的影响,通过显微镜观察凋亡细胞的形态。(3)琼脂糖凝胶电泳检测DNA荧光强度;运用流式细胞仪以检测受试细胞线粒体的膜电位变化;采用Western Blot技术检测凋亡相关蛋白Bax、Bcl-2、caspase-9、Cyt-c、Fas、Fas-L、caspa-8以及caspase-3表达变化。观察新型希夫碱锌配合物作用后裸鼠体重及体表肿瘤体积的变化。RT-PCR检测裸鼠肿瘤中相关凋亡基因的表达。结果(1)利用水杨醛、水杨酰肼、醋酸锌经水浴加热回流及沉淀最终形成新型希夫碱锌配合物;(2)CCK-8法检测细胞存活率实验结果显示,随着新型希夫碱锌配合物浓度的增高以及给药时间的延长,对MG-63人骨肉瘤细胞和MCF-7人乳腺癌细胞的增殖抑制作用逐渐增强。Annexin V-FITC法检测细胞凋亡率中新型希夫碱锌配合物能够诱导MG-63人骨肉瘤细胞凋亡,与对照组相比,10μmol/L、50μmol/L、70μmol/L的新型希夫碱锌配合物作用于MG-63人骨肉瘤细胞4h、24h、48h后表现出明显的诱导细胞凋亡的作用(P0.05)。新型希夫碱锌配合物诱导MG-63人骨肉瘤细胞凋亡同样具有时间依赖性。各组中当新型希夫碱锌配合物浓度70μmol/L作用48h诱导细胞凋亡的比例最高达29.79±1.36%,且P0.05。同样的对于MCF-7人乳腺癌细胞也有相同的趋势,其中浓度70μmol/L作用48h诱导细胞凋亡的比例最高达30.29±1.77%,且P0.05。流式细胞仪检测周期的实验中发现新型希夫碱锌配合物能够使两种肿瘤细胞的生长周期停滞与G1期,导致细胞不能由G1期向S期以及G2期移行,同时S期细胞的减少,也使肿瘤细胞的增值能力进一步降低,抑制肿瘤细胞的生长。通过倒置显微镜观察新型希夫碱锌配合物处理后的两种细胞呈现凋亡的形态学特征。(3)Western Blot检测结果显示,不同浓度的新型希夫碱锌配合物作用于MG-63人骨肉瘤细胞一段时间后,随新型希夫碱锌配合物浓度增加bax/bcl-2比值也逐渐增加。线粒体中的Cyt-c下降,胞浆中的Cyt-c上升。具有明显的浓度依赖性。并出现了明显的Cleave caspase-9和3条带。实验中随时间的延长,细胞中的Bax/Bcl-2的比值为增加。线粒体内的Cyt-c减少。胞浆中的Cyt-c增加。具有明显的时间依赖性。并出现了明显的Cleave caspase-9和3条带。在Fas、Fas-L中随希夫碱浓度的增加灰度比值分别从0.48±0.06、增加到1.83±0.90以及从0.29±0.02增加到0.85±0.07。可见具有明显的浓度依赖性,并出现了清晰的Cleaved caspase-8和3条带。随着时间的延长,在Fas、Fas-L的灰度比值也增大。具有明显的时间依赖性,也出现了清晰的Cleaved caspase-8和3条带。在MCF-7细胞中也出现了相似的结果,随新型希夫碱锌配合物浓度增加Bax/Bcl-2的比值上升,线粒体中的Cyt-c减少,胞浆中的Cyt-c增加,具有明显的浓度依赖性。并出现了明显的Cleave caspase-9和3条带。随作用时间的延长Bax/Bcl-2的比值增加。线粒体内的Cyt-c减少,胞浆中的Cyt-c增加,具有明显的时间依赖性。并出现了明显的Cleave caspase-9和3条带。Fas、Fas-L中随新型希夫碱锌配合物浓度增加灰度比值增加。可见具有明显的浓度依赖性,并出现了清晰的Cleaved caspase-8和3条带。随着时间的延长,在Fas、Fas-L灰度比值增加,具有明显的时间依赖性,也出现了清晰的Cleaved caspase-8和3条带。(4)裸鼠体内的抗肿瘤作用:新型希夫碱锌配合物能显著抑制裸鼠MG-63、MCF-7转移瘤的生长,且无肝肾功能的损伤。新型希夫碱锌配合物作用肿瘤后期中的Bax、Bcl-2、Caspase-8、Caspase-9、Caspase-3的m RNA表达随新型希夫碱锌配合物浓度的变化而变化,具有一定的量效关系。结论(1)新型希夫碱锌配合物能够诱导MG-63人骨肉瘤细胞及常见骨转移瘤MCF-7人乳腺癌细胞凋亡;(2)新型希夫碱锌配合物诱导细胞凋亡的机制与线粒体途径及死亡受体途径相关。结论
[Abstract]:The incidence of bone tumors in the background of bone tumors is not high, and only about 2% of the common tumors, but with the statistical base of up to 1.3 billion people in China, the affected population of the bone tumor has reached a large scale. At the same time, some high-incidence tumors, such as lung cancer, breast cancer, prostate cancer, and the like, are prone to bone metastasis of the tumor, so the total number of bone tumors is still quite large. At present, the treatment of bone tumor has been gradually developed from simple surgery to the direction of comprehensive treatment, especially the emergence of a new type of chemotherapy medicine, bringing the treatment of bone tumor into a new era. In the early 1960s, it has been found that the schiff base has the characteristics of anti-tumor, antibacterial and catalytic activity. In recent years, some scholars have developed metal complexes such as platinum, copper, and the like of Schiff base, and show a powerful anti-tumor effect. In this study, a novel Schiff base zinc complex was used and its effect on osteosarcoma and common bone metastases-breast cancer and its anti-swelling mechanism were studied, and a new possible solution was provided for the treatment of bone tumors. Objective To prepare a new Schiff base zinc complex and to study its anti-killing effect on human osteosarcoma cells and MCF-7 human breast cancer cells. Method (1) Synthesis of novel Schiff base zinc complex. (2) The value-added inhibition of the novel Schiff base zinc complex on the two cells cultured in vitro was determined by the CCK-8 method, the apoptosis rate of the two cells induced by the novel Schiff base zinc complex and the effect on the cell cycle were analyzed by the Annexin V-PI double-staining method, and the morphology of the apoptotic cells was observed by the microscope. (3) The fluorescence intensity of DNA was detected by agarose gel electrophoresis, and the membrane potential of the mitochondria of the tested cell was detected by flow cytometry. The expression of Bax, Bcl-2, caspase-9, Cyt-c, Fas, Fas-L, caspase-9, Cyt-c, Fas, Fas-L, caspa-8 and caspase-3 were detected by Western Blot technique. To observe the changes of body weight and body surface tumor volume of the nude mice after the action of the novel Schiff base zinc complex. The expression of related apoptosis gene in nude mice was detected by RT-PCR. Results (1) The new type of Schiff base zinc complex was formed by heating reflux and precipitation in water bath with salicylaldehyde, water Yang, and zinc acetate. (2) The experimental results of the cell survival rate of (2) CCK-8 method show that with the increase of the concentration of the novel Schiff base zinc complex and the extension of the administration time, The inhibitory effect of MG-63 human osteosarcoma cells and MCF-7 human breast cancer cells was gradually enhanced. In the Annexin V-FITC method, the novel Schiff base zinc complex in the cell apoptosis rate can induce the apoptosis of the MG-63 human osteosarcoma cells. Compared with the control group, the novel Schiff base zinc complex with the concentration of 10. mu. mol/ L,50. mu. mol/ L and 70. mu. mol/ L acts on the MG-63 human osteosarcoma cells for 4h and 24h, After 48 h, the apoptosis of the cells was significantly induced (P0.05). The novel Schiff base zinc complex has the same time-dependence on the apoptosis of the MG-63 human osteosarcoma cells. In each group, the proportion of cell apoptosis was up to 29.79% and 1.36%, and P0.05. The same trend was observed for MCF-7 human breast cancer cells, in which the ratio of cell apoptosis was up to 30.29% and 1.77% at the concentration of 70. m u.mol/ L for 48 h, and P 0.05. In the experiment of flow cytometry, it was found that the novel Schiff base zinc complex can arrest the growth cycle of the two tumor cells and the G1 phase, which leads to the inability of the cells to move from the G1 phase to the S phase and the G2 phase, while the decrease of the S-phase cells also further reduces the value-added ability of the tumor cells, And the growth of the tumor cells is inhibited. The morphological characteristics of the two cells after treatment with the novel Schiff base zinc complex were observed by an inverted microscope. (3) The results of Western Blot test showed that the new type of Schiff base zinc complex with different concentrations on the MG-63 human osteosarcoma cells increased the ratio of bax/ bcl-2 with the concentration of the new Schiff base zinc complex. Cyt-c in the mitochondria decreased and Cyt-c in the cytoplasm increased. Having a significant concentration dependence. The obvious Cleve caspase-9 and 3 bands appeared. The ratio of Bax/ Bcl-2 in the cell was increased with the increase of time in the experiment. Cyt-c in the mitochondria is reduced. Cyt-c in the cytoplasm is increased. With significant time-dependence. The obvious Cleve caspase-9 and 3 bands appeared. In the case of Fas, Fas-L, the gray ratio of the increase of the concentration of the Schiff base increased from 0.48 to 0.06, to 1.83 to 0.90, and from 0.29 to 0.02 to 0.85, respectively. Visible concentration-dependent and clear Cleared caspase-8 and 3 bands appeared. The gray-scale ratio of Fas and Fas-L was also increased with the increase of time. Clear time-dependent and clear Cleared caspase-8 and 3 bands also appeared. Similar results also appeared in the MCF-7 cells, and the ratio of Bax/ Bcl-2 increased with the concentration of the novel Schiff base zinc complex, and the Cyt-c in the mitochondria increased, and the Cyt-c in the cytoplasm increased and the concentration-dependent manner was evident. The obvious Cleve caspase-9 and 3 bands appeared. The ratio of Bax/ Bcl-2 increased with the time of action. Cyt-c in the mitochondria is reduced and Cyt-c in the cytoplasm is increased and has a significant time-dependence. The obvious Cleve caspase-9 and 3 bands appeared. The increase of the gray ratio of Fas, Fas-L with the concentration of the new Schiff base zinc complex. Visible concentration-dependent and clear Cleared caspase-8 and 3 bands appeared. With the increase of the time, the ratio of Fas and Fas-L was increased, and there was a clear time-dependence and clear Cleared caspase-8 and 3 bands. (4) Anti-tumor effect in nude mice: The novel Schiff base zinc complex can significantly inhibit the growth of MG-63 and MCF-7 metastatic tumor in nude mice, and has no damage to the liver and kidney function. The expression of Bax, Bcl-2, Caspase-8, Caspase-9 and Caspase-3 in the late stage of the tumor was changed with the change of the concentration of the novel Schiff base zinc complex. Conclusion (1) The novel Schiff base zinc complex can induce the apoptosis of the human osteosarcoma cells of MG-63 and the MCF-7 human breast cancer cells. (2) The mechanism of the novel Schiff base zinc complex to induce apoptosis is related to the mitochondrial pathway and the death receptor pathway. Conclusion
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R914.5;R96

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