【摘要】：研究背景骨腫瘤的發(fā)病率并不高,在常見(jiàn)腫瘤中僅占2%左右,但以我國(guó)高達(dá)13億人口的統(tǒng)計(jì)基數(shù)而論,骨腫瘤的患病人群仍已達(dá)到一個(gè)不容忽視的規(guī)模。同時(shí),一些高發(fā)腫瘤如肺癌、乳腺癌、前列腺癌等容易并發(fā)腫瘤的骨轉(zhuǎn)移,因此實(shí)際上總的骨腫瘤患病人數(shù)仍然相當(dāng)龐大。目前,對(duì)骨腫瘤的治療已經(jīng)從單純的外科手術(shù)逐步向綜合治療的方向發(fā)展,尤其是新型化療藥物的出現(xiàn),將骨腫瘤的治療帶入了一個(gè)新的時(shí)代。早在上世紀(jì)60年代,人們就已經(jīng)發(fā)現(xiàn)希夫堿具有抗腫瘤、抗菌、催化活性等特性,近年來(lái)有學(xué)者研制出希夫堿的鉑、銅等金屬配合物,并顯示出強(qiáng)大的抗腫瘤作用。本研究中使用一種新型的希夫堿鋅配合物并研究其對(duì)骨肉瘤以及常見(jiàn)骨轉(zhuǎn)移瘤-乳腺癌的抑制作用及其抗腫的作用機(jī)制,為骨腫瘤的治療提供一種新的可能方案。目的制備一種全新的希夫堿鋅配合物并研究其對(duì)MG-63人骨肉瘤細(xì)胞及MCF-7人乳腺癌細(xì)胞的殺傷作用并對(duì)其可能機(jī)制進(jìn)行研究。方法(1)新型希夫堿鋅配合物的合成。(2)CCK-8法測(cè)定新型希夫堿鋅配合物對(duì)體外培養(yǎng)的上述兩種細(xì)胞的增值抑制作用;采用Annexin V-PI雙染法分析新型希夫堿鋅配合物誘導(dǎo)兩種細(xì)胞的凋亡率以及對(duì)細(xì)胞周期的影響,通過(guò)顯微鏡觀察凋亡細(xì)胞的形態(tài)。(3)瓊脂糖凝膠電泳檢測(cè)DNA熒光強(qiáng)度;運(yùn)用流式細(xì)胞儀以檢測(cè)受試細(xì)胞線粒體的膜電位變化;采用Western Blot技術(shù)檢測(cè)凋亡相關(guān)蛋白Bax、Bcl-2、caspase-9、Cyt-c、Fas、Fas-L、caspa-8以及caspase-3表達(dá)變化。觀察新型希夫堿鋅配合物作用后裸鼠體重及體表腫瘤體積的變化。RT-PCR檢測(cè)裸鼠腫瘤中相關(guān)凋亡基因的表達(dá)。結(jié)果(1)利用水楊醛、水楊酰肼、醋酸鋅經(jīng)水浴加熱回流及沉淀最終形成新型希夫堿鋅配合物;(2)CCK-8法檢測(cè)細(xì)胞存活率實(shí)驗(yàn)結(jié)果顯示,隨著新型希夫堿鋅配合物濃度的增高以及給藥時(shí)間的延長(zhǎng),對(duì)MG-63人骨肉瘤細(xì)胞和MCF-7人乳腺癌細(xì)胞的增殖抑制作用逐漸增強(qiáng)。Annexin V-FITC法檢測(cè)細(xì)胞凋亡率中新型希夫堿鋅配合物能夠誘導(dǎo)MG-63人骨肉瘤細(xì)胞凋亡,與對(duì)照組相比,10μmol/L、50μmol/L、70μmol/L的新型希夫堿鋅配合物作用于MG-63人骨肉瘤細(xì)胞4h、24h、48h后表現(xiàn)出明顯的誘導(dǎo)細(xì)胞凋亡的作用(P0.05)。新型希夫堿鋅配合物誘導(dǎo)MG-63人骨肉瘤細(xì)胞凋亡同樣具有時(shí)間依賴(lài)性。各組中當(dāng)新型希夫堿鋅配合物濃度70μmol/L作用48h誘導(dǎo)細(xì)胞凋亡的比例最高達(dá)29.79±1.36%,且P0.05。同樣的對(duì)于MCF-7人乳腺癌細(xì)胞也有相同的趨勢(shì),其中濃度70μmol/L作用48h誘導(dǎo)細(xì)胞凋亡的比例最高達(dá)30.29±1.77%,且P0.05。流式細(xì)胞儀檢測(cè)周期的實(shí)驗(yàn)中發(fā)現(xiàn)新型希夫堿鋅配合物能夠使兩種腫瘤細(xì)胞的生長(zhǎng)周期停滯與G1期,導(dǎo)致細(xì)胞不能由G1期向S期以及G2期移行,同時(shí)S期細(xì)胞的減少,也使腫瘤細(xì)胞的增值能力進(jìn)一步降低,抑制腫瘤細(xì)胞的生長(zhǎng)。通過(guò)倒置顯微鏡觀察新型希夫堿鋅配合物處理后的兩種細(xì)胞呈現(xiàn)凋亡的形態(tài)學(xué)特征。(3)Western Blot檢測(cè)結(jié)果顯示,不同濃度的新型希夫堿鋅配合物作用于MG-63人骨肉瘤細(xì)胞一段時(shí)間后,隨新型希夫堿鋅配合物濃度增加bax/bcl-2比值也逐漸增加。線粒體中的Cyt-c下降,胞漿中的Cyt-c上升。具有明顯的濃度依賴(lài)性。并出現(xiàn)了明顯的Cleave caspase-9和3條帶。實(shí)驗(yàn)中隨時(shí)間的延長(zhǎng),細(xì)胞中的Bax/Bcl-2的比值為增加。線粒體內(nèi)的Cyt-c減少。胞漿中的Cyt-c增加。具有明顯的時(shí)間依賴(lài)性。并出現(xiàn)了明顯的Cleave caspase-9和3條帶。在Fas、Fas-L中隨希夫堿濃度的增加灰度比值分別從0.48±0.06、增加到1.83±0.90以及從0.29±0.02增加到0.85±0.07�？梢�(jiàn)具有明顯的濃度依賴(lài)性,并出現(xiàn)了清晰的Cleaved caspase-8和3條帶。隨著時(shí)間的延長(zhǎng),在Fas、Fas-L的灰度比值也增大。具有明顯的時(shí)間依賴(lài)性,也出現(xiàn)了清晰的Cleaved caspase-8和3條帶。在MCF-7細(xì)胞中也出現(xiàn)了相似的結(jié)果,隨新型希夫堿鋅配合物濃度增加Bax/Bcl-2的比值上升,線粒體中的Cyt-c減少,胞漿中的Cyt-c增加,具有明顯的濃度依賴(lài)性。并出現(xiàn)了明顯的Cleave caspase-9和3條帶。隨作用時(shí)間的延長(zhǎng)Bax/Bcl-2的比值增加。線粒體內(nèi)的Cyt-c減少,胞漿中的Cyt-c增加,具有明顯的時(shí)間依賴(lài)性。并出現(xiàn)了明顯的Cleave caspase-9和3條帶。Fas、Fas-L中隨新型希夫堿鋅配合物濃度增加灰度比值增加�？梢�(jiàn)具有明顯的濃度依賴(lài)性,并出現(xiàn)了清晰的Cleaved caspase-8和3條帶。隨著時(shí)間的延長(zhǎng),在Fas、Fas-L灰度比值增加,具有明顯的時(shí)間依賴(lài)性,也出現(xiàn)了清晰的Cleaved caspase-8和3條帶。(4)裸鼠體內(nèi)的抗腫瘤作用:新型希夫堿鋅配合物能顯著抑制裸鼠MG-63、MCF-7轉(zhuǎn)移瘤的生長(zhǎng),且無(wú)肝腎功能的損傷。新型希夫堿鋅配合物作用腫瘤后期中的Bax、Bcl-2、Caspase-8、Caspase-9、Caspase-3的m RNA表達(dá)隨新型希夫堿鋅配合物濃度的變化而變化,具有一定的量效關(guān)系。結(jié)論(1)新型希夫堿鋅配合物能夠誘導(dǎo)MG-63人骨肉瘤細(xì)胞及常見(jiàn)骨轉(zhuǎn)移瘤MCF-7人乳腺癌細(xì)胞凋亡;(2)新型希夫堿鋅配合物誘導(dǎo)細(xì)胞凋亡的機(jī)制與線粒體途徑及死亡受體途徑相關(guān)。結(jié)論
[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
【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】：2015
【分類(lèi)號(hào)】：R914.5;R96

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4 王強(qiáng);蛋氨酸類(lèi)希夫堿配合物的合成、表征與生物活性研究[D];中國(guó)海洋大學(xué);2011年

5 左健;氨基酸類(lèi)希夫堿配合物的合成、表征及生物活性研究[D];中國(guó)海洋大學(xué);2013年

6 閆明;新型希夫堿鋅配合物的合成及其抗骨腫瘤活性的研究[D];吉林大學(xué);2015年

7 范玉華;新型希夫堿金屬配合物的合成、表征與應(yīng)用研究[D];中國(guó)原子能科學(xué)研究院;2004年

8 趙干卿;希夫堿類(lèi)銅配合物的合成、結(jié)構(gòu)表征及其催化酚類(lèi)氧化研究[D];江蘇大學(xué);2007年

9 艾小康;新型希夫堿金屬配合物的合成、表征及熒光特性研究[D];中國(guó)海洋大學(xué);2007年

10 張鵬飛;吲哚-2，3-二酮類(lèi)希夫堿配合物的合成表征與生物活性研究[D];中國(guó)海洋大學(xué);2014年

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