聯(lián)鄰苯二酚結(jié)構(gòu)的DNA交聯(lián)劑的設(shè)計合成研究及其對黑素腫瘤細胞的選擇性殺傷作用
發(fā)布時間：2018-12-27 13:54
【摘要】：DNA作為生物體中遺傳信息的主要載體在遺傳信息的傳遞中車關(guān)重要,以DNA為靶目標的抗癌藥物的設(shè)計一直是藥物研究的熱點之一。其中,許多臨床上使用的抗腫瘤藥物的作用機理涉及到DNA的交聯(lián)。它們通過與DNA的共價結(jié)合作用將DNA雙鏈交聯(lián),阻礙DNA雙鏈的打開,使之不能進行遺傳信息的復(fù)制和傳遞,從而導致細胞的死亡達到治療癌癥的目的�？烧T導的DNA交聯(lián)劑的引入,增強了藥物的靶向性和選擇性。 本論文設(shè)計合成了十二個含不同連接基團的聯(lián)鄰苯二酚結(jié)構(gòu)的化合物,通過高碘酸鈉或者酪氨酸酶的氧化誘導,研究了它們與DNA的交聯(lián)活性。同時,鑒于惡性黑素瘤細胞中酪氨酸酶的含量高表達,我們建立了一種以聯(lián)鄰苯二酚結(jié)構(gòu)的DNA交聯(lián)劑選擇性的殺傷黑素瘤細胞的方法。具體研究內(nèi)容涵蓋以下幾點： 1.首先在體外實驗中,我們用瓊脂糖凝膠電泳實驗檢測了化合物與線性DNA交聯(lián)的能力。在高碘酸鈉的氧化誘導下,兩個酚羥基在鄰位的化合物7a-f都表現(xiàn)出了很強的DNA交聯(lián)能力,而兩個酚羥基在間位的化合物10a-f交聯(lián)能力很差。在酪氨酸酶的氧化誘導下,所有的化合物均表現(xiàn)出DNA交聯(lián)能力,兩個酚羥基在間位的化合物10a-f比兩個酚羥基在鄰位的化合物7a-f的交聯(lián)能力略好。并且,中間連有聯(lián)苯結(jié)構(gòu)的化合物10f交聯(lián)活性最好。另外,我們通過3-甲基-2-苯并噻唑酮腙(MBTH)的顏色實驗,捕捉到這類化合物在酪氨酸酶氧化誘導后有二醌的中間體的產(chǎn)生,初步提出了交聯(lián)機理。 2.我們進一步對此類聯(lián)鄰苯二酚化合物的抗癌活性進行了研究。我們用MTT法對藥物的細胞毒性進行了研究。細胞毒性實驗表明此類聯(lián)鄰苯二酚化合物對酪氨酸酶高表達的惡性黑素瘤細胞的毒性非常明顯,而對不含有酪氨酸酶的宮頸癌細胞Hela和正常卵巢細胞CHO毒性很弱。我們通過一系列的分了生物學的方法,包括在細胞內(nèi)檢測鄰苯二酚結(jié)構(gòu)的BODIPY熒光探針實驗,單細胞凝膠電泳實驗,γ-H2AX免疫熒光實驗以及通過觀測細胞核形態(tài)變化,驗證了化合物對惡性黑素瘤細胞的選擇性殺傷力主要是通過DNA損傷起作用。更重要的是,這些DNA損傷與DNA交聯(lián)有關(guān)。聯(lián)鄰二苯酚的化合物是因為能與惡性黑色素瘤細胞的細胞核中的DNA發(fā)生交聯(lián),而導致了它具有高選擇性的殺傷惡性黑色素瘤細胞的能力。
[Abstract]:As the main carrier of genetic information in organisms, DNA plays an important role in the transmission of genetic information. The design of anticancer drugs targeting DNA has been one of the hot spots in drug research. Among them, many clinical use of anti-tumor drugs related to the mechanism of cross-linking of DNA. By covalent binding with DNA, DNA double strands are linked to each other, which hinders the opening of DNA double strands and makes them unable to replicate and transmit genetic information, thus leading to cell death to achieve the purpose of cancer treatment. The introduction of inducible DNA crosslinker enhanced the targeting and selectivity of the drug. In this paper, twelve dihydroquinone compounds with different connective groups were designed and synthesized, and their crosslinking activities with DNA were studied by oxidation induction of sodium periodate or tyrosinase. At the same time, in view of the high expression of tyrosinase in malignant melanoma cells, we established a selective killing method of melanoma cells with DNA crosslinking agent with dicatechol structure. Specific research covers the following points: 1. Firstly, in vitro, agarose gel electrophoresis was used to test the crosslinking ability of the compounds to linear DNA. Under the oxidation induction of sodium periodate, two phenolic hydroxyl groups exhibited strong DNA crosslinking ability in the ortho compound 7a-f, while the two phenolic hydroxyl groups in the intermediate position compound 10a-f crosslinking ability was very poor. Under the oxidation of tyrosinase, all the compounds showed DNA crosslinking ability. The crosslinking ability of two phenolic hydroxyl compounds in the intermediate position was slightly better than that of two phenolic hydroxyl groups in the ortho position compound 7a-f. Moreover, the crosslinking activity of 10 f compound with intermediate biphenyl structure is the best. In addition, through the color experiment of 3-methyl-2-benzothiazolone Hydrazone (MBTH), we have captured the production of diquinone intermediate after the oxidation of tyrosinase, and put forward the crosslinking mechanism. 2. We further studied the anticancer activity of this kind of catechol compounds. The cytotoxicity of the drug was studied by MTT method. The cytotoxicity test showed that the toxicity of this kind of catechol compounds to malignant melanoma cells with high tyrosinase expression was very obvious, but it was weak to Hela cells without tyrosinase and CHO from normal ovarian cells. We have adopted a series of biological methods, including BODIPY fluorescence probe assay for the detection of catechol structure in cells, single cell gel electrophoresis, 緯-H2AX immunofluorescence assay and nuclear morphological changes. The selective cytotoxicity of the compounds to malignant melanoma cells was confirmed mainly through DNA damage. More importantly, these DNA damage is related to DNA crosslinking. The compound of o-diphenol is capable of crosslinking with DNA in the nucleus of malignant melanoma cells, which leads to its highly selective ability to kill malignant melanoma cells.
【學位授予單位】：武漢大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R739.5
[Abstract]:As the main carrier of genetic information in organisms, DNA plays an important role in the transmission of genetic information. The design of anticancer drugs targeting DNA has been one of the hot spots in drug research. Among them, many clinical use of anti-tumor drugs related to the mechanism of cross-linking of DNA. By covalent binding with DNA, DNA double strands are linked to each other, which hinders the opening of DNA double strands and makes them unable to replicate and transmit genetic information, thus leading to cell death to achieve the purpose of cancer treatment. The introduction of inducible DNA crosslinker enhanced the targeting and selectivity of the drug. In this paper, twelve dihydroquinone compounds with different connective groups were designed and synthesized, and their crosslinking activities with DNA were studied by oxidation induction of sodium periodate or tyrosinase. At the same time, in view of the high expression of tyrosinase in malignant melanoma cells, we established a selective killing method of melanoma cells with DNA crosslinking agent with dicatechol structure. Specific research covers the following points: 1. Firstly, in vitro, agarose gel electrophoresis was used to test the crosslinking ability of the compounds to linear DNA. Under the oxidation induction of sodium periodate, two phenolic hydroxyl groups exhibited strong DNA crosslinking ability in the ortho compound 7a-f, while the two phenolic hydroxyl groups in the intermediate position compound 10a-f crosslinking ability was very poor. Under the oxidation of tyrosinase, all the compounds showed DNA crosslinking ability. The crosslinking ability of two phenolic hydroxyl compounds in the intermediate position was slightly better than that of two phenolic hydroxyl groups in the ortho position compound 7a-f. Moreover, the crosslinking activity of 10 f compound with intermediate biphenyl structure is the best. In addition, through the color experiment of 3-methyl-2-benzothiazolone Hydrazone (MBTH), we have captured the production of diquinone intermediate after the oxidation of tyrosinase, and put forward the crosslinking mechanism. 2. We further studied the anticancer activity of this kind of catechol compounds. The cytotoxicity of the drug was studied by MTT method. The cytotoxicity test showed that the toxicity of this kind of catechol compounds to malignant melanoma cells with high tyrosinase expression was very obvious, but it was weak to Hela cells without tyrosinase and CHO from normal ovarian cells. We have adopted a series of biological methods, including BODIPY fluorescence probe assay for the detection of catechol structure in cells, single cell gel electrophoresis, 緯-H2AX immunofluorescence assay and nuclear morphological changes. The selective cytotoxicity of the compounds to malignant melanoma cells was confirmed mainly through DNA damage. More importantly, these DNA damage is related to DNA crosslinking. The compound of o-diphenol is capable of crosslinking with DNA in the nucleus of malignant melanoma cells, which leads to its highly selective ability to kill malignant melanoma cells.
【學位授予單位】：武漢大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R739.5
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