【摘要】：胰腺癌(pancreatic cancer)是一種死亡率極高的惡性腫瘤,因?yàn)樵缙谠\斷困難,所以多數(shù)患者發(fā)病時(shí)已處于癌癥晚期,并往往伴隨著癌癥的轉(zhuǎn)移。目前,以吉西他濱(gemcitabine)為主的化療是治療胰腺癌最主要的手段,但療效甚微。因此,通過(guò)引入新藥或藥物組合來(lái)提高化療效果是一種合理的選擇。DNA損傷應(yīng)答(DNA damage response,DDR)是細(xì)胞內(nèi)一套精細(xì)且復(fù)雜的信號(hào)網(wǎng)絡(luò),為時(shí)刻受到內(nèi)外源DNA損傷壓力的細(xì)胞提供保護(hù)。DDR通路涉及DNA復(fù)制、DNA損傷修復(fù)和細(xì)胞周期檢驗(yàn)點(diǎn)等通路,通過(guò)協(xié)調(diào)這些信號(hào)通路的運(yùn)行,維持基因組的穩(wěn)定性,促進(jìn)細(xì)胞的存活。ATR(ataxia telangiectasia and Rad 3 related)是DDR通路中的關(guān)鍵蛋白,它參與DNA損傷的識(shí)別,并通過(guò)激活CHK1等下游蛋白修復(fù)DNA損傷、緩解DNA復(fù)制壓力和活化細(xì)胞周期檢驗(yàn)點(diǎn)等功能。相比正常細(xì)胞,癌細(xì)胞往往更依賴于ATR活性,因此ATR被認(rèn)為是很有前景的癌癥治療靶點(diǎn)。本論文選用新開(kāi)發(fā)的高效ATR選擇性抑制劑AZ20,考察其單獨(dú)以及與吉西他濱聯(lián)合使用對(duì)胰腺癌細(xì)胞的抗腫瘤活性。在胰腺癌細(xì)胞中的結(jié)果顯示,AZ20能夠抑制細(xì)胞生長(zhǎng),但對(duì)細(xì)胞死亡的影響較小。AZ20能夠抑制ATR下游CHK1-CDC25C-CDK1信號(hào)通路的活性,同時(shí)上調(diào)DNA損傷。這些結(jié)果暗示,AZ20對(duì)細(xì)胞周期檢驗(yàn)點(diǎn)有兩個(gè)方面的影響:抑制ATR所產(chǎn)生的抑制作用和因誘導(dǎo)DNA損傷而產(chǎn)生的激活作用。依據(jù)AZ20能夠誘導(dǎo)S期和G2/M期阻滯的結(jié)果推知,AZ20對(duì)細(xì)胞周期檢驗(yàn)點(diǎn)的凈影響是上調(diào)DNA損傷所導(dǎo)致的激活作用。AZ20對(duì)DNA損傷的誘導(dǎo)將為其與DNA損傷類藥物聯(lián)用提供分子基礎(chǔ)。吉西他濱作為胰腺癌的一線化療藥物已經(jīng)被使用了接近20年,但單獨(dú)使用治療效果非常有限。因?yàn)榧魉麨I的主要作用機(jī)制是誘導(dǎo)細(xì)胞發(fā)生DNA損傷,所以我們嘗試將AZ20與吉西他濱聯(lián)用。結(jié)果表明,AZ20能增加吉西他濱的細(xì)胞毒性作用、削弱吉西他濱誘導(dǎo)的細(xì)胞周期阻滯和增強(qiáng)吉西他濱誘導(dǎo)的細(xì)胞死亡。同時(shí),AZ20能增加吉西他濱誘導(dǎo)的DNA損傷、抑制吉西他濱激活細(xì)胞周期檢驗(yàn)點(diǎn)和下調(diào)吉西他濱誘導(dǎo)的核苷酸還原酶(Ribonucleotide reductase,RNR)表達(dá)。這些結(jié)果指出,AZ20通過(guò)抑制ATR的DNA修復(fù)功能、促進(jìn)細(xì)胞周期運(yùn)行和降低RNR活性這幾個(gè)方面來(lái)上調(diào)吉西他濱誘導(dǎo)的DNA損傷,進(jìn)而增加吉西他濱誘導(dǎo)的細(xì)胞死亡。綜上所述,本論文的研究證明,AZ20能夠增強(qiáng)吉西他濱殺傷胰腺癌細(xì)胞的活性,指出通過(guò)靶向ATR來(lái)增加DNA損傷類藥物的抗胰腺癌活性是一種有效可行的治療策略。本論文的研究為AZ20的臨床應(yīng)用打下堅(jiān)實(shí)的實(shí)驗(yàn)基礎(chǔ),為胰腺癌的臨床治療提供新思路。
[Abstract]:Pancreatic cancer (pancreatic cancer) is a malignant tumor with high mortality. Because of the difficulty of early diagnosis, most of the patients were in advanced stage of cancer and often accompanied by cancer metastasis. Currently, gemcitabine (gemcitabine)-based chemotherapy is the most important treatment for pancreatic cancer, but the efficacy is minimal. Therefore, it is a reasonable choice to improve the effect of chemotherapy by introducing new drugs or combination of drugs. Damage response is a set of fine and complex signal network in cells. Providing protection for cells under constant internal and external DNA damage, the DDR pathway involves pathways such as DNA replication, DNA damage repair and cell cycle verification points, which maintain genomic stability by coordinating the operation of these signaling pathways. ATR (ataxia telangiectasia and Rad3 related is a key protein in the related pathway. It participates in the recognition of DNA damage, and restores the damage by activating downstream proteins such as CHK1, relieving the replication pressure and activating the cell cycle test points. Cancer cells tend to be more dependent on ATR activity than normal cells, so ATR is considered a promising cancer treatment target. In this study, the antitumor activity of AZ20, a highly efficient ATR selective inhibitor, was investigated on pancreatic cancer cells alone and in combination with gemcitabine. The results in pancreatic cancer cells showed that AZ20 could inhibit cell growth, but had little effect on cell death. AZ20 could inhibit the activity of CHK1-CDC25C-CDK1 signaling pathway downstream of ATR and up-regulate DNA damage. These results suggest that AZ20 has two effects on cell cycle test sites: inhibition of ATR and activation of DNA damage. According to the results that AZ20 can induce arrest of S phase and G 2 / M phase, we infer that the net effect of AZ20 on cell cycle test point is that the activation induced by up-regulation of DNA damage .AZ20 induction of DNA damage will provide a molecular basis for the combination of AZ20 and DNA damage drugs. Gemcitabine has been used for nearly 20 years as a first-line chemotherapeutic agent for pancreatic cancer, but the efficacy of gemcitabine alone is very limited. Because the mechanism of gemcitabine is to induce DNA damage in cells, we try to combine AZ20 with gemcitabine. The results showed that AZ20 could increase the cytotoxicity of gemcitabine, weaken the cell cycle arrest induced by gemcitabine and enhance the cell death induced by gemcitabine. At the same time, AZ20 could increase the DNA damage induced by gemcitabine, inhibit the gemcitabine-activated cell cycle test point and down-regulate the expression of ribonucleotide reductase (RNR) induced by gemcitabine. These results suggest that AZ20 upregulated gemcitabine-induced DNA damage by inhibiting the DNA repair function of ATR, promoting cell cycle operation and decreasing RNR activity, and then increased the cell death induced by gemcitabine. To sum up, the present study proves that AZ20 can enhance the activity of gemcitabine in killing pancreatic cancer cells. It is pointed out that targeting ATR to increase the anti-pancreatic cancer activity of DNA-damaging drugs is an effective and feasible therapeutic strategy. The research in this paper lays a solid experimental foundation for the clinical application of AZ20 and provides a new idea for the clinical treatment of pancreatic cancer.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.9

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1 任啟航;AZ20增強(qiáng)吉西他濱殺傷胰腺癌細(xì)胞活性的研究[D];吉林大學(xué);2016年

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本文編號(hào)：2135557