本文選題：卵巢癌　切入點：青蒿琥酯　出處：《山東大學》2014年碩士論文

【摘要】：卵巢癌為婦科生殖器官常見惡性腫瘤之一,死亡率居婦科腫瘤首位。目前,對于卵巢癌的標準治療是最佳減瘤手術配合鉑類化療藥物的組合治療。但由于缺乏有效的早期診斷手段和腫瘤對常規(guī)化療藥物的耐藥性,卵巢癌治療效果并不顯著,如何提高化療藥物對卵巢癌的治療效果,是醫(yī)學上亟待解決的問題。 青蒿琥酯是從中藥青蒿中提取的一種倍半萜內(nèi)酯類化合物,是目前治療重癥瘧疾的首選藥物。近年來的研究表明,青蒿琥酯可以通過升高腫瘤細胞活性氧(ROS)和DNA損傷水平,誘導細胞走向凋亡和細胞周期阻滯等多種途徑,達到抑制腫瘤生長的效果。且相比于野生型的細胞,堿基切除修復(Base excision repair, BER)和同源重組修復(Homologous recombination, HR)缺陷的細胞對青蒿琥酯更為敏感,說明青蒿琥酯誘導的DNA損傷主要由BER和HR途徑完成修復。 同源重組修復可以修復多種DNA損傷,包括雙鏈DNA斷裂和鏈間交聯(lián)。RAD51蛋白是HR修復過程中的關鍵蛋白,負責催化同源DNA鏈間的交換。RAD51在多種腫瘤中高表達,由此所導致的DNA修復能力的增強導致腫瘤細胞對放療、化療和靶向治療等多種治療產(chǎn)生耐受。因而下調(diào)RAD51蛋白表達水平有望會克服腫瘤的化療和放療耐受。本課題研究了青蒿琥酯對卵巢癌細胞的殺傷作用以及青蒿琥酯對RAD51的負調(diào)控作用。結果顯示,青蒿琥酯既能誘導卵巢癌細胞的DNA雙鏈斷裂,又能下調(diào)同源重組蛋白RAD51的表達,后一功能顯著增加了卵巢癌細胞對化療藥物順鉑的敏感性。 [研究目的] 本課題以卵巢癌細胞和小鼠體內(nèi)移植瘤為模型,研究聯(lián)合青蒿琥酯和順鉑的抗腫瘤作用及其機制。 [研究方法] 一、利用CCK-8方法檢測青蒿琥酯對人卵巢癌細胞的增殖抑制作用。 二、利用Annexin V和PI雙染法檢測青蒿琥酯處理卵巢癌細胞的凋亡情況。 三、利用流式細胞術檢測青蒿琥酯處理卵巢癌細胞后ROS水平變化情況。 四、利用平板克隆形成法檢測聯(lián)合青蒿琥酯和順鉑對卵巢癌細胞生長的抑制作用。 五、利用Western blot和Real-time PCR技術檢測青蒿琥酯處理腫瘤細胞和正常細胞RAD51, Parp-1,Ku80, XRCC1等相關損傷修復蛋白表達水平。 六、利用免疫熒光,Western blot方法檢測青蒿琥酯處理后,卵巢癌細胞γ-H2AX的焦點形成及蛋白表達。 七、利用免疫熒光檢測青蒿琥酯預處理卵巢癌細胞對RAD51焦點形成的影響。 八、利用裸鼠的體內(nèi)卵巢癌移植瘤模型,評估聯(lián)合青蒿琥酯和順鉑治療卵巢癌的效果。 九、利用RAD51高表達的卵巢癌細胞系,進一步確定青蒿琥酯的化療增敏作用由RAD51下調(diào)介導。 十、利用熒光素酶報告基因實驗檢測青蒿琥酯對RAD51啟動子活性的影響。 [實驗結果] 一、青蒿琥酯通過誘導卵巢癌細胞的凋亡抑制細胞增殖,且具有劑量依賴性。 二、青蒿琥酯誘導卵巢癌ROS水平的上升和DNA雙鏈斷裂的產(chǎn)生。 三、青蒿琥酯下調(diào)腫瘤細胞內(nèi)同源重組蛋白RAD51的表達,對正常細胞RAD51蛋白表達沒有影響。 四、青蒿琥酯處理下腫瘤細胞RAD51焦點形成受阻。 五、青蒿琥酯能增加順鉑對卵巢癌細胞的殺傷作用,聯(lián)合青蒿琥酯和順鉑對卵巢癌抑制作用具有明顯的協(xié)同效應。 六、異位高表達RAD51可降低聯(lián)合青蒿琥酯和順鉑對卵巢癌的殺傷作用。 七、青蒿琥酯增加體內(nèi)順鉑治療卵巢癌效果,聯(lián)合用藥明顯抑制了卵巢癌移植瘤的生長。 [結論] 青蒿琥酯既可誘導卵巢癌細胞ROS水平上升,DNA雙鏈斷裂的產(chǎn)生,也可下調(diào)RAD51蛋白的表達。青蒿琥酯可增加順鉑治療卵巢癌的效果。
[Abstract]:Ovarian cancer is one of the most common gynecological genital malignant tumor mortality in gynecological tumor first. At present, the standard treatment for ovarian cancer is the optimal cytoreductive surgery combined with drugs for the treatment of platinum chemotherapy. But due to the lack of effective means of early diagnosis of tumors and to the development of drug resistance of ovarian cancer, the treatment effect is not significant, how to to improve the therapeutic effect of chemotherapy for ovarian cancer, is the urgent need to solve medical problems.
Artesunate is a sesquiterpene lactone compound extracted from Artemisia annua L in, is the preferred drug for treatment of severe malaria at present. Recent studies show that artesunate can be increased through the tumor cells of reactive oxygen species (ROS) and DNA damage levels, a variety of ways to induce cell apoptosis and cell cycle arrest, inhibit tumor growth results. Compared with the wild type cells, base excision repair (Base excision, repair, BER) and homologous recombination repair (Homologous recombination, HR) - deficient cells more sensitive to artesunate, indicating that DNA damage induced by artesunate is composed of BER and HR way to complete the repair.
Homologous recombination repair can repair a variety of DNA, including DNA double strand breaks and chain crosslinking between.RAD51 protein is a key protein in HR repair process, responsible for the catalytic.RAD51 DNA chain homologous exchange between high expression in a variety of tumors, enhance DNA repair capacity resulting from the cause of tumor cells to radiotherapy, chemotherapy and targeted the treatment of various treatment tolerance. So the downregulation of the expression level of RAD51 protein is expected to overcome the resistance of tumor chemotherapy and radiotherapy. In this study the negative regulation effect of artesunate on ovarian cancer cells and the cytotoxic effect of artesunate on RAD51. The results showed that artesunate can induce ovarian cancer cells to DNA double strand breaks, and can decrease the expression of homologous recombination protein RAD51, a function significantly increased the sensitivity of ovarian cancer cells to cisplatin.
[research purposes]
In this study, the anti-tumor effects and mechanisms of artesunate and cisplatin were studied by using ovarian cancer cells and transplanted tumor in mice as a model.
[research methods]
First, the inhibitory effect of artesunate on the proliferation of human ovarian cancer cells was detected by CCK-8 method.
Two, the apoptosis of artesunate treated ovarian cancer cells was detected by Annexin V and PI double staining.
Three, the changes of ROS level after artesunate treatment of ovarian cancer cells were detected by flow cytometry.
Four, the inhibitory effect of artesunate and cisplatin on the growth of ovarian cancer cells was detected by the method of plate clone formation.
Five, we used Western blot and Real-time PCR to detect the expression levels of RAD51, Parp-1, Ku80, XRCC1 and other related damage repair proteins in artesunate treated tumor cells and normal cells.
Six, immunofluorescence and Western blot were used to detect the focal formation and protein expression of gamma -H2AX in ovarian cancer cells after artesunate treatment.
Seven, the effects of artesunate pretreated ovarian cancer cells on the formation of RAD51 focus were detected by immunofluorescence.
Eight, the effect of artesunate and cisplatin in the treatment of ovarian cancer was evaluated by using the tumor model of human ovarian cancer in nude mice.
Nine, using RAD51 highly expressed ovarian cancer cell lines, it was further determined that the chemosensitivity of artesunate was mediated by the downregulation of RAD51.
Ten, the effect of artesunate on the activity of RAD51 promoter was detected by the luciferase reporter gene test.
[experimental results]
First, artesunate inhibits cell proliferation by inducing apoptosis in ovarian cancer cells and has a dose-dependent manner.
Two, artesunate induced the rise of ROS level in ovarian cancer and the production of DNA double strand breaks.
Three, artesunate downregulated the expression of homologous recombinant protein RAD51 in tumor cells, and had no effect on the expression of RAD51 protein in normal cells.
Four, the formation of RAD51 focus in the tumor cells was blocked by artesunate treatment.
Five, artesunate can increase the killing effect of cisplatin on ovarian cancer cells. The combination of artesunate and cisplatin has a significant synergistic effect on the inhibition of ovarian cancer.
Six, heterotopic high expression of RAD51 could reduce the killing effect of artesunate and cisplatin on ovarian cancer.
Seven, artesunate increased the effect of cisplatin in the treatment of ovarian cancer, and the combined use of drugs significantly inhibited the growth of ovarian cancer xenografts.
[Conclusion]
Artesunate can induce the rise of ROS level and the production of DNA double strand breaks, and down regulate the expression of RAD51 protein. Artesunate can increase the effect of cisplatin in the treatment of ovarian cancer.

【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.31

【共引文獻】

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