青蒿琥酯通过下调同源重组蛋白RAD51增加卵巢癌细胞对化疗药物顺铂的敏感性

发布时间：2018-04-03 09:40

本文选题：卵巢癌　切入点：青蒿琥酯　出处：《山东大学》2014年硕士论文

【摘要】：卵巢癌为妇科生殖器官常见恶性肿瘤之一,死亡率居妇科肿瘤首位。目前,对于卵巢癌的标准治疗是最佳减瘤手术配合铂类化疗药物的组合治疗。但由于缺乏有效的早期诊断手段和肿瘤对常规化疗药物的耐药性,卵巢癌治疗效果并不显著,如何提高化疗药物对卵巢癌的治疗效果,是医学上亟待解决的问题。青蒿琥酯是从中药青蒿中提取的一种倍半萜内酯类化合物,是目前治疗重症疟疾的首选药物。近年来的研究表明,青蒿琥酯可以通过升高肿瘤细胞活性氧(ROS)和DNA损伤水平,诱导细胞走向凋亡和细胞周期阻滞等多种途径,达到抑制肿瘤生长的效果。且相比于野生型的细胞,碱基切除修复(Base excision repair, BER)和同源重组修复(Homologous recombination, HR)缺陷的细胞对青蒿琥酯更为敏感,说明青蒿琥酯诱导的DNA损伤主要由BER和HR途径完成修复。同源重组修复可以修复多种DNA损伤,包括双链DNA断裂和链间交联。RAD51蛋白是HR修复过程中的关键蛋白,负责催化同源DNA链间的交换。RAD51在多种肿瘤中高表达,由此所导致的DNA修复能力的增强导致肿瘤细胞对放疗、化疗和靶向治疗等多种治疗产生耐受。因而下调RAD51蛋白表达水平有望会克服肿瘤的化疗和放疗耐受。本课题研究了青蒿琥酯对卵巢癌细胞的杀伤作用以及青蒿琥酯对RAD51的负调控作用。结果显示,青蒿琥酯既能诱导卵巢癌细胞的DNA双链断裂,又能下调同源重组蛋白RAD51的表达,后一功能显著增加了卵巢癌细胞对化疗药物顺铂的敏感性。 [研究目的] 本课题以卵巢癌细胞和小鼠体内移植瘤为模型,研究联合青蒿琥酯和顺铂的抗肿瘤作用及其机制。 [研究方法] 一、利用CCK-8方法检测青蒿琥酯对人卵巢癌细胞的增殖抑制作用。二、利用Annexin V和PI双染法检测青蒿琥酯处理卵巢癌细胞的凋亡情况。三、利用流式细胞术检测青蒿琥酯处理卵巢癌细胞后ROS水平变化情况。四、利用平板克隆形成法检测联合青蒿琥酯和顺铂对卵巢癌细胞生长的抑制作用。五、利用Western blot和Real-time PCR技术检测青蒿琥酯处理肿瘤细胞和正常细胞RAD51, Parp-1,Ku80, XRCC1等相关损伤修复蛋白表达水平。六、利用免疫荧光,Western blot方法检测青蒿琥酯处理后,卵巢癌细胞γ-H2AX的焦点形成及蛋白表达。七、利用免疫荧光检测青蒿琥酯预处理卵巢癌细胞对RAD51焦点形成的影响。八、利用裸鼠的体内卵巢癌移植瘤模型,评估联合青蒿琥酯和顺铂治疗卵巢癌的效果。九、利用RAD51高表达的卵巢癌细胞系,进一步确定青蒿琥酯的化疗增敏作用由RAD51下调介导。十、利用荧光素酶报告基因实验检测青蒿琥酯对RAD51启动子活性的影响。 [实验结果] 一、青蒿琥酯通过诱导卵巢癌细胞的凋亡抑制细胞增殖,且具有剂量依赖性。二、青蒿琥酯诱导卵巢癌ROS水平的上升和DNA双链断裂的产生。三、青蒿琥酯下调肿瘤细胞内同源重组蛋白RAD51的表达,对正常细胞RAD51蛋白表达没有影响。四、青蒿琥酯处理下肿瘤细胞RAD51焦点形成受阻。五、青蒿琥酯能增加顺铂对卵巢癌细胞的杀伤作用,联合青蒿琥酯和顺铂对卵巢癌抑制作用具有明显的协同效应。六、异位高表达RAD51可降低联合青蒿琥酯和顺铂对卵巢癌的杀伤作用。七、青蒿琥酯增加体内顺铂治疗卵巢癌效果,联合用药明显抑制了卵巢癌移植瘤的生长。 [结论] 青蒿琥酯既可诱导卵巢癌细胞ROS水平上升,DNA双链断裂的产生,也可下调RAD51蛋白的表达。青蒿琥酯可增加顺铂治疗卵巢癌的效果。
[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.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

【共引文献】