本文選題：PARP抑制劑 + HR��； 參考：《南昌大學(xué)》2017年碩士論文

【摘要】：BRCA1/2是目前臨床應(yīng)用最為廣泛的聚腺苷二磷酸核糖聚合酶(poly(ADP-ribose)polymerase,PARP)抑制劑生物標(biāo)志物。然而對BRCA1/2突變的腫瘤患者,PARP抑制劑的的臨床有效率僅為30%~50%,意味著過半攜帶BRCA缺陷的患者仍面臨著該類藥物的無效治療。探索發(fā)現(xiàn)新的療效預(yù)測標(biāo)志物或標(biāo)志物組合,可用以增加PARP抑制劑臨床治療的成功率。本研究發(fā)現(xiàn)53BP1聯(lián)合BRCA1可作為預(yù)測PARP抑制劑敏感性的生物標(biāo)志物。在比較四種同源重組(homologous recombination,HR)相關(guān)基因的mRNA水平和PARP抑制劑敏感性的基礎(chǔ)上,我們選取BRCA1缺陷的乳腺癌細(xì)胞MDA-MB-436作為工具細(xì)胞株,進(jìn)行RNA干擾實驗。發(fā)現(xiàn)只有降低53BP1表達(dá),才影響細(xì)胞對PARP抑制劑的敏感性。接著我們構(gòu)建了53BP1-/-/BRCA1-/-雙缺陷細(xì)胞株,發(fā)現(xiàn)其Simmiparib的IC50是親本細(xì)胞的36.7倍,表明敲除53BP1會導(dǎo)致PARP抑制劑耐藥。53BP1缺失也減少了PARP抑制劑誘導(dǎo)的細(xì)胞周期阻滯和凋亡,并部分恢復(fù)了HR修復(fù)功能。更重要的是,敲除53BP1減弱了PARP抑制劑對裸鼠移植瘤的生長抑制作用。在雙缺陷的細(xì)胞中再表達(dá)53BP1,可以恢復(fù)PARP抑制劑的敏感性,并恢復(fù)HR相關(guān)因子至正常水平。由于超過10%的BRCA1突變的乳腺癌和卵巢癌病人不表達(dá)53BP1,因此上述發(fā)現(xiàn)提示將53BP1和BRCA1作為生物標(biāo)志物組合用于患者選擇中,有可能減少PARP抑制劑的無效治療。BRCA1/2缺陷的細(xì)胞因HR修復(fù)功能異常,對PARP抑制劑敏感。雖然PARP抑制劑對BRCA1/2突變的腫瘤可能有效,但最終仍會面臨耐藥的產(chǎn)生,從而限制其應(yīng)用。由于PARP抑制劑臨床應(yīng)用時間短,對其耐藥特性和機制了解還十分有限。為了深入研究PARP抑制劑的耐藥特點和機制,我們以MDA-MB-436細(xì)胞為模型,采用梯度遞增法構(gòu)建了四種PARP抑制劑耐藥單克隆細(xì)胞株。這些耐藥株對同類PARP抑制劑明顯耐藥,也對DNA交聯(lián)劑和拓?fù)洚悩?gòu)酶Ι抑制劑交叉耐藥。另外,PARP抑制劑幾乎不影響耐藥株的周期分布和細(xì)胞凋亡。我們采用特異性抗體研究發(fā)現(xiàn)耐藥株表達(dá)截短型的N-端BRCA1蛋白,提示PARP抑制劑的耐藥機制與BRCA1發(fā)生繼發(fā)性突變導(dǎo)致HR修復(fù)功能部分恢復(fù)相關(guān)。
[Abstract]:BRCA1/2 is the most widely used biomarker of polyadenosine diphosphate polymerase (BRCA1/2) inhibitor. However, the clinical efficacy of BRCA1/2 inhibitor in patients with BRCA1/2 mutation is only 30%, which means that more than half of the patients with BRCA deficiency still face the ineffective treatment of the drug. To explore new prognostic markers or combination of markers, which can be used to increase the success rate of clinical treatment of PARP inhibitors. In this study, we found that 53BP1 combined with BRCA1 can be used as a biomarker for predicting the sensitivity of PARP inhibitors. On the basis of comparing the mRNA level and the sensitivity of PARP inhibitor of four homologous recombination genes, we selected BRCA1 deficient breast cancer cell line MDA-MB-436 as the tool cell line and carried out RNA interference test. It was found that the sensitivity of cells to PARP inhibitors was affected only when the expression of 53BP1 was reduced. Then we constructed 53BP1-r BRCA1-r-double defective cell line and found that the IC50 of Simmiparib was 36.7 times that of parent cells, suggesting that knockout of 53BP1 led to the deletion of PARP inhibitor resistance. 53BP1 also reduced cell cycle arrest and apoptosis induced by PARP inhibitor. HR repair function was partially restored. More importantly, knockout of 53BP1 attenuates the growth inhibition of PARP inhibitors on xenografts in nude mice. The expression of 53BP1 in double deficient cells could restore the sensitivity of PARP inhibitor and restore HR related factors to normal level. Since more than 10% of breast and ovarian cancer patients with BRCA1 mutations do not express 53BP1, the findings suggest that 53BP1 and BRCA1 are combined as biomarkers for patient selection. Cells that may reduce ineffective treatment of PARP inhibitors. BRCA 1 / 2 defect are sensitive to PARP inhibitors because of abnormal HR repair function. Although PARP inhibitors may be effective against BRCA1/2 mutated tumors, they may eventually face the production of drug resistance, limiting their use. Due to the short clinical application time of PARP inhibitors, their resistance characteristics and mechanisms are still very limited. In order to study the characteristics and mechanism of drug resistance of PARP inhibitors, four PARP inhibitor resistant monoclonal cell lines were constructed using MDA-MB-436 cell model and gradient increasing method. These resistant strains were significantly resistant to similar PARP inhibitors and cross-resistant to DNA crosslinking agents and topoisomerase I inhibitors. In addition, PARP inhibitors have little effect on cell cycle distribution and apoptosis. The expression of truncated N- terminal BRCA1 protein was detected by specific antibody analysis, suggesting that the mechanism of drug resistance of PARP inhibitor is related to the partial recovery of HR repair function due to secondary mutation of BRCA1.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R979.1

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