本文選題：膠質(zhì)母細胞瘤 + 膠質(zhì)瘤起始細胞　； 參考：《廣州醫(yī)科大學》2014年碩士論文

【摘要】：膠質(zhì)母細胞瘤（glioblastoma multiforme，GBM）是成人最常見的原發(fā)性腦腫瘤，雖然以Stupp標準治療方法使膠質(zhì)瘤的療效有了明顯提高，但其5年生存率僅有9.8%[1]。目前替莫唑胺（temozolomide，TMZ）是膠質(zhì)瘤的特效化療藥物，使得新診斷的GBM中位生存期由12.1個月提高至14.6個月[2]，但是由于內(nèi)源性和獲得性耐藥，TMZ化療作用仍不理想。膠質(zhì)瘤起始細胞（GliomaInitiating Cells,GIC）是腫瘤中存在的一小部分細胞，這些細胞具有自我更新的能力，分化能力，腫瘤形成能力。這些細胞被認為可能是腫瘤形成的根源，比其他分化的腫瘤細胞對放化療更加耐藥，是導致腫瘤復發(fā)的根源[3]。因此，尋找增敏TMZ的藥物并探討化療耐藥的機制成為當前迫切的任務。目前，有許多針對腫瘤化療耐藥的藥物研究，其中有發(fā)現(xiàn)中國的傳統(tǒng)藥物雷公藤甲素（triptolide,TPL）對多種腫瘤包括膠質(zhì)瘤[4,5]、乳腺癌、胃癌、膀胱癌等[6]均有抗癌作用，其抗癌活性強。并有研究發(fā)現(xiàn)TPL能增加多種化療藥物的敏感性[7]。本研究擬探討雷公藤甲素是否能增敏替莫唑胺殺傷膠質(zhì)瘤細胞及膠質(zhì)瘤起始細胞，并探討其作用機制，揭示膠質(zhì)母細胞瘤對TMZ的化療耐藥的可能機制及雷公藤甲素的治療意義。 第一部分：雷公藤甲素增敏替莫唑胺殺傷膠質(zhì)瘤細胞 目的：探討雷公藤甲素是否能增加替莫唑胺殺傷膠質(zhì)瘤細胞的效應及其可能機制。 方法：使用CCK-8法測定TPL與TMZ對膠質(zhì)瘤細胞增殖的影響；采用Chou Talalay法評估TPL與TMZ的聯(lián)合效應；分別應用流式細胞儀檢測細胞周期技術(shù)、流式細胞儀檢測細胞凋亡技術(shù)與Western-blot技術(shù)檢測TPL與TMZ單藥或聯(lián)合作用于U87膠質(zhì)瘤細胞后的細胞周期、細胞凋亡及其相關(guān)蛋白表達的變化。 結(jié)果：TPL與TMZ作為單藥均能夠以劑量依賴性的抑制膠質(zhì)瘤細胞增殖；在U87膠質(zhì)瘤細胞系與SKMG-1膠質(zhì)瘤細胞系中，TPL聯(lián)合TMZ抑制U87細胞與SKMG-1細胞達IC50時聯(lián)合指數(shù)(combination index，CI)分別為0.76與0.866，表明TPL與TMZ具有協(xié)同作用；TPL與TMZ聯(lián)合用藥后，U87細胞凋亡比例顯著高于單藥用藥組（p0.05）；TPL與TMZ聯(lián)合用藥后，NF-κB信號轉(zhuǎn)導通路中P-I-κBα以及抗凋亡蛋白XIAP的表達明顯下調(diào)。 結(jié)論：TPL可能通過抑制NF-κB信號轉(zhuǎn)導通路的活化并進一步誘導膠質(zhì)瘤細胞凋亡來增加TMZ對膠質(zhì)瘤細胞的殺傷效應。 第二部分：雷公藤甲素增敏替莫唑胺殺傷膠質(zhì)瘤起始細胞 目的：探討雷公藤甲素是否能增加替莫唑胺殺傷膠質(zhì)瘤起始細胞的效應及其可能作用機制。 方法：使用CCK-8法測定TPL和TMZ對膠質(zhì)瘤起始細胞殺傷作用的影響，根據(jù)Chou Talalay法計算的CI評估TPL和TMZ的聯(lián)合作用，使用腫瘤球形成實驗測定藥物對膠質(zhì)瘤起始細胞的自我的更新作用的影響，使用流式細胞儀檢測技術(shù)和western-blot技術(shù)檢測藥物對膠質(zhì)瘤起始細胞凋亡作用的影響，，使用雙熒光素酶報告基因檢測系統(tǒng)檢測藥物對NF-κB轉(zhuǎn)錄活性的影響，使用western-blot技術(shù)檢測藥物對NF-κB信號通路下游蛋白、P-P65和P-I-κBα、P65核蛋白表達的影響，動物實驗觀察藥物對小鼠生存期的影響。 結(jié)果：TPL與TMZ作為單藥均能夠以劑量依賴性的抑制膠質(zhì)瘤起始細胞增殖，但是膠質(zhì)瘤起始細胞對TMZ耐藥，我們測得GSC-1與GSC-2的IC50分別為779.5umol/l和943.5umol/l。TPL能協(xié)同TMZ殺傷膠質(zhì)瘤起始細胞，即能增強TMZ的殺傷作用又能減少TMZ的使用劑量。TPL聯(lián)合TMZ能顯著抑制膠質(zhì)瘤起始細胞的自我更新能力并誘導其凋亡。TPL聯(lián)合TMZ能顯著抑制NF-κB的轉(zhuǎn)錄活性，并下調(diào)NF-κB信號通路下游蛋白、P65核蛋白、P-P65和P-I-κBα表達。在體內(nèi)實驗中，TPL聯(lián)合TMZ用藥后，小鼠遠期生存時間較單獨用藥組顯著延長 結(jié)論：TPL能協(xié)同TMZ殺傷膠質(zhì)瘤起始細胞，其作用機制可能是通過抑制NF-κB信號通路的活化并進一步誘導其凋亡所致。因此，TPL是潛在的化療增敏藥物。
[Abstract]:Glioblastoma is one of the most common primary brain tumors in adults . Although the therapeutic methods of Stupp standard have improved the efficacy of glioma , the 5 - year survival rate is only 9.8 % . At present temozolomide ( TMZ ) is a special chemotherapeutic agent for glioma , so that the median survival time of newly diagnosed patients is increased from 12.1 months to 14.6 months , but due to endogenous and acquired resistance , TMZ chemotherapy is still not ideal . Gliomamide Cells ( GIC ) is a small fraction of the cells present in the tumor . These cells have the capability of self - renewal , differentiation ability and tumor forming ability . These cells are considered to be the root cause of tumor formation , which is more resistant to chemotherapy than other differentiated tumor cells , which is the root cause of tumor recurrence . Therefore , it is an urgent task to find drugs for sensitizing TMZ and to explore the mechanism of chemotherapy resistance . At present , there are many drug researches aiming at the drug resistance of chemotherapy . Among them , it is found that the traditional Chinese medicine tripterygium root has anti - cancer effect on many kinds of tumors , including glioma , 4 , 5 , breast cancer , gastric cancer , bladder cancer , etc . It has strong anticancer activity . The purpose of this study was to investigate whether trimazole could increase the cytotoxicity of temozolomide against glioma cells and glioma starting cells , and to explore the mechanism of action , and to reveal the possible mechanism of glioma resistance to TMZ and its therapeutic significance .

The first part : The cytotoxic effect of tripterygium glycosides on glioma cells

Objective : To investigate whether the effect and possible mechanism of timozolomide on glioma cells can be increased .

Methods : CCK - 8 was used to determine the effects of tpl and TMZ on the proliferation of glioma cells .
閲囩敤Chou Talalay娉曡瘎浼癟PL涓嶵MZ鐨勮仈鍚堟晥搴旓紱
Cell cycle , flow cytometry and Western - blot were used to detect the changes of cell cycle , apoptosis and related protein expression in U87 glioma cells by flow cytometry and Western - blot .

Results : Both tpl and TMZ could inhibit the proliferation of glioma cells in a dose - dependent manner .
The combination index ( CI ) between U87 glioma cell line and SKMG - 1 glioma cell line was 0.76 and 0.866 , respectively .
Compared with TMZ , the apoptosis rate of U87 cells was significantly higher than that of single medicinal group ( p < 0.05 ) .
The expression of P - I - 魏B 偽 and anti - apoptotic protein XIAP in NF - 魏B signal transduction pathway was down - regulated after combined with TMZ .

Conclusion : The killing effect of TMZ on glioma cells may be increased by inhibiting the activation of NF - 魏B signal transduction pathway and further inducing apoptosis of glioma cells .

The second part : tripterygium galanin - sensitized temozolomide anti - glioma starting cells

Objective : To investigate whether the effect and possible mechanism of timozolomide on the starting cells of glioma cells can be increased .

鏂規(guī)硶錛氫嬌鐢–CK-8娉曟祴瀹歍PL鍜孴MZ瀵硅兌璐ㄧ槫璧峰緇嗚優(yōu)鏉

本文編號：1916598