雷公藤甲素协同替莫唑胺杀伤胶质瘤细胞及胶质瘤起始细胞

发布时间：2018-05-20 22:45

  本文选题：胶质母细胞瘤 + 胶质瘤起始细胞　； 参考：《广州医科大学》2014年硕士论文

【摘要】：胶质母细胞瘤（glioblastoma multiforme，GBM）是成人最常见的原发性脑肿瘤，虽然以Stupp标准治疗方法使胶质瘤的疗效有了明显提高，但其5年生存率仅有9.8%[1]。目前替莫唑胺（temozolomide，TMZ）是胶质瘤的特效化疗药物，使得新诊断的GBM中位生存期由12.1个月提高至14.6个月[2]，但是由于内源性和获得性耐药，TMZ化疗作用仍不理想。胶质瘤起始细胞（GliomaInitiating Cells,GIC）是肿瘤中存在的一小部分细胞，这些细胞具有自我更新的能力，分化能力，肿瘤形成能力。这些细胞被认为可能是肿瘤形成的根源，比其他分化的肿瘤细胞对放化疗更加耐药，是导致肿瘤复发的根源[3]。因此，寻找增敏TMZ的药物并探讨化疗耐药的机制成为当前迫切的任务。目前，有许多针对肿瘤化疗耐药的药物研究，其中有发现中国的传统药物雷公藤甲素（triptolide,TPL）对多种肿瘤包括胶质瘤[4,5]、乳腺癌、胃癌、膀胱癌等[6]均有抗癌作用，其抗癌活性强。并有研究发现TPL能增加多种化疗药物的敏感性[7]。本研究拟探讨雷公藤甲素是否能增敏替莫唑胺杀伤胶质瘤细胞及胶质瘤起始细胞，并探讨其作用机制，揭示胶质母细胞瘤对TMZ的化疗耐药的可能机制及雷公藤甲素的治疗意义。 第一部分：雷公藤甲素增敏替莫唑胺杀伤胶质瘤细胞 目的：探讨雷公藤甲素是否能增加替莫唑胺杀伤胶质瘤细胞的效应及其可能机制。 方法：使用CCK-8法测定TPL与TMZ对胶质瘤细胞增殖的影响；采用Chou Talalay法评估TPL与TMZ的联合效应；分别应用流式细胞仪检测细胞周期技术、流式细胞仪检测细胞凋亡技术与Western-blot技术检测TPL与TMZ单药或联合作用于U87胶质瘤细胞后的细胞周期、细胞凋亡及其相关蛋白表达的变化。 结果：TPL与TMZ作为单药均能够以剂量依赖性的抑制胶质瘤细胞增殖；在U87胶质瘤细胞系与SKMG-1胶质瘤细胞系中，TPL联合TMZ抑制U87细胞与SKMG-1细胞达IC50时联合指数(combination index，CI)分别为0.76与0.866，表明TPL与TMZ具有协同作用；TPL与TMZ联合用药后，U87细胞凋亡比例显著高于单药用药组（p0.05）；TPL与TMZ联合用药后，NF-κB信号转导通路中P-I-κBα以及抗凋亡蛋白XIAP的表达明显下调。 结论：TPL可能通过抑制NF-κB信号转导通路的活化并进一步诱导胶质瘤细胞凋亡来增加TMZ对胶质瘤细胞的杀伤效应。 第二部分：雷公藤甲素增敏替莫唑胺杀伤胶质瘤起始细胞 目的：探讨雷公藤甲素是否能增加替莫唑胺杀伤胶质瘤起始细胞的效应及其可能作用机制。 方法：使用CCK-8法测定TPL和TMZ对胶质瘤起始细胞杀伤作用的影响，根据Chou Talalay法计算的CI评估TPL和TMZ的联合作用，使用肿瘤球形成实验测定药物对胶质瘤起始细胞的自我的更新作用的影响，使用流式细胞仪检测技术和western-blot技术检测药物对胶质瘤起始细胞凋亡作用的影响，，使用双荧光素酶报告基因检测系统检测药物对NF-κB转录活性的影响，使用western-blot技术检测药物对NF-κB信号通路下游蛋白、P-P65和P-I-κBα、P65核蛋白表达的影响，动物实验观察药物对小鼠生存期的影响。 结果：TPL与TMZ作为单药均能够以剂量依赖性的抑制胶质瘤起始细胞增殖，但是胶质瘤起始细胞对TMZ耐药，我们测得GSC-1与GSC-2的IC50分别为779.5umol/l和943.5umol/l。TPL能协同TMZ杀伤胶质瘤起始细胞，即能增强TMZ的杀伤作用又能减少TMZ的使用剂量。TPL联合TMZ能显著抑制胶质瘤起始细胞的自我更新能力并诱导其凋亡。TPL联合TMZ能显著抑制NF-κB的转录活性，并下调NF-κB信号通路下游蛋白、P65核蛋白、P-P65和P-I-κBα表达。在体内实验中，TPL联合TMZ用药后，小鼠远期生存时间较单独用药组显著延长 结论：TPL能协同TMZ杀伤胶质瘤起始细胞，其作用机制可能是通过抑制NF-κB信号通路的活化并进一步诱导其凋亡所致。因此，TPL是潜在的化疗增敏药物。
[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 伪 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 .

鏂规硶锛氫娇鐢–CK-8娉曟祴瀹歍PL鍜孴MZ瀵硅兌璐ㄧ槫璧峰�嬬粏鑳炴�

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