RNA干扰AKT2对脑胶质瘤替莫唑胺化疗敏感性的影响及相关机制研究
发布时间：2018-04-02 07:18
本文选题：AKT2 切入点：胶质母细胞瘤 出处：《第二军医大学》2014年博士论文
【摘要】：成人原发性中枢神经系统肿瘤中，包括多种组织学类型的脑神经胶质瘤是最为多见的。而恶性级别最高的要数胶质母细胞瘤，GBM的标准治疗包括早期肿瘤的手术切除、口服烷化剂替莫唑胺（TMZ）化疗及辅助性放疗结合。不过，由于这些肿瘤令人难以置信的耐药和肿瘤频繁复发，胶质母细胞瘤对外科手术治疗、放化疗极其耐受，GBM的中位生存时间仅限于诊断后约15个月。除了肿瘤细胞自身对放疗、化疗的抵制，肿瘤与内环境之间的相互作用还通过参与肿瘤的抗血管生成、组织缺氧和免疫抑制。化疗药物在恶性胶质瘤的治疗中发挥重要作用。新一代烷化剂替莫唑胺是目前对恶性胶质瘤的标准化疗药物，并已被证明有着良好的临床治疗效果。替莫唑胺是经过O6-G甲基化诱导的引发DNA损伤激活的错配修复机制，然而，由于化疗药物内在性和获得性耐药现象还有血脑屏障的存在，很大程度上影响了化疗效果。 AKT作为PI3K/AKT传导途径中的关键因子，在促成肿瘤细胞生长增殖、血管的生成、增加侵袭转移的发生、抑制肿瘤的凋亡和抵制放化疗中起着非常关键的作用。大量文献报导AKT在肿瘤的发生发展及转移中扮演了重要角色，更深入的研究AKT作用的分子生物学机理并发现其与恶性癌症的相关性，有可能为癌症的基因治疗、抗肿瘤药物的开发研究提供新的思路和作用靶点。我科前期实验通过GBM基因芯片研究发现了29条基因和替莫唑胺化疗耐药相关且影响患者生存期，AKT2基因就是其中一个。近年来，一些实验研究已经表明PI3K/AKT信号传导途径与癌症化疗疗效关系比较密切。AKT活化还可能参与肿瘤放射治疗的抵抗，研究结果表明AKT2似乎是一个有效的克服胶质瘤治疗抵抗重要靶标。根据既往实验结果我们初步推测沉默AKT2基因表达可能成为临床上增加胶质瘤化疗敏感性的一种可行性的方法。本实验将分四部分研究AKT2基因沉默在胶质母细胞瘤中对替莫唑胺化疗疗效的改变及其作用机理。第一部分，RNA沉默AKT2的表达对胶质瘤U251细胞株替莫唑胺疗效的影响；第二部分，RNA沉默AKT2的表达对胶质瘤裸鼠移植瘤的替莫唑胺疗效的影响；第三部分，胶质瘤替莫唑胺化疗耐药细胞株的建立及其特性鉴定；第四部分，AKT2在胶质瘤细胞中替莫唑胺化疗抵制的作用机理研究。通过以上实验阐明AKT2基因在脑胶质瘤莫唑胺化疗耐药中的分子生物学机制，在分子水平上寻找和莫唑胺化疗耐药相关的关键基因及其作用机制，提高临床上脑胶质瘤患者对替莫唑胺的化疗敏感性。 第一部分RNA干扰AKT2的表达对胶质瘤U251细胞株替莫唑胺敏感性的影响目的：研究胶质母细胞瘤U251细胞中AKT2基因沉默对替莫唑胺化疗效果的改变。方法：AKT2基因慢病毒载体的构建，然后在体外条件下将AKT2特异性shRNA表达载体AKT2-shRNA转染至U251细胞株中。蛋白印迹、Real-time PCR实验方法测定转染shRNA后U251细胞中AKT2基因表达水平的变化，应用CCK8法检测RNA干扰AKT2后U251细胞对替莫唑胺敏感性的变化情况。 结果：转染AKT2-shRNA后U251细胞AKT2基因表达程度较U251未转染组和U251转染阴性慢病毒组都显著下降；替莫唑胺对U251细胞的半数细胞抑制浓度（IC50）从U251空白对照组的(39.72±2.41)μg/ml、阴性对照组的(39.43±2.24)μg/ml降到(27.23±1.93)μg/ml，AKT2干扰组U251细胞对TMZ药物的疗效变化有显著性差异（P＜0.05）。 结论：AKT2-shRNA能够抑制胶质母细胞瘤细胞株U251中AKT2表达，并能增加对替莫唑胺的敏感性。 第二部分RNA干扰AKT2表达对胶质瘤裸鼠移植瘤替莫唑胺敏感性的影响 目的：研究RNA沉默AKT2对脑胶质瘤移植瘤的替莫唑胺疗效的改变。 方法：首先，构建胶质瘤裸鼠成瘤模型，瘤内注射和腹腔内给TMZ药物，以替莫唑胺化疗药物和AKT2-shRNA表达载体对成瘤后的裸鼠瘤体进行联合干预治疗，进而观察并测量各组裸鼠肿瘤体积大小。Real-time PCR及免疫组化实验分别测定各组裸鼠肿瘤细胞的AKT2的表达程度， TUNEL实验测定并计算分析各组裸鼠成瘤后肿瘤组织细胞的凋亡的变化。 结果：空白对照组、替莫唑胺化疗组、TMZ+阴性对照组、TMZ+AKT2干扰组裸鼠瘤体体积分别为：(669.34±98.73) mm3、(399.86±55.26) mm3、(383.81±34.01) mm3、(297.72±41.49) mm3；瘤体质量分别为：(1.25±0.26)g、(0.72±0.11)g、(0.69±0.07)g、(0.52±0.07)g。TMZ+AKT2干扰组其肿瘤体积及瘤体质量都明显小于空白对照组、替莫唑胺组和TMZ+阴性对照组，有显著性差异（P＜0.05）。免疫组化和Real-time PCR实验表明TMZ+AKT2干扰组比其他对照组AKT2表达程度显著降低（P＜0.05）。TUNEL实验结果显示：空白对照组、替莫唑胺化疗组、TMZ+阴性对照组、TMZ+AKT2干扰组裸鼠瘤体凋亡指数分别为：(7.15±1.04)%、(25.26±2.71)%、(26.63±3.46)%、(42.81±5.97)%。TMZ+AKT2干扰组其肿瘤凋亡情况明显高于空白对照组、替莫唑胺组和TMZ+阴性对照组，结果有显著性差异（P＜0.05）。 结论: RNA干扰AKT2能够显著提高裸鼠胶质母细胞瘤对TMZ化疗的敏感性。 第三部分胶质瘤替莫唑胺化疗耐药细胞株的建立及其特性鉴定 目的：建立U251耐替莫唑胺的稳定细胞株，并对该细胞株U251/TMZ的生物学特性进行研究鉴定。 方法：通过从低药物浓度开始逐步增加U251细胞培养基中化疗药物替莫唑胺的浓度，建立对TMZ化疗耐药的胶质母细胞瘤U251/TMZ。采用四甲基偶氮唑蓝(MTT)法，计算出两种细胞株的半数细胞抑制浓度(IC50)及耐药系数(RI)。流式细胞仪测定U251和U251/TMZ两组细胞周期的分布情况。利用蛋白芯片技术对U251和U251/TMZ两种细胞株中1358中常见蛋白进行测定和分析。 结果：替莫唑胺对U251细胞株的IC50为(10.78±0.72)μg/ml，替莫唑胺对U251/TMZ的IC50为(45.42±3.17) μg/ml，细胞增殖实验所测结果计算后的耐药指数为4.21(P0.05)。流式细胞仪分析提示U251/TMZ耐药株较U251细胞株的细胞周期主要发生G2期阻滞。蛋白芯片测定结果显示bcl-2家族、caspase家族、P53等多种凋亡相关蛋白和DNA损伤修复蛋白发生明显上调或下调。 结论：成功构建了对替莫唑胺化疗耐药的胶质瘤细胞株U251/TMZ，，耐药性能十分显著而且生物学特性稳定，该细胞株适合用于胶质瘤替莫唑胺化疗耐药的研究。 第四部分AKT2在胶质瘤细胞中替莫唑胺化疗耐药的作用机制研究 目的：研究AKT2基因、多药耐药基因、MGMT等DNA损伤修复蛋白、多药耐药相关蛋白以及bcl-2、survivin、beclin-1、caspase-3等凋亡自噬相关蛋白，在胶母U251替莫唑胺(TMZ)化疗耐药中的相互关系及调控作用。 方法：利用实验的第三部分建立的耐替莫唑胺的U251/TMZ，通过蛋白芯片对U251细胞株和U251/TMZ耐药细胞株分别进行检测相关蛋白的变化，并用计算机软件进行以AKT2为中心的相关蛋白的singal-net分析。流式细胞仪测定沉默AKT2基因表达后各组细胞凋亡的改变情况。AKT2-shRNA干扰胶质母细胞瘤细胞株U251的AKT2表达，同时采用了Realtime-PCR、Western Blot实验方法，来进一步分析和评价AKT2与MDR-1、MRP-1、MGMT、bcl-2、caspase-3、P53等相关蛋白表达情况和相互调控关系。 结果: singal-net分析提示U251耐药株中AKT2基因靶向调节了bcl-2、survivin、caspase-3、beclin-1、 P53等凋亡、自噬相关蛋白，AKT2-shRNA干扰U251后其凋亡明显增加，Real-time PCR和蛋白印迹实验结果提示bcl-2、survivin、MGMT明显下调，而caspase-3、PTEN、P53、beclin-1明显上调。 结论：AKT2可能参与调控bcl-2、caspase-3、P53、survivin、beclin1等凋亡自噬相关蛋白及DNA损伤修复蛋白MGMT的表达，从而介导胶质母细胞瘤细胞株U251对替莫唑胺化疗抵抗。
[Abstract]:Adult primary central nervous system tumors, including glioma various histological types are the most common. While the malignant degree of the highest number of glioblastoma resection, standard treatment for GBM including early tumor surgery, oral alkylating agent temozolomide (TMZ) chemotherapy and adjuvant radiotherapy combined with these tumors. However, due to the incredible resistance and frequent recurrence of tumor, the surgical treatment of glioblastoma, chemotherapy is tolerance, the median survival time of GBM is only about 15 months after diagnosis. In addition to tumor cells to radiotherapy, chemotherapy resistance, the interaction between the tumor and the environment also by participating in tumor angiogenesis, tissue hypoxia and immune suppression. Chemotherapy play an important role in the treatment of malignant glioma. A new generation of alkylating agent temozolomide is currently the standard for malignant glioma Chemotherapy drugs, and has been shown to have a good clinical effect. Temozolomide is through O6-G methylation induced by mismatch repair mechanism, activation of the DNA damage however, due to the existence of intrinsic and acquired drug resistance and blood brain barrier, has great influence on the effect of chemotherapy.
AKT is a key factor in the PI3K/AKT pathway, contributed to the growth of tumor cell proliferation, angiogenesis, invasion and metastasis increased the occurrence of apoptosis and inhibition of tumor resistance plays a key role in chemotherapy. A large number of reported AKT in tumor development and metastasis plays an important role in molecular biology mechanism study on the role of AKT further and found its correlation with malignant cancer, possibly for cancer gene therapy, to provide ideas and targets of new research and development of antitumor drugs. Our previous experiments by GBM gene chip research found 29 genes and temozolomide resistance and influence the survival of the patients, the AKT2 gene is one of them. In recent years, some experimental studies have shown that the close relationship between PI3K/AKT signaling pathway and cancer chemotherapy may also be involved in the activation of.AKT tumor The resistance of radiotherapy, the results show that AKT2 appears to be an effective treatment for glioma resistance to overcome an important target. According to previous experimental results we presumed that silencing of AKT2 gene expression may become a feasible method to increase the sensitivity of glioma chemotherapy in clinic. This experiment will be divided into four parts of AKT2 gene silencing on the changes and the mechanism of temozolomide chemotherapy for glioblastoma. The first part, RNA inhibited the expression of AKT2 on glioma U251 cells to temozolomide efficacy; the second part, the expression of RNA silencing AKT2 on glioma xenografts in nude mice of temozolomide effect; the third part glioma for establishment and characterization of temozolomide chemotherapy resistant cell lines; the fourth part, AKT2 for the study of mechanism of temozolomide chemotherapy resistance in glioma cells. Through the above. Objective to elucidate the molecular biological mechanism of AKT2 gene in glioma mozolomide chemoresistance, and find molecular key genes related to resistance to mozolomide chemotherapy and its action mechanism at molecular level, so as to improve chemosensitivity of glioma patients to temozolomide in clinic.
The first part of the expression of RNA AKT2 interference effect on glioma U251 cells temozolomide Objective: To study the AKT2 glioblastoma cells U251 gene silencing on temozolomide effect change. Methods: Construction of AKT2 gene lentiviral vector, then the expression of AKT2 in vitro specific shRNA plasmid AKT2-shRNA was transfected into U251 cells. Western blot, the expression of AKT2 gene in U251 cells Real-time PCR experimental method to determine the shRNA after transfection was detected by CCK8 RNA interference AKT2 on changes of temozolomide sensitivity of U251 cells.
Results: U251 cells AKT2 gene expression level is U251 transfected group and U251 transfected negative lentivirus group were significantly decreased after transfection of AKT2-shRNA; temozolomide half cells on U251 cell inhibitory concentration (IC50) from U251 in the control group (39.72 + 2.41) g/ml, negative control group (39.43 + 2.24 g/ml (27.23) reduced to + 1.93) g/ml, AKT2 and U251 cells of interference group have difference in efficacy to TMZ drugs (P < 0.05).
Conclusion: AKT2-shRNA can inhibit the expression of AKT2 in glioblastoma cell line U251 and increase the sensitivity to temozolomide.
Effect of the second part of RNA interference AKT2 expression on the sensitivity of timozolamine in glioma xenografts in nude mice
Objective: To study the effect of RNA silencing AKT2 on the effect of temozolomide on the tumor of brain glioma.
Method: first, construct glioma in nude mice model, intratumoral injection and intraperitoneal injection of TMZ drugs with temozolomide chemotherapy and AKT2-shRNA expression vector of tumors of nude mice tumor were treated, and then observed and measured expression level of the tumor volume of size.Real-time PCR and immunohistochemical assay. The tumor cells were measured in each group of AKT2, and the determination of the calculation and analysis of the nude mice changes of apoptosis of tumor cells in the tumor after TUNEL experiment.
缁撴灉锛氱┖鐧藉鐓х粍,鏇胯帿鍞戣兒鍖栫枟缁
本文编号：1699351
本文链接：https://www.wllwen.com/yixuelunwen/shenjingyixue/1699351.html
最近更新
教材专著
