【作者】 潘强；

【导师】 杨学军；

【作者基本信息】 天津医科大学 ， 外科学， 2010， 硕士

【摘要】 胶质母细胞瘤(WHOⅣ)是颅内最常见的高恶性肿瘤,具有弥漫浸润性生长的生物学特性,预后不佳。尽管以手术为主联合放射及化学治疗的综合方案已成为治疗指南,但胶质母细胞瘤病人的预后并没有得到明显改善,平均生存时间只有12～15个月。肿瘤细胞对化疗药物的耐药性是导致治疗失败的主要原因之一,耐药涉及因素很多,其中O6-甲基鸟嘌呤-DNA甲基转移酶(O6-methylguanine-DNA methyltransferase, MGMT)与烷化剂耐药之间的关系最受关注。MGMT是一种DNA修复酶,其可在不需任何辅助因子或其他蛋白质的条件下催化DNA分子中鸟嘌呤O6位上的烷基转移至MGMT本身第145位的半胱氨酸残基上,鸟嘌呤得以复原,DNA的结构和功能得以恢复从而保护细胞免受烷化剂的损伤。高脂溶性和易于透过血脑屏障的烷化剂,被国内外公认为治疗胶质瘤的标准化疗药物。替莫唑胺(Temozolomide, TMZ)属于第二代烷化剂化疗药,是咪唑四嗪类衍生物,不经肝脏代谢,直接在脑胶质瘤组织碱性环境中分解,生成活性物质MTIC (3-methyl-triazenl-yl) imidazole-4-carboxomide, MTIC),疗效佳,副作用小,目前备受临床青睐。然而,肿瘤细胞固有的或获得性的耐药部分地限制了替莫唑胺的临床应用效果。沙利度胺(Thalidomide,THD),是谷氨酸类衍生物,具有抗血管生成作用,鉴于胶质母细胞瘤富含血管,因此沙利度胺被认为具有治疗恶性胶质瘤的潜力。在多项Ⅱ期临床试验中,替莫唑胺联合沙利度胺的化疗方案可以明显延长恶性胶质瘤病人的中位生存期,无明显的毒副作用。对沙利度胺联合替莫唑胺治疗胶质瘤的具体机制逐渐明确,在我们前期实验中发现沙利度胺可以通过激活胶质瘤细胞中PTEN的表达,抑制PI3K/AKT/mTOR信号通路的激活,促进替莫唑胺诱导的自噬及自噬性死亡,且两者联合可以通过激活caspase-3,诱导凋亡发生。然而,目前尚无替莫唑胺联合沙利度胺对替莫唑胺耐药的胶质母细胞瘤是否有作用的研究报道。本课题研究分为以下二个部分：第一部分探讨胶质瘤细胞耐药性演变规律及TMZ自身改变耐药细胞耐药特性的机理,以期为临床优化化疗方案提供依据。我们通过体外分步诱导法使人脑胶质瘤U251细胞对TMZ (0.25-16μg/mL)产生耐药；用MTT法检测耐药指数及细胞存活率；用Western blot、RT-PCR、免疫组化及免疫荧光方法检测耐药机理及增加TMZ浓度后导致胶质瘤耐药细胞生长受抑制的可能原因。历经8个月成功建立了对TMZ耐药的U251/TR, U251/TR细胞IC50 (220.87μM)是未经诱导U251细胞IC50 (33.12μM)的6.67倍(P<0.01),其耐药指数(RF)约为7；U251／TR细胞MGMT表达较未经诱导U251细胞明显增加(P<0.01)；同时本实验发现倍增替莫唑胺浓度仍可以使耐药细胞生长出现受抑制现象,U251／TR细胞MGMT检测结果显示其表达有相应下降。第二部分探讨沙利度胺联合替莫唑胺是否对U251／TR耐药细胞耐药特性有影响,并研究其可能机制。用MTT法检测耐药指数及细胞存活率；用Western blot、RT-PCR、免疫荧光等方法检测蛋白表达水平；用甲基化特异性PCR检测MGMT启动子甲基化情况。给药24小时后,双药联合组的细胞存活率明显降低,48小时后替莫唑胺组细胞存活率也开始下降,且前者降低更明显,沙利度胺组基本无下降；双药联合组及替莫唑胺组MGMT蛋白下降,两组下降水平无差异；甲基化特异性PCR结果显示启动子区甲基化情况无改变。结论：1. MGMT表达水平增高是导致U251／TR对TMZ耐药的主要机制；替莫唑胺可以通过消耗MGMT,从而改变耐药细胞耐药特性,起到自身克服耐药作用。这为临床优化节律性化疗方案提供了依据。2.沙利度胺联合替莫唑胺可以更好的抑制耐药细胞的增殖,起到较好的化疗作用,其机制不是通过改变MGMT基因启动子区甲基化状态来实现,具体机制尚需进一步研究证实。更多还原

【Abstract】 Glioblastoma (GBM WHOⅣ) is the most common and malignant intracranial tumor with diffusely infiltrative growth characteristics and dismal prognosis. The median survival of patients ranges from 12 to 15 months despite of aggressive surgery combined with radiation and chemotherapy. The highly lethal nature of this tumor partly accounts for its chemoresistance of GBM cells. The resistance of GBM cells to alkylating agents is mediated by many factors. Among these factors, the association between O6-methylguanine-DNA methyltransferase (MGMT) and chemo-resistance is more concerned. MGMT is a key enzyme in the DNA repair network, which catalyzes the transfer of the methyl group from O6-methylguanine to a cysteine residue of its active site. In this single step reaction, DNA-lesions caused by alkylating substances are repaired. MGMT subsequently is irreversibly inactivated and degraded.Although advances in chemotherapeutic drugs, alkylating agents remain the most common and effective administration in GBM, based on their lipophilic nature, which can appreciably permeate blood-brain barrier (BBB), and their intrinsic cytotoxic activities. Temozolomide (TMZ), an orally administered cytotoxic alkylating agent with broad-spectrum antitumor activity, is a bioavailable imidazotetrazine derivative of the alkylating agent dacarbazine. It penetrates BBB into the brain easily and undergoes rapid chemical conversion in the systemic circulation at physiological pH to its active compound, MTIC (5-(3-methyltriazen-l-yl)imidazole-4-carboximide), which degrades to a DNA-alkylating species, subsequently, is cytotoxic via the formation of O6-and N7-alkylguanine lesions in DNA, this procedure does not require hepatic metabolism for activation. TMZ becomes the most perspective chemotherapeutic drug against gliomas, on base that it is more effective, easier to administer and has fewer side-effects than its competitors. However, it has also failed to improve prognosis in all patients with GBM. The unsatisfactory outcome with chemotherapy has chiefly been originated from GBM tumor cells intrinsic or acquired chemoresistance.Thalidomide, a glutamic acid derivative, is considered as a potential drug to treat hyper-vascularized glioma due to its anti-angiogenesis effect. In a number of phase II clinical trials, thalidomide combined with temozolomide has been proved to prolong the median survival of patients with malignant glioma significantly, meanwhile, the side effect of this combination is not obvious.The mechanism of combination of thalidomide combined with temozolomide to treat gliomas is cleared gradually. In our previous study, we found thalidomide may inhibit PI3K/AKT/mTOR signaling pathway through activation of PTEN, a tumor suppress gene, to reinfore the induced autophagy. Meanwhile, the combination of both may induce apoptosis through activation of caspase-3. Furthermore, there is no study to examine whether the resistant characteristic of the TMZ-resistant human glioma cell line could be changed by temozolomide combined with thalidomide.The present study was divided into two parts.In the first part of this study was aimed to investigate how and why the chemoresistance to TMZ evolved in U251/TR cells, a TMZ-resistant cell line under TMZ administrator, and provide experimental evidence for optimal TMZ therapy strategy. U251/TR, was established by stepwise exposure of human U251 parental cells to TMZ induced culture for 8 months. MTT assay was used to calculate the resistance index and cell viability percentage. Western blotting(WB)、RT-PCR、immunohistochemistry (IHC) and immunofluorescence(IF) were used to detect MGMT expression for the analysis of chemoresistant evolution and its mechanism in U251 cells during the TMZ induction procedure. U251/TR showed approximately 7-fold resistance to TMZ (RF=6.67±0.53). The MGMT protein expression was significantly increased in U251/TR cells compared with the parental U251 cells (p<0.01). The growth of U251/TR cells was inhibited significantly by TMZ with double the final induced strength from day 3 to 5 compared with DMSO control group (p<0.01, for all) and MGMT protein level in those treated cells was decreased accordingly after TMZ administration (p<0.05).The second part focused on whether the resistant characteristic of the TMZ-resistant human glioma cell line could be changed by thalidomide combined with temozolomide. MTT assay was used to calculate the resistance index and cell viability percentage. Western blotting(WB)、RT-PCR、and immunofluorescence (IF) were used to detect MGMT expression for the analysis of U251/TR cells treated with temozolomide combined with thalidomide. MGMT promoter profile was analyzed by Methylation-specific polymerase-Chain-reaction analysis (MSP). Compared to negative control group, significant inhibition of proliferation was shown in TMZ+THD group after 24 h and TMZ group after 48 h. The MGMT protein levels in TMZ+THD group and TMZ group decreased significantly (p<0.01), moreover, there is no difference between both groups. We also found no change in promoter methylation in all groups.Conclusions1. Our results demonstrated that the primary mechanism of U251/TR cells resistant to TMZ is owing to increased activity of MGMT and the chemoresistance was evolving during the TMZ induction procedure. TMZ could overcome the TMZ-resistance itself by consuming MGMT steadily, which suggests that metronomic TMZ regimen could improve the chemo-sensitivity in TMZ resistant glioma clinically.2. Thalidomide combined with temozolomide could improve the cytotoxicity in U251/TR cells. The mechanism needs further evaluation in future prospective trials.更多还原