紫杉醇诱导下咽癌FaDu细胞的多药耐药性及其机制的研究

发布时间：2018-02-24 09:47

本文关键词： 下咽癌多药耐药 ABCB1 ABCG2 JNK　出处：《山东大学》2012年硕士论文　论文类型：学位论文

【摘要】：下咽癌是头颈部鳞状细胞癌中恶性程度较高的肿瘤之一,由于发病位置隐蔽,发现时多数已为中晚期,患者的5年生存率仅为30%-50%,传统的手术方法由于不能保留喉器官,导致患者的生存质量下降.以器官保留为目的的放化疗并不能提高患者生存期,5年生存率仍处于40%-60%.近年来人们提倡手术联合放化疗以提高患者生存率和生活质量,临床上比较常用的化疗药物包括顺铂(cisplatin DDP),5-氟尿嘧啶(5-fluorouracil5-FU),多柔比星(doxorabicin Dox),和长春新碱(vincristine VCR)等。但是,随着化疗药物的应用,肿瘤细胞逐渐出现了多药耐药性(multidrug resistance, MDR),从而降低了治疗效果。紫杉醇作为新一代化疗药物在晚期或复发下咽癌中被广泛应用并取得较好的疗效,研究表明其临床试验疗效高于其他传统化疗药物。尽管如此,肿瘤细胞对紫杉醇产生的耐药性仍然是其发挥作用的最大障碍。为进一步了解下咽癌细胞对紫杉醇的耐药机制,我们通过用紫杉醇诱导下咽癌细胞株FaDu的方法建立其多药耐药细胞株FaDu/T.通过比较两种细胞株间多药耐药蛋白ABCB1和ABCG2表达变化及JNK信号转导途径在这种变化中的作用,进一步揭示了下咽癌的多药耐药及调控机制,并为临床治疗下咽癌提供理论支持。目的 1.检测FaDu/T细胞的多药耐药性。 2.检测多药耐药蛋白ABCB1,ABCG2分别在FaDu和FaDu/T细胞系中的不同表达。 3.探讨JNK信号转导途径在ABCB1及ABCG2变化表达中的作用,为进-步逆转下咽癌细胞的多药耐药性奠定理论基础。材料与方法以人下咽癌细胞株FaDu为亲本细胞,采用浓度梯度递增法成功建立下咽癌细胞株FaDu的耐紫杉醇细胞株FaDu/T,紫杉醇的起始用药浓度为FaDu的IC25：5×10-9mol/L,截止实验时耐药浓度为200×10-9mol/L,整个过程持续约14个月。四甲基偶氮唑蓝法(Methyl-Thiazolyl-Tetrazolium MTT)分别检测FaDu和FaDu/T对DDP,5-FU, Dox,和VCR的多药耐药性,IC50软件分别计算出其半数致死率；多药耐药基因及蛋白ABCB1及ABCG2表达变化情况分别通过反转录-聚合酶链反应(RT-PCR), Western blotting和激光共聚焦检测；FaDu/T细胞中茴香霉素(Anisomycin)的加入前后不同时间JNK信号转导通路相关蛋白的表达变化通过Western blotting检测。结果 1. FaDu细胞的耐紫杉醇细胞株FaDu/T成功建立,FaDu/T细胞可以在含有紫杉醇的培养基中正常生长。 2.耐药细胞株FaDu/T比FaDu细胞具有更强的多药耐药性。 3.与FaDu细胞相比,FaDu/T细胞株中多药耐药基因和蛋白ABCB1的表达增加但ABCG2表达下降。 4.紫杉醇初始作用于FaDu细胞后JNK信号转导通路被激活,但是在FaDu/T细胞中JNK信号转导通路呈失活状态,MAPK信号转导通路的激活剂茴香霉素(Anisomycin)则可以使FaDu/T细胞的JNK信号通路重新激活。 5.耐药细胞株FaDu/T中加入Anisomycin时ABCB1表达下调,ABCG2的表达升高,但是在预先加入JNK特异性抑制剂SP600125的FaDu/T细胞株中加入Anisomycin时,ABCB1和ABCG2的表达无明显变化,提示JNK信号通路在下咽癌的多药耐药过程中具有重要的调控作用。结论 1.多药耐药蛋白ABCB1在FaDu/T细胞的高表达是其产生多药耐药的重要原因。 2.紫杉醇通过JNK信号转导通路影响多药耐药蛋白ABCB1和ABCG2表达 3.各种耐药蛋白交替变化表达从而对不同的化疗药物产生耐药性是导致临床肿瘤化疗失败的重要原因。
[Abstract]:Hypopharyngeal cancer is one of malignant squamous cell carcinoma of head and neck cancer incidence higher, due to the hidden location, the majority found already in the late stage, with a 5 year survival rate is only 30%-50%, the traditional surgical method due to the preservation of laryngeal organs, leading to the survival quality of the patients decreased. With the purpose of organ retention. Chemotherapy does not improve survival rate, 5 year survival rate is still in the 40%-60%. in recent years, people advocate surgery combined with radiotherapy and chemotherapy to improve survival and quality of life of patients, the clinical commonly used chemotherapeutic drugs including cisplatin (cisplatin DDP), 5- (5-fluorouracil5-FU), fluorouracil, doxorubicin and vincristine (doxorabicin Dox). (vincristine VCR). However, with the application of chemotherapy, tumor cells appeared multidrug resistance (multidrug resistance, MDR), thereby reducing the therapeutic effect of paclitaxel as a new generation. Chemotherapy pharyngeal cancer is widely used and has good efficacy in advanced or recurrent, studies show that the clinical curative effect than other traditional chemotherapy drugs. However, drug resistance of tumor cells to paclitaxel remains the biggest obstacle to play a role. The pharyngeal resistance mechanism of cancer cells to paclitaxel for further we know, through the method of paclitaxel induced hypopharyngeal carcinoma cell line FaDu in the establishment of multidrug resistance cell line FaDu/T. by comparing the two kinds of cell lines of multidrug resistance protein ABCB1 and ABCG2 expression and JNK signal transduction pathway in this kind of change in the role, further reveals the multidrug resistance and regulation mechanism pharyngeal cancer, and provide theoretical support for the clinical treatment of hypopharyngeal carcinoma.
objective
1. the multidrug resistance of FaDu/T cells was detected.
2. the multidrug resistance protein ABCB1 and ABCG2 were detected in different expressions in FaDu and FaDu/T cell lines.
3. to explore the role of JNK signal transduction pathway in the expression of ABCB1 and ABCG2, and to lay a theoretical basis for reversing the multidrug resistance of hypopharyngeal cancer cells.
Materials and methods
In human hypopharyngeal carcinoma cell line FaDu as the parental cells, the method of increasing successful establishment of hypopharyngeal carcinoma cell line FaDu in paclitaxel resistant cell line FaDu/T by concentration gradient, the initial drug concentration of paclitaxel for FaDu IC25:5 * 10-9mol/L, the cut-off resistance concentration is 200 * 10-9mol/L, the whole process lasted about 14 months. Four methyl thiazolyl tetrazolium assay (Methyl-Thiazolyl-Tetrazolium MTT) and FaDu/T FaDu detection of DDP, 5-FU, Dox, multidrug resistance and VCR, IC50 software to calculate the half lethal rate; multidrug resistance gene and protein expression of ABCG2 and ABCB1 respectively by reverse transcription polymerase chain reaction (RT-PCR), Western blotting and laser confocal detection; FaDu/T cells of anisomycin (Anisomycin) expression changes before and after the addition of different time of JNK signal transduction pathway related proteins by Western blotting test.
Result
The taxol resistant cell line FaDu/T of 1. FaDu cells was successfully established, and FaDu/T cells could grow normally in the medium containing paclitaxel.
The 2. drug resistant cell line, FaDu/T, has a stronger multidrug resistance than the FaDu cell.
3. compared with FaDu cells, the expression of multidrug resistance gene and protein ABCB1 in FaDu/T cell lines increased, but the expression of ABCG2 decreased.
4. after paclitaxel initial action on FaDu cells, JNK signal transduction pathway was activated, but JNK signaling pathway was inactivated in FaDu/T cells. The activation of MAPK signal transduction pathway, anisomycin (Anisomycin), could activate JNK signaling pathway in FaDu/T cells.
Adding 5. Anisomycin resistant cell line FaDu/T ABCB1 expression, the expression of ABCG2 increased, but the addition of Anisomycin in FaDu/T cells and pretreatment with JNK specific inhibitor of SP600125, no significant changes in the expression of ABCB1 and ABCG2, has an important role in the regulation of JNK signaling pathway in hypopharyngeal cancer multidrug resistance in the process of.
conclusion
The high expression of multidrug resistant protein ABCB1 ABCB1 in FaDu/T cells is an important cause of multidrug resistance.
2. paclitaxel affects the expression of multidrug resistant protein ABCB1 and ABCG2 through JNK signal transduction pathway
3. the alternately altered expression of various drug resistant proteins, thus producing resistance to different chemotherapeutic drugs, is an important reason for the failure of clinical tumor chemotherapy.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.63

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