【作者】 张凯茹；

【导师】 王燕； 马力； 闫惠琴；

【作者基本信息】 天津医科大学 ， 内科学， 2010， 硕士

【摘要】 研究背景：肺癌在全世界范围内的发病率和病死率居恶性肿瘤之首,其发病率逐年升高,严重影响着人类的生命健康。由于肿瘤异质性的存在,不同个体及肿瘤细胞的生物学活性存在很大差异,不同肺癌细胞对抗癌药物的敏感性也不完全相同。化学治疗是肺癌综合治疗的重要手段之一,但在化学治疗中却存在对正常组织细胞损伤的毒副作用,为了提高化疗药物对肿瘤细胞的敏感性及治疗效果,减轻化疗药物对机体的毒副反应,有必要探讨不同肿瘤细胞对化疗药物的敏感性或耐药性的分子生物学机制,为今后肿瘤的个体化治疗提供理论依据。NF-E2相关因子2 (NF-E2-related factor 2, Nrf2)是调节正常细胞产生保护性的应答反应,抵御外界不良应激刺激的关键的转录因子,包括顺铂在内的化疗药物对肿瘤细胞而言是不良刺激,在这种情况下转录因子Nrf2是否发挥对肿瘤细胞的保护作用,介导肿瘤耐药所涉及到的具体机制值得探讨。国外有研究表明Nrf2与肿瘤顺铂耐药有关,这已在卵巢癌中得以证实：Cho等人阻断Nrf2后发现耐顺铂的人卵巢癌SK-OV细胞株对顺铂的敏感性增加。然而Nrf2与肺腺癌顺铂耐药相关性的研究目前国内未见报道。我们的研究是初步探讨Nrf2与肺腺癌顺铂耐药的关系及可能的肿瘤耐药机制,为今后给予相应的干预措施逆转肺腺癌耐药、提高化疗效果提供新的理论依据。目的：我们的研究旨在测定转录因子Nrf2及其靶基因在人肺腺癌顺铂耐药细胞株(A549/DDP)及其亲本细胞株(A549)中的定量表达,从Nrf2信号传导通路角度研究肺腺癌顺铂耐药的机制,从而为个体化治疗及从基因水平开发新的耐药拮抗剂提供新的理论依据。材料与方法：1.人肺腺癌A549细胞是经手术标本传代培养获得,人肺腺癌顺铂耐药细胞系(A549/DDP)是在体外经小剂量顺铂逐渐加量诱导亲本细胞A549获得的,均由天津市肺癌研究所提供。两株细胞分别用含10%小牛血清、青霉素和链霉素各100u/ml的RPMI-1640培养基于37℃、5%CO2饱和湿度培养箱中培养,0.125%胰蛋白酶消化传代。实验所用细胞均处于对数生长期。2.用MTT法检测A549/DDP细胞的耐药性,求出耐药指数。3.采用Real-time PCR法检测目的基因的定量表达：实验分两组：A549细胞组、A549/DDP细胞组。取出等量(1×106)的细胞,抽提细胞总RNA、逆转录合成cDNA、Real-time PCR检测两组细胞中转录因子Nrf2、其信号传导通路相关基因Keap1及靶基因：GCL、NQO1、GSTP1、HO-1、MRP1、MRP2、MRP3及MRP4的定量表达,最后采用独立样本t检验分别对两组间各个基因的表达量进行比较。结果：1.细胞形态比较：倒置显微镜下A549细胞及A549/DDP细胞均贴壁生长良好,A549细胞较小,类圆形不规则,A549/DDP细胞在DDP刺激下细胞形态变得较大,呈长梭形,少数为三角细胞形态。2.本实验所用A549/DDP和A549细胞具有高度同源性,A549/DDP经冻存再复苏后耐药性质稳定。经3次独立重复MTT实验,结果表明：DDP药物浓度分别与A549/DDP细胞和亲本细胞生长抑制率成正相关(r=0.984,r=0.896),IC50分别为(8.36±0.44)ug/ml、(0.69±0.09)ug/ml,(t=29.701,p<0.001),A549/DDP细胞的耐药指数为亲本细胞的12.12倍,属于中度耐药细胞。3.Realtime PCR结果提示,耐药细胞株A549/DDP与敏感株A549相比转录因子Nrf2的表达量增加,差异有统计学意义(p<0.01)。同时伴侣分子Keap1、转录因子Nrf2的靶基因NQO1、GSTP1、GCL、HO-1、MRP4的mRNA表达增加,MRP1、MRP2及MRP3的mRNA表达降低,差别均有统计学意义(p<0.01)。结论：人肺腺癌A549顺铂耐药现象的产生可能与Keap1转录因子Nrf2及其靶基因HO-1、GCL、MRP4、GSTP1、NQO1的高表达有关,这些靶基因通过各自或联合的作用降低肺腺癌细胞凋亡、加速顺铂代谢、改变药物在细胞内的分布使顺铂远离作用靶点从而介导肺腺癌顺铂耐药。本研究在国内第一次提出Nrf2-ARE信号传导通路可能参与人肺腺癌顺铂耐药的形成及可能的耐药机制。这一发现对于从基因水平开发新的耐药拮抗剂用于肿瘤耐药的辅助治疗,避免和克服耐药具有一定临床意义。更多还原

【Abstract】 BACKGROUND:As a kind of severe diseases doing harm to human health, pulmonary cancer ranks the first place in aspects of morbidity and mortality. As the existence of heterogeneity, there is difference biologic activity in different individuals and different type of tumor cells. Different lung cancer cells have not the same sensitivity to anti-cancer drugs. Chemotherapy is the main method for treating lung cancer. Drug resistance is one of clinical major causes for low survival rate of the patients. Cis-dichlorodiamine platinum (CDDP) is effective and widely-used drug, but the result is unsatisfactory because of drug resistance. In order to decrease the harm which chemotherapeutics do to the organism and increase the sensitivity and effectiveness, we must do further research to explore the mechanism of drug resistance to tumor. As a key transcription factor in anti-oxidant reaction NF-E2-related factor 2 combines together with ARE, then regulates the expression of detoxifying enzymes, antioxidase and drug transport pumps. Both domestic and overseas researches proved that chemo-therapeutics is an obvious oxidative stress to cells, so in this condition whether transcription factor can protect lung cancer against DDP, and mediate drug resistance is a problem needing urgent management.AIM:Our Object is to study the expression and significance of transcription factor Nrf2 and its target genes in A549 cells which are resistance to cisplatin and reveal the mechanism behind it.Moreover, this provides a new direction to reverse drug resistance and have significance to avoid and overcome drug resistance of tumor.METHODS:1 Human pulmonary adenocarcinoma A549 and Cis-dichlorodiamine platinum-resistant cell lines were obtained from Tianjin Lung cancer Institutes. The cells were cultured in RPMI-1640, supplemented with 10% fetal bovine serum (FBS), 100μg/ml penicillin and 100μg/ml streptomycin. The cells were maintained at 37℃in a humidified atmosphere with 5% CO2.2 MTT was used to detect the drug resistance index of A549/DDP cells.3 Real time PCR:There are two groups:A549 and A549/DDP cell line. Total RNA was extracted using TRIzol reagents according to the manufacturer’s instructions. Isolated RNA was electrophoresed through 1.2% agarose for-maldehydegels to verify the quality of the RNA. The first strand cDNA was generated by reverse transcription. After a sufficient amount of cDNA was obtained, we performed PCR amplification using a real-time PCR cycler.RESULTS:1 We observe the morphous of A549 and A549/DDP cells through the inverted microscope. The former is smaller and spherical, while the later is bigger and spindle-shaped, minority show irregular triangle.2 A549/DDP and A549 cells in our study are of high homology. A549/DDP has stable quality of drug resistance. We perform three independent and repeated MTT essays, and it proves that the growth suppression of the two cell lines have positive correlation with DDP drug concentration(r=0.984, r=0.896).Inhibitory Concentration 50 of A549/DDP and A549 is (8.36±0.44)ug/ml, (0.69±0.09)ug/ml, (t=29.701, p<0.001). Resistance index of A549/DDP is 12.12.3 Real-time PCR shows that the mRNA expression level of transcription factor Nrf2 in A549/DDP cell line is increased compared with A549(p<0.01).At the same time there is more mRNA expression of Keap1 and the target genes of Nrf2, such as NQO1,GSTP1,GCL, HO-1,MRP4 in A549/DDP than the cells which is sensitive to DDP.CONCLUSION:It proves that the transcription factor Nrf2 and it’s target genes HO-1, GCL, MRP4, GSTP1, NQO1 are closely related with Cisplatin resistance of pulmonary adenocarcinoma. They mediate Cisplatin-resistance through decreasing apoptosis, accelerating Cisplatin metabolism and changing the distribution of Cisplatin in pulmonary adenocarcinoma cells. Our study reveals that Nrf2-ARE signal passway may be relative with the Cisplatin-resistance to pulmonary adenocarcinoma. Moreover, this provides a new direction to reverse drug resistance and have significance to avoid and overcome drug resistance of tumor.更多还原