【作者】 王启明；

【导师】 李勇；

【作者基本信息】 重庆医科大学 ， 临床口腔医学， 2011， 硕士

【摘要】 多药耐药(Multidrug Resistance,MDR)是肿瘤化疗失败的重要原因之一,因而逆转多药耐药是克服肿瘤临床耐药,提高化疗效果的重要手段。所以,能增加肿瘤细胞对化疗药物敏感性的耐药逆转剂成为了一类很有实用价值的新型药物,也是当代逆转肿瘤多药耐药研究的热点。抗肿瘤药物在细胞内有效浓度下降是MDR的主要原因,MDR与细胞膜水平上的药物摄取减少和外排增多、药物细胞内分布状况的改变、细胞解毒系统和DNA修复系统功能增强以及药物靶点的改变有关。MDR的机制众多,可以由细胞膜膜糖蛋白介导,引起药物转泄和外排;或者由细胞内某些关键酶介导,使药物在细胞内的核质比发生改变;抑或由凋亡控制基因介导,使肿瘤细胞的凋亡受到抑制;不同肿瘤具有不同的耐药表型,可以是某种耐药基因表达,也可能是多种耐药基因同时表达的结果。在治疗头颈部肿瘤时,铂类药物是化疗常用药。而肿瘤对铂类药物的耐药也时常发生,其机制与肺耐药蛋白(lung resistance protein, LRP)有关。因此逆转剂的研究与应用,特别是对不良反应较小,同时又能调节机体免疫力的中药逆转剂的研究有着重要的意义。本研究采用逐步递增卡铂浓度、体外间歇诱导法建立人舌癌耐卡铂细胞株Tca8113/CBP;噻唑蓝比色法(MTT)检测甲基莲心碱(Neferine,Nef)对Tca8113/CBP卡铂耐药性的逆转作用;RT-PCR法检测Nef作用前后Tca8113/CBP内耐药相关基因肺耐药蛋白(LRP)与成纤维细胞肌型原肌球蛋白(FMT)的表达水平;免疫荧光观察Nef作用前后细胞形态结构变化。以研究Nef对Tca8113/CBP卡铂耐药性的逆转作用。研究方法及结果1.建立人舌癌Tca8113/CBP卡铂耐药细胞株方法:使用含15%胎牛血清的RPM I-1640完全培养基,在37℃,5% CO2条件下常规培养Tca8113细胞。细胞呈贴壁生长,待生长稳定后,取对数生长期Tca8113细胞在含CBP的RPM I-1640培养液中培养,逐步提高CBP浓度间歇诱导,剂量为依次为0.1,0.2,0.4,0.8,1.2和1.6ug/ml,最终获得1株能完全耐受1.6ug/ml CBP的细胞株。细胞诱导成功后,将耐药细胞株在无药物的培养基中培养稳定传代5次后用于进行各项实验。结果:诱导的Tca8113/CBP细胞可稳定耐受1.6ug/ml的CBP,和原代Tca8113细胞比较,Tca8113/CBP细胞的耐药倍数是4.06倍(P<0.05)。2.甲基莲心碱的逆转作用RT-PCR法检测逆转前后细胞中LRP和FMT mRNA水平的改变方法:将细胞分为耐药组、逆转组和对照组(耐药组和逆转组为Tca8113/CBP细胞,对照组为Tca8113细胞),逆转组取对数生长期的Tca8113/CBP细胞,加入含有最适逆转浓度Nef的RPM I-1640培养基,作用24小时后与耐药组Tca8113/CBP和对照组Tca8113细胞采用RT-PCR法检测LRP和FMT mRNA表达水平:细胞总RNA提取后需用NanoDrop微量紫外分光光度计测得RNA浓度,半定量后再进行逆转录和PCR反应,总RNA提取、逆转录和PCR反应根据相应的试剂盒及TaqDNA聚合酶说明按常规方法进行。结果:经Nef作用24小时后逆转组Tca8113/CBP与耐药组Tca8113/CBP相比,LRP和FMT mRNA水平的表达明显降低(P<0.01)。免疫荧光观察逆转前后细胞微丝结构的改变方法:将细胞分为耐药组、逆转组和对照组(耐药组和逆转组为Tca8113/CBP细胞,对照组为Tca8113细胞),逆转组取对数生长期Tca8113/CBP,加入含有最适逆转浓度Nef的RPM I-1640培养基,作用24小时后与耐药组Tca8113/CBP和对照组Tca8113同时滴加到多聚赖氨酸包被的玻片上,在室温下静置15分钟后,用4%的多聚甲醛室温下固定20分钟,PBS清洗2次,每次10分钟;0.1%的聚乙二醇辛基苯基醚(triton X-100)室温下作用10分钟,PBS清洗2次,每次10分钟;0.5%的牛血清蛋白于37℃孵育40分钟,PBS洗2次,每次进行10分钟;5mg/L FITC-鬼笔环肽室温下染色30分钟,PBS清洗2次,每次10分钟;50%的缓冲甘油封片。上述操作应注意避光,然后在荧光显微镜下观察细胞的微丝结构。结果:Nef处理24小时后的逆转组中完全贴壁的Tca8113/CBP细胞质内F-actin分布趋于有序均匀,荧光亮度明显减弱,接近于对照组Tca8113细胞的水平。研究结果表明:Nef对Tca8113/CBP的卡铂耐药性有逆转作用,其机制可能与Nef干预Tca8113/CBP细胞内LRP和FMT表达,增加Tca8113/CBP细胞内卡铂的含量,改变微丝结构,降低细胞粘附能力,干扰细胞增殖周期有关更多还原

【Abstract】 MDR (Multidrug Resistance, MDR) is an important reason for failure of cancer chemotherapy. In order to enhance the results of chemotherapy, it is very important to overcome the MDR of the drug i.e. achieve a reversal of MDR of the tumor. To realize this, certain new reversal drugs have to be discovered, and researchers all over the world are working towards this end. It won’t be wrong to say that this is quite a popular field of research these days.The main reason for MDR is the decrease in the effective concentration of the Anticancer drugs in the tumor cells. MDR is induced because the concentration of the drug at the cell membrane level is reduced. The drug uptake and efflux is hampered because of the increase in the intracellular distribution of drugs, which changes the cell detoxification systems and DNA repair systems, by changing the nuclear-cytoplasmic ratio. MDR is generally induced due to interference in the cell membrane. The glycoprotein mediated membrane causes leakage in the efflux of the drug transfer. MDR may also be mediated by some key intracellular enzyme which changes the nuclear-cytoplasmic ratio. It may also be controlled by apoptosis, by inhibition of the tumor cell apoptosis; different tumors have different resistance phenotype, which can be a resistance gene or it could be the expression of multiple drug resistance genes simultaneously in results. In the treatment of head and neck cancer, the platinum drugs are the most commonly used chemotherapy drugs. The tumor resistance to platinum drugs are frequent, because of the mechanism of the lung resistance protein (lung resistance protein, LRP). This is because MDR is LPR mediated. It has been found that the use of traditional Chinese herbal drugs to regulate the body’s immune system, shows lower side effects, and this can also significantly reverse the risk of MDR, as compared to the commonly used allopathic drugs.In this study, the concentration of carboplatin in vitro is gradually increased and induced to set up intermittent resistance to carboplatin in human tongue cells Tca8113/CBP. Tetrazolium assay (MTT) is used to test neferine (Nef) on the Tca8113/CBP Carboplatin Reversal of drug resistance. RT-PCR assay is used before and after Tca8113/CBP to test Nef resistant genes within the lung resistance protein LRP and fibroblast muscle-type tropomyosin expression of FMT. Immunofluorescence is used to observe the cell morphology before and after the Nef structural changes, to study the Nef on Tca8113/CBP carboplatin drug resistance reversal effect.Research methods and results1. The establishment of Carboplatin resistance in human tongue cancer cells Tca8113/CBPMethods: a medium of RPM I-1640 was prepared containing a 15% bovine fetal serum at 37℃and 5% CO2 Tca8113 cells under normal culture. Cells were adherent to growth and stable and the logarithmic growth phase began in the culture medium. The concentration of CBP was gradually increased to induce intermittent doses 0.1,0.2,0.4,0.8, 1.2 and 1.6ug/ml, ultimately the cells can fully withstand 1.6ug/ml CBP cell lines. Cells used for the experiment were successfully drug resistant and stable after 5 generations. Results: The cells can be induced to stable tolerance of 1.6ug/ml Tca8113/CBP the CBP, and primary Tca8113 comparison, Tca8113/CBP multiple drug resistance is 4.06 times (P <0.05).2. Neferine role reversalRT-PCRMethods: The cells were divided into resistant groups, reversal group and control group (resistance group and the reversal group Tca8113/CBP cells Tca8113 cells in the control group), resistant group’s logarithmic growth phase Tca8113/CBP, induced with the optimal concentration of Nef in the RPM I-1640 medium, which reversed the resistant group cells for 24 hours. All the three group cells detected by RT-PCR expressed levels of LRP and FMT mRNA: first, the tRNA was extracted from all the three groups. The NanoDrop machine was used to detect the concentration of the RNA, using the UV spectrophotometer. After this, the RT-PCR was started according to the corresponding instructions on the RT-PCR kit and TaqDNA polymerase enzyme, according to conventional methods. Results: after induction of Nef for 24 hours the reversal group Tca8113/CBP was compared with the resistant group Tca8113/CBP, LRP expression was significantly reduced, and FMT was also reduced.ImmunofluorescenceMethods: The cells were divided into resistant group, reversal group and control group (drug group and the reversal group Tca8113/CBP cells Tca8113 cells in the control group), resistant group’s logarithmic growth phase Tca8113/CBP, induced with the optimal concentration of Nef in the RPM I-1640 medium, which reversed the resistant group cells for 24 hours. All the three groups were dropped onto poly-lysine-coated glass slides. Maintained at room temperature for 15 minutes. With 4% paraformaldehyde fixed at room temperature for 20 minutes. It was washed with PBS 2 times, each time for 10 minutes. 0.1% octyl polyethylene glycol phenyl ether (triton X-100) at room temperature for 10 minutes. It was washed it PBS 2 times, each 10 minutes; 0.5% bovine serum albumin at 37℃incubated for 40 minutes. It was washed with PBS 2 times for 10 minutes. 5mg / L FITC-phalloidin staining at room temperature for 30 minutes. It was washed with PBS 2 times , every 10 minutes; then 50% glycerol buffer was mounted. For all the above procedures, a dark field should be maintained. it is then observed under a fluorescent microscope, for the cells microfilament structure. Results: 24 hours after the induction of Nef the reversal of the group resulted in completely adherent to the slides. F-actin distribution tends to be orderly and uniform fluorescence intensity was significantly reduced, which was found to be close to the level of the control group.The results show that: Nef on Carboplatin resistant Tca8113/CBP has reversed the role of drug resistance, which may be intracellular. This caused the expression of LRP and the FMT to increase intracellular content of carboplatin, change microfilament structure and reduce cell adhesion and cell cycle related interference.更多还原