【作者】 张隽开；

【导师】 王忠裕；

【作者基本信息】 大连医科大学 ， 中西医结合临床， 2008， 博士

【摘要】 肝癌细胞的多药耐药性(multidrug resistance,MDR)形成是导致肝癌化疗失败的主要原因之一。肝癌细胞一旦获得多药耐药性,则在对同一类型的药物产生抗药性的同时,对结构和作用机制不同的抗肿瘤药物均产生交叉耐药性。多药耐药性形成直接关系到肝癌患者对化疗的反应和预后。经研究现已发现了导致MDR产生的多个表征蛋白及相关靶点,并据此研究出多种相应的化疗增敏药物及治疗方法,但均因各种原因限制了其应用。MDR产生机制复杂,其中化疗诱导MDR产生的上游信号机制仍需进行大量的探索工作。近年研究发现,在血红素代谢过程中,由血红素加氧酶(heme oxygenase-1,HO-1)催化产生的多种物质均具有细胞保护作用。HO-1在多种实体性肿瘤中均有高表达,因其具有保护细胞凋亡的作用,提示HO-1可能是抑制肿瘤生长的新靶标。本课题以Bel/FU肝癌耐药细胞株为研究对象,应用氯化高铁血红素(Hemin)、锌原卟啉(zinc protoporphyrinⅨ, ZnPPⅨ)、小分子RNA干扰及中药黄芪影响HO-1的表达,通过研究血红素加氧酶在肝癌细胞多药耐药中的影响及相关信号分子机制,为逆转肿瘤多药耐药寻找新的作用靶点。论文一血红素加氧酶对Bel/FU肝癌耐药细胞株多药耐药性的影响目的:评估血红素加氧酶-1(Heme Oxygenase,HO-1)对Bel/FU肝癌耐药细胞株化疗敏感性的影响,探讨血红素加氧酶对肝癌细胞多药耐药性可能的影响机制。方法:①:将Bel/FU细胞接种于96孔培养板,分三组,一组设为空白对照组,另外两组分别加入60uM的Hemin,10uM的ZnPPⅨ进行诱导24小时后,分别加入不同浓度梯度的ADM、MMC、5-FU化疗药物作用24小时,MTT法检测细胞对各化疗药物敏感性;②:分别加入不同浓度梯度的Hemin(15umol/L,30umol/L,60umol/L,120umol/L,240umol/L)及ZnPPⅨ(2umol/L,6umol/L,10umol/L,14umol/L,18umol/L)诱导培养24小时后,RT-PCR检测血红素加氧酶HO-1(heme oxygenase-1,HO-1),MDR-1(multidrug resistance,MDR-1),谷胱甘肽-S-转移酶GST-π(Glutathione S-transferase,GST-π)及β-actin核酸水平表达量;Western blot检测P-糖蛋白(P-glycoprotein,P-GP),GST-π及β-actin蛋白质水平表达量;③:设空白对照组后,分别加入Hemin(60umol/L,240umol/L)和ZnPPⅨ(10umol/L,18umol/L)诱导,应用流式细胞仪测定细胞内罗丹明123的潴留以评估P-GP的功能。结果:①:Hemin作用于Bel/FU细胞诱导24小时后,各化疗药物(5-FU,ADM及MMC)半数抑制浓度(IC50)与空白对照组比较各P值均<0.01,化疗药物半数抑制浓度均明显增加;ZnPPⅨ干预组表现则相反,细胞活性降低,各化疗药物半数抑制浓度均显著降低;②:Hemin作用于Bel/FU细胞诱导24小时后,与空白组比较,血红素加氧酶HO-l在核酸水平的表达量明显增加(F=71.513,其中2umol/L组P=0.029,余均<0.01),MDR-1mRNA及GST-πmRNA的表达量、P-GP及GST-π的蛋白表达量也显著增加(各P值均<0.01),且均呈剂量依赖趋势;ZnPPⅨ干预组表现则相反,各指标均显著降低,但也表现出剂量依赖趋势;③:罗丹明潴留试验结果显示:与空白组比较,Hemin干预组罗丹明荧光曲线明显左移,且浓度增高曲线左移越明显,提示细胞内罗丹明潴留减少;而ZnPPⅨ干预组则表现为曲线右移,出现相反的结果。结论:影响HO-1的表达可改变肝癌耐药细胞株的化疗敏感性,这种变化是通过影响MDR-1、GST-π核酸及蛋白质水平的表达,同时改变了P-糖蛋白药物外排功能实现的;提示HO-1可作为逆转肿瘤多药耐药的潜在作用靶点。论文二RNA干扰HO-I基因对Bel/FU肝癌耐药细胞株阿霉素化疗敏感性的影响目的:应用siRNA干扰技术特异性地将HO-1基因表达下调后,观察Bel/FU肝癌耐药细胞株阿霉素化疗敏感性的变化及影响机制。方法:设计与制备针对HO-1及β-actin的小分子RNA。①:按正常细胞组、空白对照组、干扰组及阳性对照组,将Bel/FU肝癌耐药细胞接种于六孔板,各组分别加入正常培养基、空脂质体转染培养基、含针对HO-1的siRNA脂质体培养基、含针对β-actin的siRNA脂质体培养基,作用48小时后,RT-PCR检测HO-1、β-actin mRNA表达量,Western blot检测P-GP,β-actin蛋白质水平表达量;②:按正常细胞组、空白对照组、干扰组,应用流式细胞仪测定细胞内罗丹明123的潴留并评估P-GP的功能;③:按正常细胞组、空白对照组、干扰组,分别加入不同浓度梯度的阿霉素,MTT法测定体外阿霉素化疗敏感性。结果:①:siRNAs作用48小时后,干扰组HO-1 mRNA表达被干扰未见表达,而正常细胞组、空脂质体转染组(空白对照组)、阳性对照组未见明显影响;β-actin mRNA则在阳性对照组无表达,其余各组无影响;Western blot显示:与正常培养组及各对照组相比,P-糖蛋白表达量下降;②:罗丹明潴留试验结果显示:与正常培养组及空白对照组相比,siRNAs干扰组罗丹明荧光曲线明显右移,提示细胞内罗丹明潴留增加;正常培养组及空白对照组曲线基本重叠,未见明显差异;③: Bel/FU细胞培养48小时后,与正常组相比,RNA干扰组细胞活性降低,阿霉素半数抑制浓度(IC50)显著降低,而空白对照组与正常组相比无明显差异。结论:应用siRNAs抑制HO-1的表达后,多药耐药细胞株Bel/FU细胞对阿霉素的化疗敏感性增强,这种增敏作用是通过下调P-糖蛋白表达,降低P-糖蛋白罗丹明外排功能而实现的。论文三黄芪对Bel/FU肝癌耐药细胞株化疗敏感性的影响目的:评估黄芪注射液对肝癌耐药细胞化疗敏感性的影响,结合黄芪对血红素加氧酶的调节作用探索可能的影响机制。方法:黄芪注射液作用于Bel/FU肝癌化疗耐药细胞株,从三个方面观察作用前后化疗敏感性的变化:应用MTT法检测细胞株对5-Fu,ADM及MMC作用后的存活率;应用免疫细胞化学检测化疗耐药表征蛋白P-糖蛋白表达量的变化;应用流式细胞术罗丹明外排试验检测黄芪注射液对细胞株P-糖蛋白功能的影响。应用RT-PCR方法检测黄芪注射液作用前后血红素加氧酶(HO-1)核酸水平表达量的变化。结果:①:黄芪注射液作用于Bel/FU细胞诱导24小时后,黄芪各浓度组化疗药物半数抑制浓度(IC50)与空白对照组比较各P值均<0.01,化疗药物半数抑制浓度均显著降低;②:应用8ul/mL黄芪注射液作用于Bel/Fu细胞后,免疫细胞化学检测显示黄芪组(vs对照组) P-糖蛋白阳性表达明显减少(Wilcoxon W值为398.500,Z值为-2.363,近似P值为0.018<0.05);流式细胞仪罗丹明潴留试验结果显示,黄芪组罗丹明荧光曲线明显右移,细胞化疗药物外排功能降低; Bel/FU细胞HO-lmRNA的表达水平按HO-1cDNA/β-actin黄芪组为0.316±0.08,对照组为0.624±0.173,HO-1mRNA的表达显著减少(P<0.01)。结论:黄芪注射液可增强肝癌耐药细胞株Bel/FU的化疗敏感性,下调化疗耐药表征蛋白P-糖蛋白表达,降低P-糖蛋白药物外排功能,实现这种作用的同时伴随有HO-1mRNA表达的减少。更多还原

【Abstract】 Multidrug resistance (MDR) is a significant obstacle to providing effective chemotherapy to many Hepatic Carcinoma patients.Once the cells gain MDR,they will describe the phenomenon of simultaneous resistance to a broad of unrelated drugs,which affect the chemotherapeutic effectiveness and prognosis of the patients.Some molecular targets and proteins are gradually found,but the mechanisms of mutidrug resistance are not yet fully understood.Much effort needed has been extended to advance the mechanism in cancer cells,especially in the upstream signal pathways.In recent years,there reported some products in the degradation of heme catalyzed by heme oxygenase-1(HO-1),which can protect the cells from apoptosis. Elevated HO-1 expression and activity was found in various tumor tissues. The studies suggest that HO-1 exerts a role in controlling cell growth in a cell-specific manner which could be a new molecular target and upstream mechanism of MDR.In our studies,some methods are adopted to affect the HO-1 expression in Drug-resistant Hepatic Carcinoma Bel/FU Cells,then explores the effect and relevant mechanism of Heme Oxygenase-1 on chemosensitivity of the cells.Part one Effect of Heme Oxygenase on Multidrug Resistance of Drug-resistant Hepatic Carcinoma Bel/FU Cell LineObjective:To study the effect and relevant mechanism of Heme Oxygenase-1 on chemosensitivity of drug-resistant hepatic carcinoma Bel/FU cell line.Methods:①:Bel/FU cells were collected and planted in 96 well plates,cells were divided into three groups,two induced separately with 60uM Hemin and 10uM ZnPPⅨ,the other planted in normal condition.After several gradient concentrations of drugs(ADM,MMC,5-FU) are added into the wells,the drug sensitivity test of Bel/FU cells was carried out with MTT assay.②:After induced with different gradient concentrations of Hemin(15umol/L,30umol/L,60umol/L,120umol/L,240umol/L)and ZnPPⅨ(2umol/L, 6umol/L,10umol/L,14umol/L,18umol/L)for 24h,heme oxygenase-1 (HO-1),multidrug resistance-1(MDR-1) and Glutathione S-transferase(GST-π)mRNA level were determined by RT-PCR technique;P-glycoprotein(P-GP),GST-πprotein level were detected by Western blot analysis;Rhodamine123 retention fluorescent intensity measured by flow cytometry was used to detect the function of P-GP.Results:After Bel/FU cells being dealt with Hemin for 24h,there were significant differences in 50% inhibiting concentration (IC50) of chemical drugs between control groups and contrast group.IC50 of control groups increased obviously. Meanwhile,the mRNA expression of HO-1 and MDR-1 or GST-πin the cells of control group were observably higher than those in contrast group,of which were both consistent with the dose-effect curve. P-GP and GST-πprotein level expression appeared the analogous results. The curve of Rhodamine123 retention fluorescent intensity in the control cells removed left award from the place of contrast group,which indicated that the efflux pump function of P-GP was strengthened.The control groups by ZnPPⅨ,however,showed a contrary result. Lineal correlation analysis showed that the HO-1mRNA levels were significantly correlated with mRNA and protein expression of MDR-1 and GST-π.Conclusions:Inpacting heme oxygenas-1 can influence the chemosensitivity of Bel/FU cell line,which relates with its action in up(or down)-regulation of MDR-1 and GST-πexpression and efflux pump function of P-GP.The enzyme of HO-1 may be a new target of reversing multidrug resistance in Hepatic Carcinoma cells. Part two Effect of HO-1 Small Interfering RNA on Chemosensitivity of Adriamycin in Drug-resistant Hepatic Carcinoma Bel/FU Cell LineObjective:To investigate the chemosensitivity of Adriamycin and reverse mechanism of drug resistance in hepatic carcinoma Bel/FU cell line after heme oxygenase-1 being blocked with siRNAs.Methods:Design and prepare the small interfering RNAs aiming at HO-1 andβ-actin gene.The cells were divided into 4 groups as normal group(only in nutritive medium),blank contrast group(medium contain blank liposome),RNA intefering group(medium contain HO-1 siRNAs liposome)and negative group(medium containβ-actin siRNAs liposome).After intubating for 24 hours, HO-1 andβ-actin mRNA level were determined by RT-PCR technique;P-GP,GST-πprotein level were detected by Western blot analysis;Rhodamine123 retention fluorescent intensity measured by flow cytometry was used to detect the function of P-GP.In additively, different gradient concentrations of Adriamycin were added into plant wells,then MTT assay was carried out to detect the chemosensitivity of Bel/FU cells.Results:After Bel/FU cells being dealt with different methods for 24h, HO-1 mRNA level almost disppeared in RNA intefering group while other groups had no change; P-GP protein level by Western blot analysis deceased in RNA intefering group contrasted with those of another three groups; The curve of Rhodamine123 retention fluorescent intensity in the control cells removed right award from the place of contrast group,which indicated that the efflux pump function of P-GP was weakened; MTT assay indicated that the cell activity decreased significantly and 50% inhibiting concentration (IC50) of Adriamycin decreased obviously in the control group. Conclusions:After Bel/FU cells being dealt with HO-1 siRNAs liposome, the Adriamycin chemosensitivity of Drug-resistant Bel/FU cells increased,which achieved by down-regulation of protein level expression and efflux pump function of P-GP. Part three Effect of Astragulus Injection on Chemosensitivity of Drug-resistant Hepatic Carcinoma Bel/FU Cell LineObjective:To study the effect and relevant mechanism of Astragulus injection on chemosensitivity of drug-resistant hepatic carcinoma Bel/FU cell line.Methods:The drug sensitivity test of Bel/FU was carried out with MTT assay.The distribution of P-glucoprotein(P-GP) in the cells were determined by immunohistochemical stainning.Rhodamine123 retention fluorescent intensity measured by flow cytometry was used to detect the function of P-GP. HO-1mRNA level was determined by RT-PCR technique.Results:After Bel/FU cells being dealing with Astragulus injection for 24h,there were significant differences in 50% inhibiting concentration (IC50) of chemical drugs between control groups and contrast group(P<0.01).IC50 of control groups decreased obviously.Immunohistochemistry showed that P-GP expression were significantly lower in the cells treated with 8ul/ml Astragulus injection than those of contrast group(Z=-2.363, P=0.018).The curve of Rhodamine123 retention fluorescent intensity in the control cells removed right award from the place of contrast group,which indicated that the efflux pump function of P-GP was weakened.Meanwhile,the mRNA expression of HO-1 in the cells of control group were observably lower than those of in contrast group(0.316±0.08vs0.624±0.173, P<0.01).Conclusions:Astragulus injection can enhance the chemosensitivity of Bel/Fu cell line,which may relate with its action in decreasing protein expression and efflux pump function of P-GP concomitant with the decrease of HO-1mRNA level.更多还原