【作者】 刘晔；

【导师】 郭慕依； 张志刚； 刘学光；

【作者基本信息】 复旦大学 ， 病理学， 2009， 博士

【摘要】 顺铂(cisplatin, CP),即顺式—二氨二氯合铂,是最有效的肿瘤化疗药物之一,已在临床应用30多年,其抗癌谱广,目前已被广泛应用于治疗睾丸癌、卵巢癌、膀胱癌、头颈部肿瘤、宫颈癌、小细胞及非小细胞肺癌等。然而,由于该药对肾脏可发生累积性毒性损伤作用,在接受大剂量CP治疗的肿瘤病人中,约1／5病例可发生急性肾功能衰竭的严重后果,因此其临床应用受到极大的限制。CP的肾毒性作用机制复杂多样,细胞凋亡始终是极为重要的机制之一,过去对凋亡机制的研究主要集中在天冬氨酰酶(caspases)依赖途径。凋亡诱导因子(apoptosis-inducing factor, AIF)是一种位于线粒体内膜的黄素蛋白。在某些促凋亡信号刺激下,AIF经蛋白水解剪切从线粒体内膜上脱落经开放的线粒体通透性转换孔道(mitochondrial permeability transition pore, MPTP)被释放,继而发生细胞核转位。AIF在核内可直接引起纯化的细胞核染色质聚集和DNA片断化,无需依赖caspases的级联反应,是非caspase依赖的凋亡途径中的重要蛋白分子。近年来CP通过非caspase依赖如AIF凋亡通路诱导细胞凋亡的现象受到关注,然而对此过程及相关机制尚有争议,并无统一而明确的说法。研究表明AIF上游的重要调节因子是多聚腺苷二磷酸核糖聚合酶{[poly (ADP-ribose) polymerase], PARP}。PARP是一种具有促进细胞损伤修复和增加细胞凋亡双重作用的核内蛋白。当某些严重损伤造成PARP过度激活,会引起AIF的核转位,继而造成细胞凋亡：而PARP的抑制剂如3-AB则能抑制AIF核转位。但是关于该调节机制在CP肾毒性机制中的作用,尤其是体内研究仍较缺乏。本课题通对体外(CP作用于肾小管上皮细胞株HK-2)及体内(CP大鼠肾损伤模型)实验,观察CP对HK-2细胞致坏死和凋亡作用,以及对胞质胞核AIF蛋白、核酸表达的影响；再通过细胞保护剂氨磷汀(amifostine,AF)、Z-VAD-FMK、AIF靶向siRNA以及PARP抑制剂3-AB应用,观察对CP肾毒性损伤形态学改变及对细胞凋亡率影响；运用3-AB进一步研究AIF介导的凋亡通路的调节因子与可能机制,从而为进一步防治CP肾毒性损伤的临床实践提供新的理论和实验基础。第一部分顺铂(CP)对HK-2细胞的毒性作用目的观察顺铂(CP)对HK-2细胞的毒性作用方法不同浓度(0～200μM)和时间(0～24hr)CP作用于HK-2细胞。用台盼蓝染色检测细胞活度,流式细胞仪检测细胞坏死与凋亡情况。Western Blot检测不同时间CP作用后活化的caspases的表达。用显微镜观察和基因组DNA电泳检测保护剂氨磷汀对CP作用下HK-2细胞的影响。结果CP导致HK-2细胞活度降低,与剂量和时间呈显著性相关。细胞凋亡率呈一定剂量(0～100μM)和时间(0～24hr)依赖性,分别在100μM(CP作用24hr)和24hr(50μM CP)时达高峰,分别为对照组的5.2倍和5.0倍。CP作用能激活细胞caspase-3,8,9的表达。CP作用6hr时,caspase-8,9开始活化,18hr时分别是12hr时的8.1倍和3.0倍。CP作用12hr时,caspase-3开始活化,18hr和24hr时,其表达分别是12hr的10.5倍和93.7倍。AF对CP致HK-2细胞的坏死和凋亡有明确的保护作用。小结CP对HK-2细胞毒性作用具有明显的时间和剂量依赖性；较高浓度CP作用,细胞以直接坏死为主要表现,而较低浓度CP作用,细胞则以凋亡为主；CP作用于HK-2细胞,其caspase-8,9和caspase-3先后发生活化；AF能减轻CP致HK-2细胞损伤,对细胞具有保护作用。第二部分AIF通路介导CP诱导HK-2细胞凋亡的研究目的研究AIF是否介导CP诱导HK-2细胞凋亡方法采用Western印迹法和荧光实时定量RT-PCR分别检测不同浓度(0～200μM)和时间(0～12hr)CP作用下,体外培养HK-2细胞的胞质和胞核AIF蛋白及AIF mRNA表达。采用免疫荧光染色检测CP作用下HK-2细胞AIF表达分布。采用TUNEL法和流式细胞仪检测泛caspase抑制剂和AIF靶向siRNA对CP作用下HK-2细胞凋亡的抑制作用。结果HK-2细胞经不同浓度和时间CP的作用,其胞质AIF (cAIF)、胞核AIF(nAIF)及其mRNA表达均有不同程度增加。cAIF表达在CP浓度25μM作用12hr和CP浓度50μM作用3hr起均有升高,其差异分别是对照组的2.3倍(P<0.05)和1.7倍(P<0.01)；nAIF表达呈一定浓度和时间依赖性,在CP浓度150μM作用12hr和CP浓度50μM作用9hr达高峰,其差异分别为25μM组的4.3倍和3hr组的3.7倍。nAIF表达与PARP剪切片断(cleaved-PARP)表达趋势基本一致。AIF mRNA表达在CP浓度50μM作用0～9hr呈梯度增加。免疫荧光结果显示,经CP作用,部分HK-2细胞发生AIF核转位。泛caspase抑制剂(Z-VAD-FMK)和AIF-siRNA对CP诱导细胞凋亡抑制率分别为60.1%和39.2%,两者联合应用对其抑制作用更明显。小结CP上调HK-2细胞AIF核酸和蛋白表达,nAIF和cleaved-PARP表达呈显著的剂量依赖性,nAIF表达具一定时间依赖性；CP诱导AIF发生从胞质线粒体到核的转位；AIF siRNA干扰和Z-VAD-FMK均可部分抑制CP诱导的HK-2细胞凋亡,两者联合应用作用加强。第三部分3-氨基苯甲酰胺(3-AB)对CP肾毒性损伤的保护作用目的探讨3-氨基苯甲酰胺(3-AB)对CP肾毒性损伤的保护作用及可能机制方法将HK-2细胞分为4组：对照组、CP组、CP+3-AB组、3-AB组,Western Blot分别检测细胞PAR、μ-calpain、AIF蛋白表达,Hoechst33258染色和流式细胞仪检测细胞凋亡。将细胞分为5组：对照组、CP组、CP+3-AB组、3-AB组,CP+3-AB+LY294002组,Western Blot检测磷酸化Akt(p-Akt)表达。SD大鼠分成4组：正常对照组、CP处理组、3-AB保护组和AF保护组,HE染色显示3-AB、AF对肾毒性作用的影响,AIF免疫组化染色显示肾小管上皮AIF表达变化,Western Blot检测皮髓交界肾组织PAR、μ-calpain、AIF蛋白表达。结果体外实验：与对照组相比,CP组PAR、62KD和57KD AIF表达增加,μ-calpain表达减少(提示活化),3-AB能减少PAR和57KD AIF表达,抑制μ-calpain活化；3-AB能明显减少CP导致的凋亡现象,对CP作用的HK-2细胞有保护作用；3-AB能进一步增加CP作用下p-Akt表达。体内实验：3-AB和AF能减轻CP导致的大鼠肾损伤,3—AB能减少近端小管上皮细胞AIF表达；与正常对照组相比,CP处理组PAR、62KD和57KD AIF表达增加,μ-calpain表达减少,3-AB能减少PAR和57KD AIF表达,抑制μ-calpain活化。小结PARP抑制剂3-AB可抑制CP作用下PAR形成、μ-calpain活化,及截断型AIF的表达；3-AB对CP诱导的HK-2细胞凋亡有抑制作用；3-AB和AF均能减轻CP的大鼠肾毒性作用；3-AB对CP作用下HK-2细胞PI3K/Akt通路有进一步活化作用。结论1.较高浓度CP作用,细胞以直接坏死为主,较低浓度CP作用,细胞以凋亡为主；CP引起HK-2细胞凋亡既通过死亡受体途径,也通过线粒体途径。2.AIF参与介导CP诱导HK-2细胞凋亡。3.3-氨基苯甲酰胺(3-AB)对CP肾毒性损伤具有保护作用,与其减少PAR形成、抑制μ-calpain活化,减少截断型AIF的表达及激活PI3K/Akt通路可能有关。更多还原

【Abstract】 IntroductionCisplatin (cis-diamminedichloroplatinum) is one of the most effective anticancer chemotherapeutic drugs. It has been used over 30 years in the clinic and has a broad spectrum of anticancer efficacy. Cisplatin has been widely used for the treatment of testicular, ovarian, bladder, head and neck, cervical, nonsmall and small cell lung carcinoma, etc. However, because of its accumulative toxicity to the kidney—there are approximate 20% cisplatin treated cases would complicate acute renal failure—its clinical application was limited. The mechanism of nephrotoxicity induced by cisplatin is complicated and varied. Cellular apoptosis is always an important one and researches on it mostly focused on caspases dependent pathways.Apoptosis-inducing factor (AIF) is a flavoprotein located on inner membrane of the cellular mitochondria. Reacting to some apoptotic stimuli, AIF is released from the inner membrane by a cleavage process and shifts to cytosol throuogh mitochondrial permeability transition pore (MPT), it then translocates to the nucleus. AIF could result in condensation of chromatin and break into large segments, which do not need capases cascade, so AIF is an important protein in caspase independent apoptotic pathway. In recent years, the phenomenon that CP induced apoptosis may mediated by AIF was paid more attention. However, the process and associated mechanisms are still obsure and largely in contradiction. Some research revealed that an important upstream regulatory factor of AIF is poly (ADP-ribose) polymerase (PARP). PARP is a nuclear protein which has dual function of facilitating cellular injury repair and promoting cellular apoptosis. Severe injury to cell would cause extensive activation of PARP and AIF nuclear translocation, which result in cellular apoptosis.3-aminobenzamide (3-AB), an inhibitor of PARP could eliminate the nuclear translocation of AIF. However, how this mechanism is functioned in CP induced nephrotoxicity, especially the research in vivo is rarely reported.In this study, experiments in vitro (treating HK-2 cell with CP) and in vivo (inducing nephrotoxicity by CP in rat) were performed. We investigated CP induced cellular necrosis and apoptosis and evaluated its effect on AIF mRNA, cytosol and nuclear AIF protein. Amifostine, Z-VAD-FMK, AIF siRNA and 3-AB were used as inhibitor to examine their effect on morphology change of nephrotoxicity and cellular apoptotic percentage. We also explore some regulatory factor of AIF dependent apoptotic pathway and their possible mechanisms to provide new academic and pratical base for further prevention and treatment of CP nephrotoxicity.PartⅠThe toxic effect of CP on HK-2 cellObjectives To investigate the toxicity of cisplatin (CP) on HK-2 cellMethods HK-2 cells were exposed to various concentrations (0-200μM) of CP for different times (0-24hr). Trypan blue staining was used to detect cell viability. Flow cytometer was applied to evaluate the necrosis and apoptosis percentage. Activated caspases expression after CP treatment with different times was measured by western blot. The protective effect of amifostine (AF) on CP treatment to HK-2 cells was assessed qualitatively and quantitatively by microscopic observation and DNA ladder.Results CP induced decreases of cell viability were significantly correlated with treating dose and time. Apoptotic percentage presented dose (0～100μM) and time (0～24hr) dependent manner, which was peaked at 100μM (CP treated for 24hr) and 24hr (CP treated with 50μM), and were 5.2 fold and 5.0 fold of control group. Exposure to CP treatment activated caspase-3,8,9. Caspase-8 and-9 was activated from 6hr and reached 8.1 and 3.0 folds, respectively, of 12hr group for 18hr. Caspase-3 was activated from 12hr and reached 10.5 and 93.7 folds of 12hr group for 18hr and 24hr respectively. AF was evidenced to be protective to necrosis and apoptosis of CP treated HK-2 cells.Conclusion The toxicity of CP to HK-2 cell presented to be dose and time-dependent. High dose CP mainly resulted in direct cellular necrosis and low dose mainly resulted in apoptosis. Caspase-8,-9 and-3 were activated in order with CP treatment. AF alleviated cellular injury and was protective against apoptosis.PartⅡAIF mediated HK-2 cell apoptosis induced by CPObjectives To investigate whether CP induced HK-2 cell death was mediated by AIF.Methods Cultured HK-2 cells were exposed to various concentration (0～200μM) for different times (0～12hr). Western blot and real time RT-PCR were applied to detect AIF protein and mRNA expression. Immunofluorescence was used to examine AIF distribution in HK-2 cells. The suppressive effect of pan-caspases inhibitor (Z-VAD-FMK) and AIF-siRNA on apoptosis of HK-2 cells was assessed qualitatively and quantitatively by TUNEL staining and flow cytometer.Results HK-2 cells exposed to various concentrations (0～200μM) of CP for different times (0-12hr) show increases of cytosolic AIF (cAIF), nuclear AIF (nAIF) and its mRNA level. As for cAIF, a 2.3-fold increase (P<0.05 vs control of non-treatment) was revealed at 25μM for 12hr and 1.7-fold (P<0.01 vs control) at 50μM for 3hr. Increase of nAIF expression showed a concentration and time-dependent manner, reached peaks at 150μM CP treatment for 12hr and 50μM CP treatment for 9hr, which were of 4.3 folds (P<0.005 vs 25μM group) and 3.7 folds (P<0.05 vs 3hr group). The expression of nAIF was consistent with cleaved-PARP expressive pattern. Real-time RT-PCR revealed that the AIF mRNA expression was increased when cells were treated with 50μM CP and its effect reached a peak at 9hr. Immunofluorescence demonstrated that AIF shift from cytosol to nuclei in some cultured HK-2 cells treated with CP. Adding Z-VAD-FMK and AIF-siRNA to CP treated HK-2 cells, the apoptotic percentages were reduced by 60.1% and 39.2% respectively. The inhibitory effect was even more significant with their combinative application.Conclusion CP up-regulated the expression level of AIF protein and transcription. The expression of nAIF and cleaved-PARP showed dose dependent manners and the expression of nAIF was also time dependent. CP induced AIF to shift from cytosolic mitochondria to nulei. Both AIF-siRNA and Z-VAD-FMK partially inhibited apoptosis of HK-2 and the effect was enhanced with combinative application.PartⅢThe protective effect of 3-aminobenzamide (3-AB) on nephrotoxicity induced by CPObjectives To investigate whether 3-aminobenzamide (3-AB) has protective effect on CP induced nephrotoxicity and the possible mechanism.Methods Cultured HK-2 cells were divided into four groups:control group, CP group, CP+3-AB group,3-AB group. Western Blot was used to detect PAR [poly (ADP-ribose)],μ-calpain and AIF protein expression. Hoechst 33258 staining and flow cytometer was performed to evaluate apoptosis. Western Blot was also used to detect phospho-Akt (p-Akt). SD rats were divided into four groups:control group, CP treated group,3-AB treated group and AF treated group. HE staining was applied to show the effect of 3-AB and AF. Immunohistochemistry was performed to detect AIF expression in renal tubules. Western Blot was used to detect PAR,μ-calpain and AIF protein expression in renal cortico-medullary junction.Results In vitro The expression of PAR,62KD AIF and 57KD AIF were all increased andμ-calpain was decreased (indicating activation) in CP group compared with control group. 3-AB reduced PAR and 57KD AIF expression and inhibitedμ-calpain activation.3-AB significantly reduced apoptosis induced by CP and show protective effect to HK-2 cells. 3-AB further enhanced p-Akt expression induced by CP. In Vivo Both 3-AB and AF alleviated CP induced renal injury.3-AB reduced AIF expression in proximal tubular cells. Compared with control group, the expression of PAR,62KD AIF and 57KD AIF were all increased andμ-calpain was decreased in CP treated group.3-AB treatment could reduce PAR and 57KD AIF expression and inhibiteμ-calpain activation.Conclusion PARP inhibitor 3-AB inhibited CP induced PAR formation, activation ofμ-calpain, and the expression of truncated AIF.3-AB had inhibitory effect on apoptosis of HK-2 cell induced by CP. Both 3-AB and AF alleviated nephrotoxicity induced by CP. 3-AB further activated PI3K/Akt signalling pathway induced by CP.Conclusions1. High dose CP mainly results in direct cellular necrosis and low dose mainly results in apoptosis. Both death receptor pathway and mitochondrial pathway are involved in HK-2 cell apoptosis induced by CP.2. AIF mediates HK-2 cell apoptosis induced by CP.3.3-aminobenzamide (3-AB) has protective effect on CP induced nephrotoxicity, which may be associated with its effect of reduction of PAR, inhibition ofμ-calpain, reduction of truncated AIF and activation of PI3K/Akt pathway.更多还原