氧化损伤与修复机制在黄曲霉毒素相关原发性肝癌的发生与化疗反应性中的作用

【作者】 苏智雄；

【导师】 黎乐群；

【作者基本信息】 广西医科大学 ， 肿瘤学， 2007， 博士

【摘要】 第一部分黄曲霉毒素B1对原发性肝癌患者外周血淋巴细胞氧化损伤的实验研究目的通过低浓度黄曲霉毒素B1处理体外培养外周血淋巴细胞,了解黄曲霉毒素B1对体外培养淋巴细胞氧化损伤及修复机制的影响;同时探讨潜在性功能性DNA修复基因多态性在其中的作用。方法采取10例健康人和20例肝癌患者外周血淋巴细胞作为研究对象,流式细胞技术检测不同剂量黄曲霉毒素B1诱导的DNA氧化损伤水平,同时测定细胞内超氧化物岐化酶(SOD)、脂质过氧化损伤产物-丙二醛(MDA)、羟自由基(OH~-)及活性氧含量,以了解黄曲霉毒素B1对体外培养淋巴细胞抗氧化能力、脂质过氧化损伤及氧化水平的影响。此外,运用RT-PCR检测黄曲霉毒素B1诱导后淋巴的DNA损伤修复酶基因P53、P21、hOGG1、APE、PARP的表达,并对APE、hOGG1基因分型。采用基因组学与毒理学相结合、基因结构和基因功能相结合的方法考察潜在性功能性DNA修复酶多态性。结果1.低浓度黄曲霉毒素B1干预后,淋巴细胞8-羟基鸟嘌呤(8-oxoG)染色阳性率和荧光强度均高于空白组,肝癌组的淋巴细胞8-oxoG染色阳性率和荧光强度均高于对照组(P<0.05)。2.上清液总超氧化物岐化酶T-SOD活力,随着AFB1浓度的增加而降低,各剂量组与空白组相比,T-SOD含量差异有显著性(P<0.01);上清液OH~-含量,随着黄曲霉毒素B1浓度的增加而逐渐增加,最高剂量组与空白组相比,OH~-含量差异有显著性(P<0.05)。经偏相关分析结果,8-oxoG与OH~-成显著正相关,相关系数为0.3812,P=0.038,8-oxoG与T-SOD成显著负相关,相关系数为-0.5539,P=0.001。3.20μg/ml AFB1处理时,P53、P21、hOGG1、APE和PARP的mRNA表达水平最高,与空白组相比有统计学差异(P<0.05)。4.携带APE Glu148Asp位点变异基因型(GC和CC)个体的DNA损伤高于野生型基因型(GG)的个体,两者之间差异有统计学意义(P<0.05)。结论1.黄曲霉毒素B1暴露后肝癌患者外周血淋巴细胞的氧化损伤比正常人为高。2.黄曲霉毒素B1暴露后淋巴细胞的损伤主要由黄曲霉毒素B1诱导产生过多的OH~-所致。3.黄曲霉毒素B1暴露后淋巴细胞内8-oxoG水平与修复酶APE基因型相关。携带变异基因型APE 148Asp者黄曲霉毒素B1诱导后淋巴细胞的DNA损伤较重。第二部分DNA修复酶hOGG1和PARP在原发性肝癌和肝硬化组织中差异表达的临床病理意义目的通过检测DNA修复酶hOGG1及PARP在原发性肝癌(hepatocellularcarcinoma,HCC)及肝硬化组织中的表达,探讨hOGG1和PARP在HCC和肝硬化组织中的表达特点及其与临床病理因素的关系。方法应用EnVision免疫组化法检测41例非HCC正常肝组织、99例HCC、51例HCC癌旁肝硬化组织中hOGG1和PARP表达情况。结果在HCC组织中hOGG1和PARP在细胞核与细胞质中均可表达,其中hOGG1细胞核阳性分度在正常组、肝硬化组、HCC组之间依次增高(P<0.001),阳性表达率分别为29.3%、52.9%、77.8%;PARP细胞核阳性分度在正常组、肝硬化组、HCC组之间依次降低(P<0.001),阳性表达率分别为70.7%、56.9%、29.3%。多因素分析显示:hOGG1蛋白在细胞核表达与ALT有关(相对危险度为1.022,P=0.041),hOGG1蛋白在细胞浆表达与总胆红素有关(相对危险度为1.155,P=0.018),PARP蛋白在细胞浆表达与外周血白细胞数(相对危险度为0.757,P=0.037)有关。结论在HCC发生过程中hOGG1蛋白在肝细胞核中的表达可能随着病程进展而增强,PARP蛋白在肝细胞核中的表达可能随着病程进展而降低,可能反应了从肝硬化到肝癌进程中肝细胞内氧化应激状态的改变。第三部分术前化疗对DNA修复酶hOGG1及PARP在原发性肝癌组织中表达的影响目的通过检测DNA修复酶hOGG1及PARP在原发性肝癌及癌旁组织中的表达,探讨术前化疗对损伤修复机制的影响。方法应用EnVision免疫组化法检测41例正常肝组织、187例(术前未做化疗患者99例和术前行经肝动脉插管化疗患者88例)原发性肝癌患者的肝癌组织和癌旁组织中hOGG1、PARP的表达。结果正常肝组织、术前化疗组与术前未化疗组的肝癌组织hOGG1蛋白细胞核表达阳性分度依次递增,三者之间差异有统计学意义(均P<0.05);正常肝组织、术前化疗组与术前未化疗组的肝癌组织PARP蛋白细胞核表达阳性分度依次递减,三者之间差异有统计学意义(均P<0.05)。PARP癌旁组织阳性表达强度(P=0.038)、ALT(P=0.001)、病理分级(P=0.040)是肿瘤复发的危险因素,外周血淋巴细胞数(P=0.026)、化疗(P=0.049)是肿瘤复发的保护因素。结论1.术前化疗可以杀伤对氧化损伤敏感的肝癌细胞,表现为未化疗组癌组织hOGG1蛋白表达高于化疗组癌组织,未化疗组癌组织PARP蛋白表达低于化疗组癌组织。2.经过术前化疗药物筛选的DNA损伤修复能力加强的癌细胞如果逃避了手术切除可能成为术后复发的来源。3.PARP癌旁组织阳性表达强度高的患者,肝癌复发的可能性增加,PARP也许可以成为提示肝癌患者预后的新指标。更多还原

【Abstract】 ObjectiveTo investigate the status of oxidative stress and repair,and the role of potentially functional polymorphisms of DNA damage repair genes in in vitro cultured lymphocytes exposed to low concentration of Aflatoxin B1(AFB1).MethodsPeripheral blood lymphocytes were collected from ten healthy volunteers and 20 hepatocellular carcinoma(HCC)patients.Oxidative DNA damage levels in lymphocytes exposed to various concentration of AFB1 were examed using flow cytometry.Intracellular superoxide dismutase(SOD),lipid peroxidation products-malondialdehyde(MDA),hydroxyl radicals(OH)and reactive oxygen species(ROS)were also determined.In addition,the expression of DNA repair enzymes(P53、P21、hOGG1、APE and PARP)was analyzed by RT-PCR,and the genotypes of APE and hOGG1 were determined by PCR-CTPP assay.Results1.After AFB1 treatment,8-oxoG levels in the lymphocytes were higher than that in the controls.In contrast to control group,HCC cases had higher levels of 8-oxoG in lymphocytes(P＜0.05).2.Total SOD content in the supernatant was reversly associated with,while the OH~- content in the supernatant increased with the AFB1 concentration. Meanwhile,8-oxoG levels in lymphocytes were associated with both OH~-(r=0.3812,P=0.038)and T-SOD content(r=-0.5539,P=0.001). 3.There were highest levels of mRNA of P53,P21,hOGG1,APE and PARP in lymphocytes at concentration of 20μg/ml AFB1 incubation(in contrast to the blank controls,P＜0.01).4.Individuals carrying APE Glu148Asp or APE Asp148Asp genotypes showed higher levels of DNA damage than those carrying APE Glu148Glu genotypes (P＜0.05).Conclusion1.After AFB1 exposure,the peripheral blood lymphocytes in HCC patients revealed severe oxidative damage than that in health controls.2.After exposure to low concentrations of aflatoxin B1,lymphocytes damage may mainly induced by excessive production of OH~-.3.APE 148Asp genotype is associated with higher level of oxidative damage in this in vitro model thus might have etiology implication for AFB1-related hepatocarcinogenesis. AIMTo explore the expression and clinicopathological implications of human 8-oxoGuanine DNA glycosylase-1(hOGG1)and of poly ADP-ribose polymerase(PARP)in hepatocellular carcinoma(HCC)and cirrhotic livers.METHODShOGG1 and PARP expression was detected by immuneohistochemical EnVision method on liver tissues of non-HCC controls(n=41),cirrhotic(n=51)and HCC patients(n=99).RESULTSThere were three types of hOGG1 and of PARP positive staining in HCCs: nuclear,cytoplasmic and mixed.The expression levels of hOGG1 in nucleus was significantly higher in HCCs than that in hepatocirrhosis and controls,and significantly higher in hepatocirrhosis than that in controls(P＜0.01).The expression levels of PARP in nucleus was significantly lower in HCCs than that in hepatocirrhosis and controls,and significantly lower in hepatocirrhosis than that in controls(P＜0.01).The expression of hOGG1 and of PARP was not associated with the clinicopathological factors in HCCs(P＞0.05).Multiplicity indicated that hOGG1 expression in nucleus was related to ALT(OR=1.022, P=0.041),hOGG1 expression in cytoplasm was related to T bili(OR=1.155, P=0.018),PARP expression in cytoplasm was related to peripheral blood leucocytes(OR=0.757,P=0.037). CONCLUSIONDuring hepatocarcinogenesis,the expression pattern of hOGG1 and PARP in liver cell nucleus might reflect the status of oxidative stress in each stage thus has etiology implications. ObjectiveTo explore the effect of preoperative chemotherapy on DNA repair enzyme of human 8-oxoGuanine DNA glycosylase-1(hOGG1)and of poly ADP- ribose polymerase(PARP)in hepatocellular carcinoma(HCC)patients.MethodshOGG 1 and PARP expression was detected by immuneohistochemical EnVision method in tissues of normalliver(n=41),HCC and the surrounding liver(n=187, preoperative chemotherapy n=88,without preoperative chemotherapy n=99).ResultsThe expression levels of hOGG1 in nucleus was significantly higher in HCCs without preoperative chemotherapy than that undergoing preoperative chemotherapy and normal livers,and significantly higher in HCCs undergoing preoperative chemotherapy than that in normal livers(P＜0.05).The expression levels of PARP in nucleus was significantly lower in HCCs without preoperative chemotherapy than that undergoing preoperative chemotherapy and normal liver,and significantly lower in HCC undergoing preoperative chemotherapy than that in normal livers(P＜0.05).COX multivariate prognosis analysis indicated that the risk factors for HCC to recur were the positive degree of PARP,ALT,the histological grade of HCC.(all P＜0.05)and the protective factor was the counting of lymphocyte(all P＜0.05). ConclusionChemotherapy increases the ability of DNA repair in HCC tissues.To go through screening,the HCC cells with potent capability of DNA repair might be the sourse of recurrence after operation if they escape both preoperative chemotherapy and surgical resection.The PARP expression was the risk factor for HCC relapse.更多还原