【作者】 刘志明；

【导师】 李国坚；

【作者基本信息】 广西医科大学 ， 普通外科学， 2013， 博士

【摘要】 目的：研究宁夏银川地区、广西南宁、柳州和桂林不同纬度地区人群AFB1暴露水平及其与DNA氧化损伤、修复的关系。材料和方法：收集宁夏银川地区、广西南宁、柳州及桂林地区304例肝癌患者手术切除术后的病理石蜡肝组织标本,以四地区肝血管瘤和胆道结石患者190例病理石蜡肝组织标本为对照。采用IHC测定肝组织中AFB1-DNA加合物表达水平和8-OHdG、hOGG1、MGMT和PARP-1的表达水平；运用PCR-RFLP方法检测南宁、柳州和桂林三地肝组织的P53基因突变热点。并以广西南宁市226例HBsAg(-)成年男性作为研究对象,通过设计问卷调查其饮食习惯,采用ELISA法检测其血浆AAA水平、PCC水平和尿液中AFB,与8-OHdG水平。结果：1.宁夏银川地区、南宁、柳州和桂林四地区HCC患者AFB1-DNA加合物阳性率分别为：36%(18/50)、59.5%(47/79)、43.8%(39/89)和17.4%(15/86),其中南宁地区高于桂林和宁夏银川地区,差异有统计学显著性(P<0.05)。四地对照组AFB1-DNA加合物阳性率分别为：15.5%(9/58)、16.4%(9/55)、10.8%(4/37)和12.5%(5/40),无统计学显著性差异(P>0.05)。2.南宁、柳州和桂林三地区HCC组8-OHdG的阳性水平分别为50.6%(40/79)、51.7%(46/89)和48.8%(42/86),无显著性差异(P>0.05),三地对照组8-OHdG的阳性水平分别为55.2%(32/58)、54.5%(30/55)和64.7%(24/37),无显著性差异(P>0.05)。3.P53基因第249位点密码子突变检测：254例HCC患者肝组织中,共有55例检测出P53基因249位点密码子突变,突变率为21.7%(P<0.001)。南宁、柳州、桂林三地区HCC肝组织中P53基因249位密码子突变率分别为26.6%(21/79)、24.7(22/89)和14.0%(12/86)(P>0.05),突变率差异无统计学意义。P53基因突变率在AFB1-DNA加合物阳性表达者高于AFB1-DNA加合物阴性表达者,差异有统计学意义(χ2=8.076,P=0.004)。4.南宁、柳州和桂林三地区HCC组hOGG1的表达水平分别为：75.7%(60/79)、67.4%(60/89)和60.5%(52/86)；三地正常对照组分别为：53.5%(31/58)、47.3%(26/55)和37.8%(14/37)。三地HCC组hOGG1的阳性表达高于正常对照组(P<0.05)。三地HCC组和正常对照组,南宁地区表达水平和平均光密度值最高,差异有显著性(P<0.05)。hOGG1表达水平与AFB1-DNA加合物表达在南宁地区的HCC组和正常对照组的均存在相关性(P<0.05),而在柳州和桂林地区各组中未发现有统计学显著相关性(P>0.05)。5.南宁、柳州和桂林三地区HCC组MGMT的表达水平分别为：51.4%(40/79)、51.7%(46/89)和47.7%(41/86);三地正常对照组分别为：82.8%(48/58)、76.3%(42/55)和75.7%(28/37)。三地HCC组的MGMT表达水平和平均光密度值均低于正常对照组(P<0.05)；三地HCC组和正常对照组的MGMT表达水平和平均光密度值无显著差异(P>0.05),MGMT表达与AFB1-DNA加合物表达在三地各组中均未发现有统计学显著相关性。6.南宁、柳州和桂林三地区HCC患者PARP-1的表达水平分别为：89.9%(71/79)、76.4%(68/89)和60.5%(52/86),其差异有统计学意义(χ2=19.200,P<0.05)。三地正常对照组分别为：46.6%(27/58)、60.0%(33/55)和40.5%(15/37),三地对照组之间,PARP-1表达差异没有统计学意义(χ2=3.800, P=0.150)。PARP-1表达水平与AFB1-DNA加合物水平相关性分析显示：HCC组PARP-1表达水平与AFB1-DNA表达水平呈正相关(χ2=17.400,P=0.001),两者在正常对照组中相关性分析未达统计学显著性水平(χ2=0.852,P=0.356)。7.单因素及多因素分析结果显示职业、吸烟、喝酒、饮茶、在外就餐频率、食用粮油有无包装及米面保存时间显著影响南宁地区成年男性AFB1的暴露水平,其中职业、吸烟、食用散装粮油和蔬菜及水果的摄入显著影响人群的氧化应激水平。结论：1.广西南宁、柳州、桂林和宁夏银川四地区HCC人群肝组织中AFB1-DNA加合物水平存在地域差异,广西桂中南地区HCC人群AFB1暴露水平高于桂北地区及宁夏银川地区。广西三地人群普遍存在AFB1暴露的证据,提示广西地区HCC的发生与人群中AFB1的暴露有关,复杂的生活相关因素造成人群AFB1的暴露,可能增加广西地区人群患HCC风险。2.广西南宁、柳州、桂林三地区间人群肝组织8-OHdG水平无明显差异。AFB1暴露与8-OHdG表达水平无明显的相关性。3.广西南宁、柳州和桂林三地肝癌人群的hOGG1表达水平高于正常人群,三地人群中肝细胞DNA氧化损伤修复水平存在差异。在较高的AFB,暴露水平下,hOGG1表达水平与AFB1-DNA加合物水平密切相关。4. MGMT修复蛋白表达缺失造成DNA损伤修复功能下降可能在肝癌的发生过程中起到重要作用。5. PARP-1修复蛋白在肝癌人群中的表达水平高于正常人群；PARP-1的表达水平在南宁、柳州、桂林三地呈依次递减趋势。6.P53基因“突变热点”广泛存在于广西HCC患者体内,与AFB,的暴露关系密切。7.南宁地区成年男性AFB1的暴露水平和氧化应激水平受环境因素影响。更多还原

【Abstract】 Objective:To investigate the levels of Aflatoxin B1(AFB1) DNA adducts in the human liver tissues from Nanning, Liuzhou and Guilin cities in Guangxi, Yinchuang city in Ningxia, and to explore the association between AFB1exposure and DNA damage repair.Methods:Formalin-fixed and paraffin-embeded liver tissues were collected from304cases of hepatocellular carcinoma (HCC) patients from Yinchuang of Ningxia province, Nanning, Liuzhou and Guilin cities of Guangxi province. One hundred ninty cases of liver tissue samples were collected from hemageoma and biliary calculi patients as controls. Immunohistochemistry method was used to detected the levels of AFB1DNA adducts,8-OHdG, hOGG1, MGMT and PARP-1. Polymerase Chain Reaction-restriction fragment length polymorphism assay (PCR-RFLP) was applied to detected the expression of P53gene. Life-style information was collected by questionnaire from226HBsAg(-) adult males in Nanning, their levels of AAA, PCC in plasma and AFB1,8-OHdG in urine were detected by ELISA.Results:1. In HCC group, the positive levels of AFB1-DNA adducts were59.5%(47/79) in Nanning,43.8%(39/89) in Liuzhou,17.4%(15/86) in Guilin and36%(18/50) in Yinchuang of Ningxia province. Higher levels of AFB1-DNA adducts in Nanning was found when it was compared with that in Guilin and Yinchuang (P<0.05). But in control group, no significant difference was found among them (P>0.05). The positive levels of AFB1-DNA adducts were15.5%(9/58) in Nanning,16.4%(9/55)in Liuzhou、10.8%(4/37)in Guilin and12.5%(5/40) in Yinchuang of Ningxia province, respectivly.2. In HCC group, the positive levels of8-OHdG were50.6%(40/79) in Nanning,51.7%(46/89) in Liuzhou,48.8%(42/86) in Guilin. No significant difference was observed among them (P>0.05). In control group, no significant difference was found among them, either (P>0.05). The positive levels of8-OHdG were55.2%(32/58),54.5%(30/55) and64.7%(24/37) in Nanning, Liuzhou and Guilin, individually.3. In HCC group, the positive rate of "hot spot mutation " of P53gene was21.7%(55/254)(P<0.001). In Nanning, Liuzhou and Guilin, the rate of "hot spot mutation " of P53gene were26.6%(21/79)、24.7(22/89)和14.0%(12/86), respectively. No statistically different was found among them (P>0.05) Furthermore, compared with subjects which AFB1-DNA adducts was negative, higher rate of "hot spot mutation" of P53gene was observed in patients which AFB1-DNA adducts was positive (x2=8.076, P=0.004)4. In HCC group, high levels of hOGGl were75.7%(60/79)、67.4%(60/89) and60.5%(52/86) in Nanning, Liuzhou and Guilin, respectively. Compared with normal control group, the highest level of expression of hOGG1was in Nanning (P<0.05). In HCC and control group of Nanning, positive correlation was found between hOGG1and AFB1-DNA adducts (P<0.05). But no statistically significant association was observed between Liuzhou and Guilin (P>0.05)5. For levels of MGMT in HCC group,51.4%(40/79),51.7%(46/89) and47.7%(41/86) were found in Nanning, Liuzhou and Guilin city, no significant difference was found among them. Their corresponding control group was 82.8%(48/58)、76.3%(42/55) and75.7%(28/37) of MGMT, no significant difference was observed among them. But in these three cities, higher expressions of MGMT was found in control groups when compared with their corresponding HCC group (P<0.05). No statistically significant association was observed between levels of MGMT and valves of AFB1-DNA adducts(P>0.05).6. In HCC group, expressions of PARP-1were89.9%(71/79),76.4%(68/89) and60.5%(52/86) in Nanning, Liuzhou and Guilin city. Statistical difference was found (%2=19.200, P<0.05).Their corresponding control groups were46.6%(27/58),60.0%(33/55) and40.5%(15/37). No significant difference was found(χ2=3.800, P=0.150). Positive correlation was observed between PARP-1and valves of AFB1-DNA adducts in HCC groups (x2=17.400, P=0.001), but not in control groups (x2=0.852, P=0.356)7. Univariate and multivariate analysis showed that occupation, smoking, alcohol, tea, dinning-out, food grain and oil whether the packaging and save time of rice significantly affect the level of AFB1exposure in adult males in Nanning city. Moreover, occupation, smoking, consumption of bulk grain and oil,vegetables and fruits intaking, significantly affect the level of oxidative stress in the participants.Conclusions:1. There are regional differences of AFB1-DNA adducts among Yinchuang of Ningxia province, Nanning, Liuzhou and Guilin cities of Guangxi province. AFB1exposure level in the southern part of Guangxi province is higher than those in the northern part of Guangxi and Yinchuang of Ningxia province. AFB1exposure is universal in Guangxi province. These data suggest that AFB1exposure contributes to HCC prevalence in Guangxi province. Moreover, complex life-style factors caused the AFB1exposure may increase the risk of HCC in Guangxi population.2. For levels of8-OHdG in liver tissues, no significant difference was found among Nanning, Liuzhou and Guilin cities of Guangxi province. The AFB1exposure failed to show a statistically significant association with8-OHdG expression.3. Compared with normal control, higher levels of hOGG1was observed in patients with HCC in Nanning, Liuzhou and Guilin cities of Guangxi province. hOGG1showed a close association with AFB1-DNA adducts in high AFB1exposure situation.4. Declining of DNA damage repair function which resulted from deletion of MGMT repairing protein expression may play an important role in the pathogenesis of HCC.5. The level of PARP-1expression in HCC was significant higher than those in healthy control. A declining tendency was found in Nanning, Liuzhou and Guilin group for the level of PARP-1.6. The high frequency "hotspot mutation" of P53gene in HCC patients in Guangxi, play an important role in AFB1exposured popualtion.7. The level of AFB1exposure and oxidative stress in adult males in Nanning city were affected by environment.更多还原