【作者】 胡建莉；

【导师】 伍钢；

【作者基本信息】 华中科技大学 ， 肿瘤学， 2010， 博士

【摘要】 我国是全世界食管癌发病率最高的国家,年平均死亡率为14.59/10万,占全部恶性肿瘤发病率和死亡率的第四位。我国食管癌的病理与欧美不同,以鳞癌为主,占97.6%。食管癌的发生发展是一个涉及多因素、多阶段、多基因变异积累及相互作用的复杂过程,与长期吸烟、饮酒、亚硝胺类和霉菌摄入、膳食中缺乏维生素及微量元素、热损伤等因素有关,而饮酒是食管鳞癌(ESCC)发病的主要危险因素。尽管饮酒所带来的一系列医学问题已经引起全世界的普遍关注,但饮酒行为与食管癌易感性间的相互关系至今还未能引起足够重视。流行病学资料表明,慢性酒精消耗是上消化道癌(口腔癌,口咽癌,下咽癌,喉癌,食管癌)的强烈致癌因素。我国是酒类生产和消费大国,嗜酒者占一般人群14.3%。饮酒已经成为公关或社交中必不可少的一种手段。酒精的致癌效应涉及到多项机制：乙醇的局部作用、增加的乙醛结果、活性氧族(ROS)的增加、前致癌物质的活化等。慢性饮酒诱导氧化应激后ROS明显增加,而血红素加氧酶-1(HO-1)是主要的抗氧化酶,在组织和细胞的氧化应激和炎症反应中起到重要的调节作用。很多恶性肿瘤中HO-1呈高表达水平,同时伴随抗凋亡的能力提高、促进肿瘤的血管生成、导致肿瘤的转移和扩散、对治疗的敏感性降低等。然而这些变化与食管癌的关系如何,国内外鲜有研究,特别是HO-1在食管鳞癌中的表达及其与其他一些重要蛋白的共同关系(如EGFR, VEGF等),再者氧化应激后HO-1的表达水平如何,是否与饮酒后易感食管癌有关联,如何关联,都需进一步开展大样本的流行病学研究证实。此外,疾病关联的遗传研究发现,遗传可变性能影响HO-1对外源性应急的反应,个体不同的应激反应能力导致HO-1反应有很大的个体差异。深入研究个体的HO-1基因型不仅可以阐明其与疾病发生的相关性,还可为其临床应用的可行性提供理论上的依据。HO-1基因5’端启动子区域(GT)n重复多态性和T(-413)A SNP被认为是潜在的功能性多态性位点,对应激因素诱导的HO-1反应具有调节作用,从而可能在疾病的发生过程中起重要作用。那么饮酒与抗氧化酶HO-1的基因多态性有怎样的关系?HO-1启动子(GT)n重复序列多态性与食管鳞癌易感的关系?HO-1(GT)n双核甘酸多态性和HO-1 SNP的关系?目前国内外还未见阐明。本课题初步探讨了中国男性饮酒、HO-1基因(GT)n重复序列多态性与食管鳞癌易感性的关系,以期为其预防和临床治疗提供科学依据。目的：描述新诊断ESCC病人的流行病学特点,了解ESCC与饮酒、吸烟、膳食营养、体力活动、遗传等因素的关系,阐明不同的饮酒方式(饮酒量、饮酒类型、饮酒年限等)与ESCC发生的危险性。方法：采用病例-对照研究方法,以问卷调查表的形式调查新诊断ESCC病人283例和正常对照组538例饮酒、吸烟等各项指标。多元回归方法估计比值比(OR)及95%可信区间(95%CI)。结果：ESCC组中平均年龄、中度饮酒、重度饮酒、吸烟人群比例均高于对照组人群,P<0.001。所有类型的酒类品种都使ESCC的风险增加。轻度、中度、重度饮酒患ESCC的风险逐渐升高,OR分别为1.48、4.03、7.14。中度、重度饮啤酒者患ESCC的风险是增加的,ORs分别为4.19和8.83(P=0.000)。饮白酒的轻度、中度、重度组患ESCC的风险是逐步升高的,OR分别为2.33、3.96、6.29(P值分别为0.007、0.000、0.000)。饮酒年限超过40年的患食管鳞癌的风险是最高的,OR=6.31(P=0.000)。停止饮酒年限低于5年比仍然饮酒者发生食管鳞癌的风险增加,OR=3.27(P<0.001)。结论：ESCC的危险性与饮酒是强烈相关的。嗜酒特别是白酒,证实是有害的。戒酒后对身体也没有保护作用。目的：从第一部分病例对照研究的病例组中选取143例,检测食管鳞癌组织中血红素加氧酶-1(HO-1)、乏氧诱导因子-1α(HIF-1α)、表皮生长因子受体(EGFR)和血管内皮生长因子165b(VEGF165b)的表达并探讨这四种蛋白与饮酒、临床病理特征的关系及它们在食管鳞癌发生发展中的作用。方法：应用免化SP法检测HO-1、HIF-1α、EGFR和VEGF165b的表达。结果：食管鳞癌组织中HO-1、HIF-1α、EGFR和VEGF165b的阳性表达率分别为40.6%、43.4%、58%和13.3%；饮酒程度与HO-1阳性表达率呈负相关性(P=0.001),与HIF-1α和EGFR阳性表达率呈正相关性,与VEGF165b表达无相关性。HO-1阳性表达率与组织学分级呈正相关性(P=0.001),但与临床分期、纵隔淋巴结的转移没有明显的相关关系(P>0.05)。HIF-1α阳性表达率与分期、纵隔淋巴结转移及组织学分级呈正相关性(P<0.05)。EGFR阳性表达率与分期、纵隔淋巴结转移呈正相关性(P<0.05),但与分化程度没有相关性(P>0.05)。VEGF165b与分期、纵隔淋巴结转移及肿瘤分化程度无相关性(P>0.05)。HO-1阳性表达率与HIF-1α、EGFR阳性表达率呈正相关关系(P值均<0.001),与VEGF165b阳性表达率没有明显的相关性(P=0.206)。HIF-1α与EGFR呈正相关关系(P=0.040),与VEGF165b呈负相关关系(P=0.035)。EGFR与VEGF165b没有相关性(P=O.131)。结论：HO-1、HIF-1α、EGFR和VEGF165b可能与ESCC的发生发展相关；饮酒可能影响ESCC中HO-1、HIF-1α、EGFR的表达；HO-1、HIF-1α、EGFR及VEGF165b相互间存在相关性。HO-1、VEGF165b可能成为治疗的新靶点。目的：以第二部分的143例ESCC病人为研究对象,调查分析中国男性食管鳞癌患者饮酒状况；初步评价食管鳞癌个体HO-1表达水平与饮酒的关系；进一步探讨HO-1基因启动子区域两处多态性：(GT)n和T(-413)ASNP与饮酒者发生食管鳞癌的相关性,并对两处基因多态性的功能相关性进行初步探讨。方法：病例对照研究方法调查143例食管鳞癌患者及264例正常对照者。采用流式细胞仪检测食管鳞癌组和对照组个体外周血单核细胞(peripheral blood mononuclear cells, PBMCs) HO-1表达水平；提取外周血细胞DNA,采用遗传分析仪RT-PCR法对HO-1(GT)n微卫星多态性进行片段分析,采用TaqMan探针法行HO-1T(-413)ASNP基因分型；用Hardy-Weinberg遗传平衡吻合度检验方法对各基因型进行遗传平衡检验；用连锁不平衡分析软件(linkage disequilibrium analysis, LD A)对两处多态性位点进行连锁分析,并用PHASE2.0软件构建单体型；校正年龄、吸烟等因素后,分别评价这两处多态性位点以及单体型与饮酒者发生食管鳞癌的关联；并结合PBMCs胞内HO-1表达水平探讨这两处多态性位点的功能相关性。结果：(1) PBMCs HO-1表达水平：ESCC病人PBMCs胞内HO-1表达水平较正常对照组明显增高(56.8±12.05 MFIvs 35.4±22.70 MFI), P<0.05。(2)病例组和对照组的特征比较：ESCC组的年龄较对照组偏高(61.27±10.42岁vs58.05±10.00岁), ESCC组饮酒的频度和饮酒量都比对照组明显增多,P<0.001。(3)饮酒、HO-1(GT)n微卫星多态性与食管鳞癌发生的关联研究：所有的研究人群中(GT)n重复次数中23次和30次为主要的等位基因片段；ESCC组比对照组有高“L”等位基因(55.6%vs 43.7%),低“S”等位基因频率(44.4%vs56.3%), OR=1.610, 95%CI 1.21-2.15,P=0.001; ESCC组比对照组有高的携带“L”的等位基因型(S/L+L/L型)(79.7%vs 65.9%),其患ESCC的风险高,OR=2.03,95%CI 1.26-3.29,P=0.004,调整年龄、吸烟、饮酒状况后的OR为2.21(95%CI 1.30-3.78,P=0.004)。调整年龄和吸烟状态后,按照HO-1基因启动子区域(GT)n等位基因型分析了男性饮酒的五种状态(从不饮酒、曾经饮酒、轻度饮酒、中度饮酒、重度饮酒)与ESCC发病风险的关系：比较ESCC组和对照组,中度、重度饮酒组携带“L”等位基因的基因型(S/L和L/L型)比携带S/S基因型患ESCC的风险比轻度饮酒组/从不饮酒组/曾经饮酒组明显增高(中度饮酒组的OR=3.53[95%CI 1.28-9.73, P=0.015],重度饮酒组的OR=3.24[95%CI 1.14-9.23, P=0.028],轻度饮酒组的OR=1.11[95%CI 0.42-2.94,P=0.827],从不饮酒组的OR=1.15[95%CI 0.15-8.60, P=0.892],曾经饮酒组的OR=NC)。(4) T(-413)ASNP与ESCC发生的关联研究:ESCC组和对照组人群T(-413)A SNP位点各等位基因和各等位基因型的分布无显著性差异；校正年龄、吸烟等因素后的回归分析显示,携带T/A及A/A基因型相对携带T/T基因型人群发生ESCC的危险性未见显著性差异。(5)连锁不平衡分析及单体型构建：HO-1 (GT)n微卫星多态性与T(-413)A SNP位点的D’值为0.71,表示两多态性位点存在较强的连锁程度；构建的4种单体型(ST、LT、SA和LA)中,ST和LA是主要的单体型；校正年龄、吸烟后,与ST单体型相比,LT单体型与食管鳞癌的发病相关,OR值为1.75(95%CI 1.17-2.93,P=0.006)。(6)功能学评价：校正年龄、吸烟因素后,ESCC组和对照组人群PBMCs胞内HO-1表达水平随着(GT)n-L等位基因数目的增多而降低,L/L携带者PBMCs HO-1表达水平最低；在ESCC组,L/L基因型携带者比S/S基因型携带者HO-1表达水平显著降低(41.27±7.48MFIvs75.65±8.32MFI), P=0.015。T(-413)ASNP位点的不同基因型对ESCC人群中PBMCs HO-1表达水平没有明显的影响,T/T、T/A和A/A三种基因型携带者中PBMCs HO-1表达水平在ESCC组和对照组中均无显著性差异,P值分别为0.557和0.538。在轻度和中度饮酒组,L/L基因型PBMCs胞内HO-1表达比S/S基因型PBMCs胞内HO-1表达显著降低是有统计学意义的,P值分别为0.011和0.026,但这一趋势在重度饮酒组没有差异。结论：(1) HO-1(GT)n微卫星多态性位点是通过调节个体的HO-1表达水平与中国汉族男性饮酒者易感ESCC相关联：长(GT)n片段(L≥25次)携带者HO-1表达水平比短(GT)n片段(S<25次)携带者HO-1表达水平降低,其患ESCC的风险性增加。(2)重度、中度饮酒者比轻度、不饮酒者长(GT)n片段(L≥25次)携带者HO-1表达水平比短(GT)n片段(S<25次)携带者HO-1表达水平更降低,其患食管鳞癌的风险性可能增高。(3)(GT)n微卫星多态性与T(-413)ASNP位点具有相互连锁的关系,连锁后的单体型TL与ESCC发病风险相关。(4)HO-1基因启动子区域(GT)n微卫星多态性位点在调节HO-1功能方面可能起主导的作用。(5)(GT)n微卫星多态性可以作为ESCC的新型遗传标记,为ESCC的临床预防和治疗提供了理论上的指导作用。更多还原

【Abstract】 Objective:To describe the epidemiology of the newly diagnosed cases of esophagus squamous carcinoma (ESCC) and explore the correlation between ESCC and alcohol drinking and other potential carcinogen.Methods:A case-control study was conducted with 283 newly diagnosed cases and 538 normal controls by questionnaires.Results:Average age, proportion of moderate drinkers, heavy drinkers and smokers in the ESCC cases were higher than the controls (P＜0.001). All types of alcohol consumption increase the risk of ESCC. The risk of ESCC increased gradually with light, moderate and heavy drinkers, OR=1.48,4.03,7.14. The risk of ESCC at the moderate and heavy beer drinkers increased, OR=4.19 and 8.83 (P=0.000). The risk of developing ESCC increased gradually with the light、moderate and heavy white spirit drinkers, OR=2.33,3.96,6.29 (P=0.007,0.000,0.000). The individuals who had drunk for more than 40 years were at the highest risk of ESCC, OR=6.31 (P=0.000). Compared with the drinkers who still drunk, the drinkers who had given up drinking for less than 5 years were at a higher risk of ESCC, OR=3.27 (P＜0.001). Conclusions:Alcohol drinking plays an important role in risk of ESCC. Alcohol drinking, especially drinking white spirit has harmful effect on developing ESCC, but drinking a little beer doesn’t. Giving up drinking doesn’t protect the body from developing ESCC. Keywords:esophageal carcinoma; epidemiologic study; alcohol drinking; danger factorObjective:The aim of this work was to detect expression of HO-1, HIF-1α, EGFR and VEGF165b in esophagus squamous carcinoma (ESCC) tissues, and then discuss their correlations with alcohol drinking and clinical pathological features in development of ESCC.Methods:Immunohistochemistry SP method was used to detect the expression of HO-1, HIF-1α, EGFR and VEGF165b in 143 ESCC tissues.Results:Positive expression rates of HO-1, HIF-1α, EGFR and VEGF165b in ESCC tissues were 40.6%,43.4%,58%and 13.3%respectively; Extend of drinking showed negative correlation with expression rate of HO-1 (P=0.001), and positive with expression rate of HIF-1αand EGFR. Expression rate of HO-1 was positive correlation with the histological grade (P=0.001), but no correlation with the clinical stage and mediastinal lymph node metastasis (P>0.05). Positive HIF-1αexpression was correlation with clinical stage、mediastinal lymph node metastasis and differentiation of tumor (P＜0.05). Positive expression of EGFR showed correlation with clinical stage and mediastinal lymph node metastasis (P＜0.05), but no correlation with the differentiation of tumor (P>0.05). The expression rate of VEGF165b showed no correlation with clinical stage、mediastinal lymph node metastasis and differentiation of tumor (P＞0.05). The expression rate of HO-1 showed positive correlation with the expression rates of HIF-1α、EGFR (P＜0.001), but no correlation with VEGF165b (P=0.206). HIF-1αexpression rate showed positive correlation with EGFR (P=0.040), and negative correlation with VEGF165b(P=0.035). There was no correlation between EGFR and VEGF165b (P=0.274).Conclusions:The expression of HO-1, HIF-1α, EGFR and VEGF165b play an important role in the occurrence and developement of ESCC. Alcohol drinking is a significant risk factor of ESCC and has great effort on the developing ESCC by influencing the expression of HO-1, HIF-1α, EGFR. The expression of HO-1 shows correlation with HIF-1αEGFR and VEGF165b.HO-1 and VEGF165b may be a potential therapy target.Objective:To evaluate correlation between alcohol drinking and expression of HO-1, and correlation between alcohol drinking and HO-1 gene promoter polymorphism along with risk of esophageal squamous cell carcinoma(ESCC) on Chinese males and study junction of (GT)n and T(-413)ASNP.Methods:Case-control study was performed in 143 ESCC patients and 264 cancer-free controls. Intracellular HO-1 expression in PBMCs was detected by flow cytometry. Genomic DNA was extracted from venous blood samples, HO-1(GT)n microsatellite polymorphism was examined by PCR-based genotyping and DNA sequencing. HO-1 T(-413)A SNP was detected by TaqMan probes method. Hardy-Weinberg equilibrium indicated an absence of discrepancies between genotype and allele frequencies. Linkage disequilibrium (LD) between these two polymorphisms was evaluated by linkage disequilibrium analysis program (LDA) and the haplotypes were built using PHASE2.0 program. The correlation between the polymorphism sites and haplotype with the risk of ESCC of the Chinese male alcohol drinkers were valued after adjusted and the functional correlation between the two polymorphisms was discussed with the expression of PBMCs HO-1.Results:(1) Compared with the controls, HO-1 expression in PBMCs in the ESCC was higher (56.8±12.05 MFI vs 35.4±22.70 MFI, P＜0.05).(2) Age, Frequency and consumption of alcohol drinking showed a significant difference between the cases and the controls. P＜0.001.(3) Distribution of the numbers of (GT)n repearts was bimodal, with two main peak located at 23 and 30 GT repeats. The cases carried a high L (55.6%vs 43.7%) and a low S (44.4% vs 56.3%) allele frequencies compared with the controls. The cases which carried a higher "S/L+L/L" (79.7%vs 65.9%) allele frequency were at a high risk of developing ESCC, OR=2.03 (95%CI 1.26-3.29, P=0.004), adjusted OR=2.21 (95%CI 1.30-3.78, P=0.004). After adjusting for age and smoking status, we estimated the risk for esophageal cancer in five drinking categories (never/rare, ex-drinkers, light, moderate, and heavy) by HO-1 (GT)n genotype. When subjects were analyzed according to alcohol consumption, the adjusted ORs for S/L and L/L compared with S/S were higher for heavy and moderate drinkers (in heavy drinkers, OR=3.24,95%CI 1.14-9.23, P=0.028; and in moderate drinker, OR=3.53,95%CI 1.28-9.73,P=0.015) than light/never/ex-drinkers (OR 1.11,95%CI 0.42-2.94, P=0.827/OR 1.15,95%CI 0.15-8.60,P=0.892/ORNC).(4) The T(-413)A SNP alleles and their distribution between cases and controls had no significant difference; The risk of developing ESCC between the T/A and A/A carriers and the T/T carriers had no significant difference after adjusted.(5) The D’value between HO-1(GT)n microsatellite polymorphism and T(-413)A SNP was 0.71, which showed a strong linkage. Compared with the ST haplotype, the LT haplotype was associated with the developing of ESCC after adjusted, OR=1.75 (95%CI 1.17-2.93, P=0.006).(6) The expression of PBMCs HO-1 in the cases and controls decreased with the increasing of the numbers of (GT)n-L allele after adjusted. The HO-1 expression in PBMCs of L/L carrier was the lowest. The HO-1 expression in PBMCs of the L/L carriers in the cases was manifestly lower than the S/S carriers(41.27±7.48 MFI vs 75.65±8.32 MFI), P=0.015. There was no significant difference between HO-1 expression in PBMCs in the cases and the T(-413)A SNP genotypes. The expression of PBMCs HO-1 of the T/T、T/A and A/A carriers in the cases and controls had no significant difference, P=0.557 and 0.538. The expression of PBMCs HO-1 of the L/L carriers in the light and moderate drinking groups was obviously lower than the S/S carriers, P=0.011 and 0.026, but it was not found in the heavy drinking groups.Conclusions:(1) The HO-1(GT)n microsatellite polymorphism is related to the developing ESCC of the Chinese male alcohol drinkers by regulating the expression of HO-1:the expression of HO-1 of L allele carriers is lower than S allele and has a higher risk of developing ESCC.(2). HO-1 expression in PBMCs with L(GT)n in heavy and moderate drinkers is obviously lower than with S(GT)n in light drinkers and non-drinkers, and the former may have a higher risk of developing ESCC.(3) There is a linkage between HO-1(GT)n microsatellite polymorphism and T(-413)A SNP; LT haplotype was associated with the developing of ESCC.(4) HO-1(GT)n microsatellite polymorphism may play an important role in regulating function of HO-1.(5) HO-1(GT)n microsatellite polymorphism serves as a novel genetic marker of ESCC, which could have guided significance for ESCC clinical prevention and treatment.更多还原