【作者】 操敏；

【导师】 李琦； 张宗德；

【作者基本信息】 北京市结核病胸部肿瘤研究所 ， 内科学， 2008， 博士

【摘要】 目的:通过对34例正常对照者血清和113例原发性肺癌患者的血清、手术切除肿瘤组织和肿瘤远端正常肺组织的抗氧化酶和氧化代谢产物的测定,并分析组织中SODmRNA及其蛋白表达量,观察肺癌患者氧化/抗氧化失衡的特点,探讨肺癌患者氧化/抗氧化失衡的影响因素及可能的作用机制,为补充或完善肺癌发生和发展机制及肺癌的综合治疗提供依据。方法:用比色法测定患者的氧化/抗氧化酶的量及活性,包括:总抗氧化能力(total antioxidant capacity,TAOC)、总超氧化物歧化酶(total superoxide dismutase,TSOD)(包括MnSOD和CuZnSOD)、谷胱甘肽过氧化物酶(glutathione peroxidase,GPx)、过氧化氢(hydrogen peroxide,H2O2)、丙二醛(malonaldehyde,MDA),用半定量PCR和Western blot法测定患者组织中MnSOD和CuZnSOD的mRNA和蛋白表达量。结果:㈠肺癌患者氧化/抗氧化失衡的特点:1.与正常对照组相比,肺癌患者血清中TAOC、TSOD、MnSOD和GPx活性明显降低,CuZnSOD、H2O2、MDA明显增高(p<0.01)。正常人血清中仅MnSOD酶活性与MDA呈负相关,TAOC与H2O2呈正相关(p<0.01)。肺癌患者血清中H2O2与CuZnSOD、MnSOD、TAOC具有相关性,而MDA仅与GPx具有相关性(P<0.05或0.01)。2.与肿瘤远端正常肺组织相比,肺癌患者癌组织中的TAOC、TSOD、CuZnSOD、GPx活性增高,H2O2、MDA降低(p<0.01),MnSOD无改变(p>0.05)。肿瘤远端正常肺组织中的TSOD和MnSOD活性与H2O2具有相关性,GPx和MnSOD活性与MDA间具有相关性(p<0.05或0.01)。癌组织中的TSOD、CuZnSOD、MnSOD、GPx及TAOC均与MDA具有相关性,MnSOD酶活性及TAOC与H2O2间具有相关性,TAOC与TSOD、CuZnSOD、MnSOD、GPx间具有相关性(p<0.05或0.01)。㈡肺癌患者氧化/抗氧化失衡的影响因素:1.与肺鳞癌组相比,肺腺癌组血清中TSOD和CuZnSOD活性降低,H2O2增高;肺腺癌组癌组织中的氧化代谢产物H2O2、MDA增高(p<0.05或0.01)。2.不同细胞分化程度、血CEA正常与增高、CKH阴性和阳性、CKL阴性和阳性的肺癌患者血清和癌组织中氧化/抗氧化指标在两组间比较差异不显著(p>0.05)。3.不同TNM分期的肺癌患者血清中的氧化/抗氧化指标间差异不显著(p>0.05);Ⅲ+Ⅳ期肺癌患者癌组织中的MnSOD活性较Ⅰ+Ⅱ期患者增高(p<0.05)。4.免疫组化CEA阳性的肺癌患者血清GPx活性较阴性者降低,癌组织中的SOD活性增高(p<0.05)。5.P63阳性的肺癌患者血清MDA较阴性患者增高(p<0.01)。癌组织中的氧化/抗氧化指标在组间比较差异不显著(p>0.05)。6.TTF-1阴性和阳性的肺癌患者血清的氧化/抗氧化指标间差异不显著(p>0.05)。TTF-1阳性的肺癌患者癌组织中的CuZnSOD和MDA高于TTF-1阴性的肺癌患者(p<0.05)。7.分别以正常对照组血清和肿瘤远端正常肺组织各项指标的平均值±1倍标准差作为异常分界值,⑴肺腺癌组的血清TSOD<54.66 U/ml的发生率为50%、癌组织TSOD>76.86 U/mg的发生率为38%,高于肺鳞癌组(p<0.05或0.01)。Ⅲ+Ⅳ期组、TTF-1阳性组的癌组织TSOD>76.86U/mg的发生率分别为38.3%和51.7%,经卡方检验有统计学意义(p< 0.01)。Logistic回归显示,病理组织学类型、TNM分期与组织TSOD的异常发生密切相关(p< 0.05)。⑵鳞癌组的血清CuZnSOD>32.89 U/ml的发生率为85.2%,高于腺癌组。TTF-1阳性组的癌组织CuZnSOD>53.67 U/mg的发生率为51.7%,高于TTF-1阴性组(p<0.05或0.01)。⑶Ⅲ+Ⅳ期组的癌组织MnSOD>31.52U/mg的发生率为25.5%,高于Ⅰ+Ⅱ期组(p< 0.01)。⑷癌组织GPx>20.62AU的发生率在低分化组达100%,在CEA阳性组为44.1%,经卡方检验有统计学意义(p< 0.01)。⑸腺癌组的血清H2O2>102.42 mmol/L和组织H2O2<24.78 mmol/g的发生率均>40%,鳞癌组的组织H2O2<24.78mmol/g发生率为66.7%,经卡方检验有统计学意义(p< 0.01)。⑹组织MDA<0.36nmol/mg的发生率在肺鳞癌组为44.4%,在CEA阴性组和TTF-1阴性组分别为52.2%和50.0%,经卡方检验有统计学意义(p< 0.01)。Logistic回归显示,TTF-1与组织MDA的异常发生密切相关(p< 0.05)。⑺血清和癌组织的TAOC的异常率在各组间差异均无显著性(p>0.05)。㈢肺癌患者癌组织和肿瘤远端正常肺组织的MnSOD和CuZnSOD的mRNA及蛋白表达量的分析:1.与肿瘤远端正常肺组织相比,肺癌患者癌组织中MnSOD mRNA和蛋白表达量均降低,CuZnSOD mRNA和蛋白表达量均增高(p<0.01)。2.MnSOD、CuZnSODmRNA和CuZnSOD的蛋白表达量在不同细胞类型组、不同组织分化程度组、不同TNM分期组、血CEA正常和增高组、免疫组化CKH、CKL、CEA、P63阴性和阳性组间差异均无显著性(p>0.05)。MnSOD的蛋白表达量仅在TTF-1阳性组高于TTF-1阴性组(p<0.05)。癌组织的MnSODmRNA低表达量率在肺鳞癌组可达94.1%,高于腺癌组(P<0.01)。MnSOD蛋白的低表达量率在低分化肺癌组为36.4%,高于中+高分化组(P<0.01)。结论:㈠肺癌患者存在着氧化/抗氧化失衡,其特点表现为:1.血清中,除CuZnSOD外,抗氧化酶活性降低,氧化代谢产物增高。其中,TSOD降低与MnSOD降低相伴随,抗氧化酶与氧化代谢产物间具有一定的相关性。显示血清抗氧化酶活性异常及氧化代谢产物蓄积与肺癌发生或存在有关。2.癌组织中,除MnSOD外,抗氧化酶活性高于肿瘤远端正常肺组织,氧化代谢产物含量较低。其中,TSOD的增高与CuZnSOD的增高相伴随。肺癌组织中TAOC和氧化代谢产物及MDA和抗氧化酶间均呈正相关,MnSOD与H2O2也呈正相关。显示癌组织中因各种因素诱导产生的抗氧化酶活性增高,尤其是CuZnSOD的增高可能与肺癌的发生和发展相关。㈡肺癌患者氧化/抗氧化失衡主要与下列因素有关:1.肺癌病理类型:与肺鳞癌患者相比,肺腺癌患者血清总SOD和CuZnSOD酶活性降低,H2O2增高。而其腺癌组织中的氧化代谢产物H2O2、MDA增高。2.TNM分期:Ⅲ+Ⅳ期肺癌患者癌组织中的MnSOD活性及TSOD和MnSOD异常率较Ⅰ+Ⅱ期肺癌患者增高。显示MnSOD在肺癌发展中的作用。3.肿瘤标记物:免疫组化CEA阳性的患者血清GPx增高,p63表达阳性的患者血清MDA的含量增高,TTF-1表达阳性的患者癌组织中CuZnSOD和MDA均较阴性组增高,提示CEA、p63、TTF-1可能分别为GPx、CuZnSOD和脂质过氧化反应的诱导物之一。㈢肺癌组织中的MnSODmRNA和蛋白表达量低于肿瘤周围正常肺组织,而CuZnSODmRNA和蛋白表达量高于肿瘤周围正常肺组织。其中,CuZnSOD的蛋白含量与酶活性在癌组织中的改变相一致。此外,肺鳞癌组织中MnSODmRNA的低表达率高于腺癌,低分化肿瘤的MnSOD蛋白的低表达率高于中、高分化肿瘤。提示不同细胞类型可造成MnSODmRNA表达的差异,而MnSOD蛋白的低表达与肿瘤分化程度相关。更多还原

【Abstract】 Objects:The changes of oxidants and antioxidants in the activities,the expression of mRNA and protein were evaluated in 57 serum,113 lung tumor and 58 tumor-free lung tissues collected between the years 2006 and 2007 from 113 individuals with surgically resectable lung cancer.To elucidate the factors that have the effect on the imbalance of the oxidant and antioxidant system,to explore the mechanism that induce the imbalance,to consummate the mechanism of the onset,development and treatment of lung cancer.Methods:The activities of TAOC,TSOD,MnSOD,CuZnSOD,GPx,H2O2 and MDA were determined by chromometry.MnSOD,CuZnSOD mRNA and protein expression were evaluated by reverse transcription polymerase chain reaction and Western analysis in tumor,as well as corresponding control tissues.Resulst:1.The cha- racteristics of the oxidants and antioxidants in the patients with lung cancer were:①To compare with the controls,the activities of TAOC,TSOD,MnSOD and GPx were lower obviously in serum,CuZnSOD activity and H2O2,MDA were higher(p< 0.01).In the control serum MnSOD activity was negativly correlated to MDA, TAOC activity was positivly correlated to H2O2(p<0.01).In the patients’serum H2O2 had the correla- tion with the activities of CuZnSOD,MnSOD and TAOC.MDA was correlated to GPx activity(P<0.05 or 0.01).②To compare with the level of these indexes in the tumor- free tissues,the activities of TAOC, TSOD, CuZnSOD, GPx were higher, H2O2 and MDA were lower in the tumor tissues(p<0.01), the activity of MnSOD was similar be- tween the groups(p>0.05).In the tumor-free tissues H2O2 was correlated to the activi- ties of TSOD and MnSOD,MDA was correlated to the activities of GPx and MnSOD (p<0.05 or 0.01). In the tumor tissues the activities of TSOD,CuZnSOD,MnSOD,GPx and TAOC had the correlation with MDA, the activities of MnSOD and TAOC had the correlation with H2O2,TAOC activity was correlated to the activities of TSOD, CuZnSOD,MnSOD,GPx(p<0.05 or 0.01).2.The factors that had the effects on the imbalance of the oxidants and antioxidants in the patients with lung cancer were:①To compare with the group of the squamous cell carcinoma,the activities of TSOD and CuZnSOD were lower and H2O2 was higher in the serum of the group of adeno- carcinoma; while H2O2,MDA were higher in the tumor tissues of the group of adeno- carcinoma (p<0.05 or 0.01).②The activities of oxidants/antioxidants in the serum and tumor tissues were not correlated to the cell differentiation, serum CEA,CKH and CKL immunohistochemical staining (p>0.05).③There was no relation between the activities of oxidants/ antioxidants and the TNM stages in the serum(p>0.05).MnSOD activity in the tumor tissues of stageⅢ+Ⅳwas higher than that of stageⅠ+Ⅱ(p< 0.05).④The indexes of immunohistochemical staining:⑴To compared with the group of the negative CEA,GPx activity was lower in the serum and SOD activity was high- er in the tumor tissues of the group with the positive CEA (p<0.05).⑵MDA was higher in the serum of the group with positive P63 than that of the group with negative P63 (p<0.01).⑶The activities of CuZnSOD and MDA in the tumor tissues of the group with positive TTF-1 were higher than those of the group with negative TTF-1(p<0.05).⑤The mean values plus or minus standard deviation of the indexes in the control serum and tumor-free tissues were used as the dividing margin to discriminate the normal or the abnormal value.⑴The incidence of lower TSOD in the serum of the group of adenocarcinoma was 50%,the incidence of higher TSOD in the tumor tissues was 38%,which were higher than the incidence of the group of squa- mous cell carcinoma(p<0.05 or 0.01).The incidence of higher TSOD in the tumor ti- ssuees of the group of stageⅢ+Ⅳand positive TTF-1 was 38.3% and 51.7% separa- tly (p<0.01).There was closed relation between the pathohistology,TNM classification and abnormal ratio of TSOD in the tumor tissues by Logistic regression analysis(p< 0.05).⑵The incidence of higher CuZnSOD in the serum of the group of squamous cell carcinoma was 85.2%.The incidence of higher CuZnSOD in the tumor tissues of the group with positve TTF-1 was 51.7 %(p<0.05 or 0.01).⑶The incidence of higher Mn- SOD in the tumor tissues of the group of stageⅢ+Ⅳwas 25.5%(p< 0.01).⑷The incidence of higher GPx in the tumor tissues of the group of low differentiation was 100%, the incidence of higher GPx in the tumor tissues of positive CEA was 44.1% (p<0.01).⑸The incidence of higher H2O2 in the serum of the group of adenocar- cinoma and lower H2O2 in the tumor tissues was more than 40%, and the incidence of lower H2O2 in the tumor tissues of the group of squamous cell carcinoma was 66.7% (p< 0.01).⑹The incidence of lower MDA in the tumor tissues of the group of squam- ous cell carcinoma was 44.1%, the incidence of lower MDA in the tumor tissues of the group with negative CEA and TTF-1 was 52.2% and 50% separately (p<0.01). There was closed relation between the TTF-1 and abnormal ratio of MDA in the tum- or tissues(p<0.05).⑺There was no significant difference in the groups with abnormal incidence of TAOC in the serum and the tumor tissues.3.The results of the expression of MnSOD and CuZnSOD mRNA and protein in tumor and tumor-free tissues are that:①There was less MnSOD mRNA and protein in tumor than in tumor-free lung.There was more CuZnSOD mRNA and protein in tumor than in tumor-free lung tissue(p<0.01).②There was no relation between the expression of MnSOD,CuZnSOD mRNA and protein and the pathohistology,histology differentiation,TNM stages,CEA in the serum,immunohistological staining CKH,CKL,CEA,P63(p>0.05).MnSOD prot- ein expression in the group with positive TTF-1 was higher than that in the the group with negative TTF-1(p<0.05).The incidence of lower MnSOD mRNA was 94.1% in squamous cell carcinoma(p<0.01).The incidence of lower MnSOD protein was 36.4% in the low differentiation.(p<0.01).Conclusion:㈠The characteristics of the imbalance between the oxidants and antioxidants in the patients with lung cancer are included that:1.In the serum the levels of antioxidants are higher except CuZnSOD and the oxidants are lower.The lower activity of TSOD is companied with that of MnSOD. The activities of some antioxidants are correlated to the contents of oxidants.It can be concluded that the abnormal activities of antioxidants and the accumulation of oxid- ants are correlated to the onset and development of lung cancer.2.The activites of antioxidants are higher in tumor than in tumor-free lung except MnSOD,while the contents of oxidants are lower.The higher TSOD activity is companied with higher CuZnSOD activity.The activities of some antioxidants are correlated to the contents of oxidants. It can be concluded that antioxidants activities induced by many factors are higher in the tumor tissues,especial CuZnSOD activity,and are correlated to the onset and development of lung cancer.㈡The factors that have the relation with the im- balance of oxidants/antioxidants are included that:1.The cell type of pathology.To compared with the squamous cell carcinoma,the activities of TSOD and CuZnSOD in serum are lower,H2O2 is higher,while H2O2 and MDA are higher in the tumor tissues of the adenocarcinoma.2.TNM stage.The activity of MnSOD and the incidence of higher TSOD and MnSOD in the tumor tissues of stageⅢ+Ⅳis higher than that of stageⅠ+Ⅱ.These demonstrate the effect of MnSOD on the development of lung cancer3.Tumor markers.By immunohistochemical staining,GPx activity is higher in the serum of the patients with positive CEA,MDA content is higher in the serum of the patients with positive p63, CuZnSOD activity and MDA is higher in the tumor tissues of the patients with positive TTF-1 than that of the patients with negative TTF-1.These demonstrate that CEA,p63,TTF-1 may be the inducers of GPx, CuZnSOD and lipid peroxidants.㈢To compare with the levels of the indexes in the tumor-free tissues,the expression of MnSOD mRNA and protein is lower,the expression of CuZnSOD mRNA and protein is higher in the tumor tissues.The higher expression of CuZnSOD protein is coincident with the change of its activity.The incidence of the lower MnSOD mRNA expression in the tumor tissues of the squm- ous cell carcinoma is higer than that of the adenocarcinoma.The incidence of the lower MnSOD protein expression in the low differentiation is higher than that in the middle and high differentiation.It can be illustrated that the cell type of pathology can cause the different expression of MnSOD mRNA,the lower expression of MnSOD protein is correlated to the degree of the differentiation.更多还原