【作者】 陈罡；

【导师】 罗殿中；

【作者基本信息】 广西医科大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 目的诱捕受体3(Decoy receptor 3,DcR3)是肿瘤坏死因子受体超家族的新成员之一,在多种恶性肿瘤中呈现过表达。本文通过研究原发性肝细胞癌(hepatocellular carcinoma,HCC)组织及肝癌细胞系中DcR3 mRNA和蛋白的表达情况及其与临床病理特征之间的关系,阐明DcR3在肝癌发生、发展及复发转移过程中的作用;同时探讨DcR3表达与肝癌细胞凋亡的关系;并且观察将DcR3中和性抗体加入表达该基因的肝癌细胞系后,肝癌细胞生长增殖、运动迁移、凋亡及对顺铂敏感性等各种细胞生物学特性的变化。材料与方法①应用半定量逆转录-聚合酶链反应RT-PCR方法,检测DcR3 mRNA在69例肝癌及癌旁组织中的表达和相对含量,分析DcR3 mRNA的相对含量与肝癌临床病理特征的关系。②125例HCC、48例癌旁、64例肝硬化组织和25例正常肝组织构建肝癌组织微阵列。应用免疫组织化学方法(EnVision法)检测DcR3蛋白的表达水平,并分析其与HCC临床病理特征的关系。③应用半定量RT-PCR,免疫组织化学,脱氧核糖核酸末端转移酶介导的缺口末端标记(TUNEL)及DNA电泳技术检测39例HCC及癌旁肝组织中DcR3mRNA及蛋白的表达和凋亡情况,并分析DcR3表达与细胞凋亡的关系。④RT-PCR分析人肝癌细胞系HepG2,HepB3,SMMC-7721,bel-7402,bel-7405,癌旁肝细胞系QSG-7701及正常肝细胞系HL-7702 DcR3的mRNA水平;免疫细胞化学检测各个细胞系的DcR3蛋白表达水平;将DcR3中和性抗体加入肝癌细胞系中,通过MTT检测不同浓度DcR3中和性抗体作用于不同肝癌细胞存活率,挑选实验细胞株及DcR3中和性抗体最佳作用时间和作用浓度;免疫细胞化学检测DcR3中和性抗体加入后肝癌细胞DcR3蛋白表达差异;通过绘制细胞生长曲线以及集落形成实验来分析生长增殖特性;流式细胞仪(FCM)检测该抗体对细胞周期的影响;通过FCM,TUNEL染色,DNA ladder,电镜观察分析其对凋亡的影响;通过划痕实验分析DcR3对肝癌细胞迁移能力的影响;检测加入DcR3中和性抗体后hepG2对化疗药物顺铂敏感性的改变。结果①69例原发性肝细胞癌组织中,67例标本可扩增出426bp的条带,DcR3mRNA的阳性表达率为97.10%,明显高于癌旁组织DcR3 mRNA的阳性表达率76.81%(53/69,P＜0.01);癌组织中DcR3 mRNA相对含量明显高于癌旁组织(P＜0.01);序列分析发现其两例病例序列mRNA与源序列比较均有4个不同的碱基改变;DcR3 mRNA表达与转移复发和肿瘤大小有关,与年龄、性别、分化程度、临床分期、有无肝硬化、AFP、门脉癌栓、包膜浸润、肿瘤结节及HBsAg无明显相关。②组织微阵列利用率为96.56%(253/262);HCC组织中的DcR3蛋白的阳性率73.33%(88/120)明显高于癌旁54.17%(26/48,P=0.017)、肝硬化组织37.10%(23/62,P=0.000)以及正常肝组织8.70%(2/23,P=0.000)。癌旁组织和肝硬化组织中的DcR3蛋白阳性率明显高于正常肝组织(P=0.000,P=0.005);HCC中临床TNM分期Ⅰ,Ⅱ期DcR3阳性率61.76%(42/68)明显低于Ⅲ,Ⅳ期88.46%(46/52,P=0.001);HCC中无转移组DcR3阳性率58.82%(20/34)明显低于转移组96.67%(29/30,P=0.000);DcR3表达率在AFP≥400μg/L组80.82%(59/73)、有门脉癌栓组91.30%(42/46)、包膜浸润组84.34%(70/83)和多个肿瘤结节组89.13%(41/46)分别明显高于AFP＜400μg/L组61.70%(29/47,P=0.021)、无门脉癌栓组62.16%(46/74,P=0.000)、无包膜浸润组48.65%(8/37,P=0.000)及单个肿瘤结节组63.51%(47/74,P=0.002)。DcR3表达与年龄、性别、分化程度、有无肝硬化及肿瘤直径无关。③39例肝癌组织中,细胞凋亡指数(AI)及DNA ladder阳性率均明显低于癌旁组织(P=0.000);肝细胞凋亡与转移、临床分期和包膜浸润有关,与年龄、性别、分化程度、有无肝硬化、AFP、门脉癌栓、肿瘤大小、肿瘤结节及HBsAg无关。相关性分析显示AI与DcR3 mRNA及蛋白表达均呈负相关(r=-0.34,P=0.002;r=-0.679,P=0.000),与DNA ladder阳性率呈正相关(r=0.62,P=0.000)。DcR3 mRNA与蛋白表达呈正相关(1=0.419,P=0.000)。④5种肝癌细胞系中均有DcR3mRNA和蛋白的表达,癌旁肝细胞系QSG-7701及正常肝细胞系HL-7702均无DcR3mRNA和蛋白的表达;选择HepG2为实验靶细胞,DcR3中和性抗体最佳实验浓度为0.8mg/L,作用时间48小时;加入DcR3中和性抗体后,HepG2肝癌细胞株DcR3蛋白表达明显减弱;加入DcR3抗体的细胞其生长增殖能力和集落形成能力均显著下降(P＜0.01);DcR3中和性抗体使细胞周期阻滞在G1/S期(P＜0.01)。同时凋亡明显增加(P＜0.01);细胞迁移能力也明显被抑制(P＜0.01);加入DcR3中和性抗体后顺铂作用下肝癌细胞细胞增殖存活率明显下降(P＜0.01)。结论①原发性肝细胞癌组织和肝癌细胞系中,DcR3 mRNA和蛋白均表达增高。DcR3 mRNA和蛋白的高表达在原发性肝细胞癌的发生、发展及转移复发过程中可能发挥重要的作用;②原发性肝细胞癌DcR3 mRNA表达产物可能存在碱基改变;③应用组织芯片大规模高效检测临床组织样本是可行的,具有快速、方便、经济、准确的特点;④DcR3的高表达可抑制凋亡;⑤DcR3中和性抗体能显著抑制肝癌细胞的生长和迁移并促进凋亡的发生并且能增加对顺铂的敏感性,提示该基因在肝癌的发生发展、凋亡及浸润转移中发挥重要作用,并有望在临床治疗中广泛应用。更多还原

【Abstract】 ObjectiveDecoy receptor 3(DcR3),a newly identified member of the tumor necrosis factor receptor(TNFR)super-family,is over-expressed in many various kinds of malignant tumors.The objective of the research is to investigate the expression of DcR3 mRNA and protein in hepatocellular carcinoma(HCC)tissues and HCC cell lines and their correlations with clinicopathologic features,and to elucidate its role in tumorigenesis,development,recrudescence and metastasis, as well as to explore the relationship between the expression of DcR3 and the apoptosis in HCC.Then to observe the change of the biological characteristics(growth and proliferation,movement and transference,apoptosis,sensitivity to CDDP)in the hepatocellular carcinoma cell line with positive DcR3 expression adding the neutralization antibody DcR3.Materials and Methods①The expression of DcR3 mRNA was detected by semi-quantitive reverse transcription-polymerase chain reaction(RT-PCR)in 69 cases of HCC and the adjacent-tumor liver tissues.The relationship between the relative content of DcR3 mRNA and clinicopathological features of HCC was analyzed.②The tissue microarrays comprised 125 cases of HCC tissues, 48 adjacent-tumor liver tissues,64 liver cirrhosis tissues and 25 normal liver tissues,Immunohistochemistry(EnVision method)was employed to detect the expression of DcR3 proteins.Statistic analysis was made to figure out the correlation between the expression and the clinicopathological features of HCC③The mRNA and protein expression of DcR3 were detected in 39 cases of HCC and their para-tumor tissues by RT-PCR and immunohistochemistry,respectively.The apoptosis was evaluated by the method of terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL)and DNA agarose electrophoresis.Statistic analysis was made to figure out the correlation among the DcR3 expression,apoptosis and the clinicopathological features of HCC.④Human hepatocellular carcinoma cell lines:HepG2,HepB3, SMMC-7721,bei-7402,bel-7405:liver cell line of para- tumor tissues QSG-7701 and normal liver cell line HL-7702 were harvested without any stimulation.The mRNA expression of DcR3 was detected by RT-PCR and the expression of DcR3 protein was detected by immunocytochemistry.The neutralization antibody DcR3 was added into different liver cancer cell lines and the cell livability rate was detected to decide the cell line for the following experiments and also to search the best working time and concentration of the neutralization antibody DcR3 by MTT assay.The difference of the DcR3 protein expression in hepatocellular carcinoma cell line was checked by immunocytochemistry after adding the neutralization antibody DcR3.The cellular proliferation was examined by cell growth curve and colony formation assay;The changes of cell cycle of cells were analyzed by flow cytometry(FCM);The apoptosis was explored by FCM, DNA ladder,TUNEL and electron microscope;The transferring ability was investigated by the wound healing test;And the sensitivity to Cisplatin(CDDP)of the hepatocellular carcinoma cell line was detected by MTT assay with neutralization antibody DcR3.Results①In 69 cases of HCC tissues,426 bp bands were detected in 67 cases,the positive rate of DcR3 mRNA expression was 97.10%,which is significantly higher than that in adjacent-tumor liver tissues 76.81%(53/69,P＜0.01).The relative content of DcR3 mRNA in HCC was significantly higher than that in the adjacent-tumor liver tissues (P＜0.01).Sequencing analysis showed 4 different point mutations in the DcR3 gene mRNA of HCC in two cases of positive amplification products.The expression was associated with metastasis and the tumor size but had no correlation with age,gender,differentiation grades,clinical stages,cirrhosis,AFP,portal vein tumor embolus,capsular infiltration,tumor nodes or HBsAg.②There were 253 cases that could be used in the tissue microarrays(utilization rate was 96.56%,253/262);The positive rate of DcR3 in HCC tissues was 73.33%(88/120),significantly higher than that in the adjacent-tumor liver tissues 54.17%(26/48, P=0.017),cirrhosis tissues 37.10%(23/62,P=0.000)and the normal liver tissues 8.70%(2/23,P=0.000).The positive rate of DcR3 in adjacent-tumor tissues and cirrhosis tissues were both significantly higher than that in the normal liver group(P=0.000,P=0.005); The positive rate of DcR3 in HCC tissues in the clinical TNMstageⅠ,Ⅱwas 61.76%(42/68),obviously lower than that in the stageⅢ,Ⅳ88.46%(46/52,P=0.001);The positive rate of DcR3 in the cases without metastasis within 20 months was 58.82%(20/34), significantly lower than that in the group with metastasis 96.67% (29/30,P=0.000);The positive rates DcR3 in the groups of AFP≥400μg/L 80.82%(59/73),portal vein tumor embolus 91.30% (42/46),capsular infiltration 84.34%(70/83)and multiple tumor nodes 89.13%(41/46)were significantly higher than that in the groups of AFP＜400μg/L 61.70%(29/47,P=0.021),without tumor embolus 62.16% (46/74,P=0.000),with no capsular infiltration 48.65%(8/37, P=0.000)and single node 63.51%(47/74,P=0.002).It was not associated with patients’ age,sex,histological classification, cirrhosis or tumor size.③In 39 cases of HCC tissues,the apoptosis index(AI)and the positive rate of DNAladder in HCC were both significantly lower than that in the non-cancerous group(P=0.000).The AI was related to metastasis,clinical TNM stages and capsular infiltration but had no correlation with age,gender,histological differentiation grades,cirrhosis,plasma AFP levels,portal vein tumor embolus, tumor sizes,number of the tumor nodes or HBsAg.The relative analysis showed that there were negative correlations between AI and the expression of DcR3 mRNA and protein in HCC(r=-0.34,P=0.002; r=-0.679,P=0.000).There was positive correlation between the positive rate of DNA ladder and the expression of DcR3 mRNA and protein in HCC(r=0.62,P=0.000).The expression of DcR3 mRNA was positively related to the expression of protein(r=0.419,P=0.000).④The all of 5 kinds of human hepatocellular carcinoma cell lines expressed DcR3 in mRNA and protein,while QSG-7701 and HL-7702 did not.HepG2 was chosen as the target cell lines for the experiments with the best concentration of neutralization antibody DcR3 0.8mg/L, best time 48h.The DcR3 protein expression became weakening in HepG2 after the DcR3 neutralization antibody DcR3;The cell viability, growing speed and colony formation ability were decreased obviously by adding DcR3-Ab(P＜0.01).The progression of the cell cycle was arrested in Gl/S-phase remarkably induced byDcR3-Ab(P＜0.01); The apoptosis was also significantly increased(P＜0.01);The transferring ability was significantly inhibited(P＜0.01)and the livability of the liver cells with CDDP was decreased by DcR3-Ab.Conclusions①DcR3 mRNA and protein are both over-expressed in HCC tissues and cell lines.The over-expression of DcR3 mRNA and protein may play an important role in the process of carcinogenesis,progression, recrudescence and metastasis in HCC;②Point mutation of the DcR3 gene might exist in HCC;③Tissue microarrays techniqueis a feasible,rapid,economic and accurate approach for screening clinical tissue specimens on a large scale;④The over-expression of DcR3 might inhibit the apoptosis of HCC cells;⑤DcR3 neutralization antibody can restrain the growth and movement,accelerate the apoptosis of the HCC cells,meanwhile increase the sensitivity to CDDP.The DcR3 gene plays an important role in origination,progression and metastasis of liver cancer and it is hopeful to be applied in the future clinical therapy to hepatocellular carcinoma.更多还原