Preliminary Study of Oxidative Stress in Human Hepatocellular Carcinoma and Adjacent Normal Liver Tissues

OBJECTIVE The antioxidative system in human hepatocellular carcinoma was investigated. METHODS The activities of cytosolic catalase (CAT), superoxide dismu-tase, glutathione peroxidase (GSH-Px), glutathione S-tranferase and levels of reduced glutathione, total protein thiols and malondialdehyde were assayed in 10 cases of hepatocellular carcinoma and adjacent normal liver. RESULTS Hepatoma tissues showed higher activities of CAT, GSH -Px and lower content of total antioxidative capacity compared to adjacent normal liver tissue (P<0.05). CONCLUSION These findings suggest that the antioxidative defense-related enzymes and antioxidants are largely regulated in hepatoma cells. However, the mechanism which is not clear requires further investigation.     

Expression of cell cycle regulator genes in KB, a human squamous cell carcinoma cell line, after irradiation

DNA damage induced by irradiation causes overexpression of the p53 gene, and subsequently the upregulation of p53 downstream genes involved in cell cycle modification. Irradiated malignant cells which possess wild-type p53 have been known to undergo G1 arrest due to p21/Cip1/Waf1 upregulation. Other p53 downstream genes related to the modification of the cell cycle such as gadd45 may cause G2 arrest. Many of the genes which regulate the cell cycle progression have been identified, including the G1 phase specific ink4 family of cyclin-dependent kinase inhibitors (CDK-I), another group of CDK-Is, which affect the cyclin-CDK complexes ubiquitously, and S/G2 accelerator genes. The sequential changes in these cell cycle regulator genes after irradiation has not been clarified. We analyzed the appearance of the apoptotic fraction and cell cycle perturbation after irradiation using KB, a human squamous cell carcinoma line derived from oral floor, and examined the alteration of gene expression for cell cycle regulator genes. The KB cells proceeded to undergo apoptosis in a time and dose dependent manner after irradiation and showed G2 arrest accompanied by upregulation of p53, ubiquitous CDK-Is, and S and G2 accelerator genes.     

Expression of ras and myc oncogenes in human hepatocellular carcinoma and non-neoplastic liver tissues

An immunohistochemical assay was used to assess expression of ras p21 and myc p62 oncogene products in human hepatocellular carcinoma (HCC) and non-neoplastic liver tissues. The monoclonal antibodies Y13 259 and Myc1-9E10, specific for ras p21 and myc p62 oncoproteins, were employed on paraffin-embedded sections. Most HCCs showed enhanced ras p21 and myc p62 expression, as indicated by staining intensity. Cirrhotic livers revealed increased myc p62 and occasionally increased ras p21 expression. HBsAg+ hepatocytes showed intense immunostaining for ras p21. Fibrotic, cholestatic, fetal and normal adult liver did not present enhancement of oncoprotein production. We suggest that combined over-expression of ras and myc oncoproteins may be important for the malignant phenotypic alteration in human HCC.     

CD44在肝细胞癌及癌周组织中的表达及其意义

 目的 研究CD44s及CD44v6蛋白在人肝细胞癌组织中表达及其意义。方法 应用免疫组化方法检测42例人肝细胞癌及其癌周组织与10例正常肝组织标本的CD44s及CD44v6表达情况。结果 CD44s及CD44v6在肝癌组织中表达明显高于癌周及正常肝组织，与门静脉内癌栓、包膜浸润及分化程度密切相关（分别为P＜0.01，P＜0.05，P＜0.05）。生存时间2年及2年以上组CD44s及CD44v6阳性表达分别为17.6 %（3／17），5.8 %（1／17），生存时间短于2年分别为52.6 %（10／19），42.2 %（8／19）（P＜0.05）。结论 肝细胞癌组织中CD44的表达与细胞分化、浸润转移密切相关，其阳性表达有助于判断预后。     

大肠癌生长抑制基因ST13在恶性肿瘤及其相邻正常组织中的表达

目的 研究恶性肿瘤及其相邻正常组织中ＳＴ１３基因的表达差异。方法 本研究采用逆转录聚合链反应酶标免疫法（ＲＴ－ＰＣＲ－ＥＬＩＳＡ）对５７例恶性肿瘤（其中大肠癌２０例，胃癌１１例，乳腺癌１０例，肝癌９例，肺癌７例）及其相邻正常组织中ＳＴ１３基因的表达进行检测。结果 ＳＴ１３基因在大肠癌，胃癌中呈低表达趋势，平均值，大肠癌１．１９６，相邻正常组织０．９０２，Ｐ＝０．０９。胃癌０．９０９，相邻正常组织０     

细胞分裂周期7蛋白在肝癌组织的表达及其对肝癌细胞增殖和侵袭的影响

目的 检测细胞分裂周期7(CDC7)蛋白在人肝癌组织的表达,探讨CDC7过表达对肝癌细胞增殖和侵袭的影响。方法 实时定量聚合酶链式反应(Real-time PCR)检测CDC7 mRNA在肝癌组织和癌旁组织的表达,检测CDC7 mRNA在肝癌细胞系和正常肝脏细胞中的表达。Western blot检测CDC7蛋白在肝癌组织和癌旁组织中的表达,检测CDC7蛋白在肝癌细胞系和正常肝脏细胞中的表达水平。利用慢病毒载体构建稳定过表达CDC7的肝癌HCCLM3和SMMC 7721细胞株。细胞克隆实验和CCK-8法检测CDC7过表达对肝癌细胞增殖的影响。Transwell实验和划痕实验检测CDC7过表达对肝癌细胞侵袭迁移的影响。结果 与癌旁组织相比,CDC7 mRNA和蛋白在肝癌组织中表达水平显著升高(P＜0.001);与正常肝细胞相比,肝癌细胞系的CDC7 mRNA和蛋白表达水平显著增高(P＜0.001);CCK-8法、细胞克隆实验说明CDC7过表达会明显增强肝癌细胞的增殖能力;划痕实验、Transwell实验说明CDC7过表达会明显提高肝癌细胞的侵袭能力。结论 CDC7蛋白在人肝癌组织高表达,其过表达促进肝癌细胞的增殖和侵袭能力。     

肝癌组织CT抗原基因CSAG1表达及体外对肝癌细胞生长的影响

目的:探讨CSAG1(chondrosar-coma associated gene 1)基因在肝癌组织中的表达及其对肝癌细胞生长增殖的影响,为了解肝癌发生的分子机制奠定理论基础.方法:利用半定量RT-PCR技术检测CSAG1基因在12例肝癌和癌旁组织中的表达差异;RNA干扰技术沉默肝癌细胞株(Focus)内CSAG1基因的表达;利用CCK-8、克隆形成实验以及流式细胞仪分别定量分析内源性CSAG1基因被沉默前后的Focus细胞生长曲线、克隆形成能力以及细胞周期的变化.结果:与癌旁组织相比较,CSAG1基因在75%(9/12)肝癌组织中的表达量明显上调;沉默肝癌细胞株Focus中内源性CSAG1基因表达后,Focus细胞的生长明显减慢,克隆形成能力明显降低,S期细胞明显减少.结论:CSAG1可能是一个新的肝癌相关的促进基因,对其进行深入的研究有可能发现肝癌发病的新机制,也有可能发现肝癌治疗的新靶点.     

Expression and localization of vascular endothelial growth factor receptors in human hepatocellular carcinoma and non-HCC tissues

Flt-1 (VEGF receptor-1) and KDR/Flk-1 (VEGF receptor-2) are the high-affinity receptors for the angiogenesis factor, vascular endothelial growth factor (VEGF). VEGF expression has been confirmed in human hepatocellular carcinoma (HCC), and VEGF is thought to be involved in the angiogenesis within HCC tissues. However, expressions of VEGF receptors in HCC have not been reported. We immunohistochemically examined expressions and localizations of Flt-1 and KDR in 28 surgically resected HCC tissues. In non-cancerous area, Flt-1 and KDR were mainly found in macrophages including Kupffer cells; both receptors were found in vascular endothelial cells in the portal veins and arteries within portal tracts; and KDR was also found in some sinusoidal endothelial cells. In cancerous area, Flt-1 and KDR were found in some macrophages, and also in the endothelial cells of intratumoral blood vessels. In 25 moderately and/or poorly differentiated HCCs, KDR expression in the blood space endothelial cells was clear and continuous in 20 cases, and focal in 5 cases. These results suggest that there would be an angiogenesis mechanism via VEGF/Flt-1 or VEGF/KDR in HCC, and the VEGF/KDR system would take a more important role.     

Distinct hepatitis B virus integration patterns in hepatocellular carcinoma and adjacent normal liver tissue

Infection by the hepatitis B virus (HBV) is one of the main etiologies of hepatocellular carcinoma (HCC). During chronic infection, HBV DNA can integrate into the human genome, and this has been postulated as a possible mechanism of HBV‐induced HCC. In this study we used 2199 HBV integration sites from Dr.VIS v2.0 and mapped them to the human genome (hg19) to obtain viral integration sites (VIS) related to protein‐coding and non‐protein‐coding genes. In total, we found 1,377 and 767 VIS within close proximity to protein coding genes and noncoding genes, respectively. Genes affected more than two times included 23.1% of protein‐coding genes and 24.7% of long noncoding RNAs (lncRNA). Only 4.8% of VIS were shared between HCC and non‐tumor tissues. HBV integrations were more common in chromosomes 5, 8, 10, and 19 in HCC tissue and chromosomes 1 and 2 in non‐tumorous tissue. The number of integration sites on each chromosome correlated with the number of fragile sites in non‐tumorous tissue but not in HCC tissue. Functional enrichment analysis of the protein‐coding genes containing or in close proximity to HBV integration sites in HCC tissue showed an enrichment of cancer related gene ontology terms. Additionally, the most frequently associated lncRNA genes were related to telomere maintenance, protein modification processes, and chromosome localization. Thus, HBV may have preferred integration sites in the human genome that serve a critical role in HCC development. These results show that HCC treatment may benefit from the development of next generation anti‐viral therapies.     

Transforming growth factor-alpha in human hepatocellular carcinoma and coexpression with hepatitis B surface antigen in adjacent liver.

BACKGROUND. Hepatitis B virus (HBV) infection is closely associated with the development of hepatocellular carcinoma (HCC) in many patients, but the mechanisms by which HBV contributes to HCC are not known. Transforming growth factor-alpha (TGF-alpha), a regulator of growth and regeneration in rat liver that can be found in high levels in some human cancers, theoretically could play such an intermediate role in the development of HCC. METHODS. The expression of TGF-alpha and its relation to the HBV antigens were evaluated in human HCC and adjacent nontumorous livers from 33 patients from the United States and China using immunoperoxidase staining of paraffin-embedded sections. RESULTS. TGF-alpha was detected in HCC from 27 of 33 (82%) patients; the frequencies were similar in patients from the United States and China. TGF-alpha was detected in HCC more frequently in patients whose adjacent nontumorous livers had detectable hepatitis B surface antigen (HBsAg) and/or hepatitis B core antigen (HBcAg) than in those whose adjacent livers lacked HBsAg and HBcAg. Detection of TGF-alpha was not affected by tumor size, histologic type, or grade. TGF-alpha was detected in adjacent nontumorous livers from 31 of 33 patients (94%). Coexpression at a high intensity of TGF-alpha and HBsAg in the same hepatocytes could be demonstrated by specific staining of consecutively cut sections for 17 of 33 patients (52%). CONCLUSIONS. TGF-alpha is expressed at a high level in 82% of human HCC. Localization of HBsAg within the same hepatocytes as TGF-alpha suggests a possible interaction between HBV and TGF-alpha during hepatocarcinogenesis in humans. Stimulation of TGF-alpha expression could be part of a chain of events by which HBV contributes to the development of HCC in some patients.     

Glucocorticoid receptors in hepatocellular carcinoma and adjacent liver tissue

Glucocorticoid and progesterone receptors, tyrosine aminotransferase, gamma-glutamyltransferase and alpha-fetoprotein levels were determined in human hepatocellular carcinoma (HCC) and adjacent liver tissues. Glucocorticoid receptor was present in seven of ten HCC samples, values ranged from 1.9 to 66.8 fmol/mg protein. Progesterone receptor was present in two of ten HCC samples with values of 1.7 and 7.2 fmol/mg protein, respectively. In the adjacent liver tissues, no measurable progesterone receptor was found and only one sample had glucocorticoid receptor with a value of 3.0 fmol/mg protein. The increase of glucocorticoid receptor in HCC samples was coincident with a decreased level of tyrosine aminotransferase and an increased level of gamma-glutamyltransferase. No correlation was found among glucocorticoid receptor level, serum or tissue alpha-fetoprotein levels. The presence of glucocorticoid receptors in HCC suggest that hormones may play an important role in the formation of hepatoma, and hormonal therapy may be useful for patients with HCC.     

β-catenin,β-TRCP在肝癌和癌旁组织中的表达

目的：研究β-catenin、β-TRCP在肝细胞癌和癌旁组织中的表达特点及相互关系。方法：β-catenin和β-TrCP的表达是用免疫组化方法在石蜡包埋的肝癌和癌旁组织切片进行染色。结果：77％的肝癌中β-catenin呈明显阳性表达，其中51％呈过度表达。癌旁组织中表达较癌组织低，但表达水平与癌组织的表达水平成正相关。β-TRCP在肝细胞癌和癌旁组织中的表达基本一致，有20％表达减弱或缺失，其中18％为β-eatenin过度表达者。结论：β-catenin在癌旁组织中的表达水平与肝癌组织的表达水平有关。有18％的肝癌β-catenin过度表达可能是由于β-TRCP减弱或缺失造成。     

头颈部鳞癌及癌旁组织端粒酶活性研究

研究原发头颈部鳞癌及相应癌旁组织中端粒酶活性表达,并探讨其作为头颈部鳞鳞癌分子生物学标志物的可能性。方法：采用ＴＲＡＰ－ＰＣＲ－ＥＬＩＳＡ法对３２例原发头颈癌鳞癌及１５例相癌组织进行端粒酶活性的定性和定量检测。结果：原发头癌鳞癌组端粒酶活性值明显高于癌旁组。     

IL-17在原发性肝癌组织中的表达及其临床意义

目的:探讨IL-17在原发性肝癌组织中的表达及其临床意义。方法:23例原发性肝癌患者、9例肝良性肿瘤患者均为本院2009年3月至2010年12月的住院患者,采集手术切除肝癌组织、相应癌旁组织以及良性肿瘤组织标本。ELISA检测患者血清中IL-17表达水平,real-time PCR检测组织中IL-17、ROR-γt mRNA的表达水平,免疫组化检测IL-17蛋白在肝癌组织中的表达。结果:肝癌患者血清IL-17表达水平明显高于良性肿瘤对照组。Real-time PCR检测结果显示,肝癌组织中IL-17、ROR-γt mRNA的表达明显高于癌旁和正常肝组织(P<0.05)。免疫组化结果显示,肝癌组织及癌旁组织中IL-17表达均高于正常肝脏组织,且肝癌组织中表达明显高于癌旁组织。结论:IL-17在肝癌组织中高表达,有可能作为肝癌诊断及治疗的新靶点。     

肝细胞癌组织Smad4基因表达及其临床意义的研究

目的:拟通过检测Smad4基因在肝细胞癌(HCC)中的表达,初步探讨TGFβ-Smad信号通路中Co-Smad(Smad4)与肝细胞癌的发生和发展之间的可能关系.方法:采用免疫组化ABC法及原位杂交法(ISH)检测41例肝细胞癌组织切片中癌与癌旁Smad 4蛋白和mRNA的表达,5例外伤性肝破裂手术切除标本作为正常对照.比较HCC组与对照组及HCC与癌旁Smad 4蛋白和mRNA表达的差异,并进行统计分析.结果:正常对照组Smad 4蛋白和mRNA均呈阳性表达;HCC组织Smad4蛋白阳性率为48.8%(20/41),癌旁组织中为78.0%(32/41),两者比较差异有统计学意义,P<0.01;HCC组织Smad4 mRNA阳性卒为51.2%(21/41),癌旁组织中为73.1%(30/41),两者比较差异有统计学意义,P<0.05;与正常肝组织比较,HCC组织中Smad4蛋白和mRNA阳性表达均显著降低,P<0.05;HCC组织Smad4 mRNA阳性表达在病理分级Ⅰ、Ⅱ级与Ⅲ、Ⅳ级之间差异有统计学意义,P<0.05.结论:Smad 4基因表达缺失可能在肝细胞癌的发生和发展中发挥作用.     

细胞分裂周期相关蛋白5在肝细胞肝癌中的表达及临床意义

目的 探讨细胞分裂周期相关蛋白5(CDCA5)、细胞外调节蛋白激酶(ERK)在肝细胞肝癌中的表达及临床意义.方法 取260例原发性肝细胞肝癌患者的肝细胞肝癌组织,取52例肝脏良性病变患者的正常肝组织作为对照.蛋白质印迹法(Western blot)检测CDCA5、ERK、细胞周期蛋白依赖性激酶1(CDK1)、细胞周期蛋...     

黑色素瘤抗原MAGE-B亚家族基因在肝细胞癌中的表达

目的　检测黑色素瘤抗原MAGE B基因 (MAGE B1,MAGE B2 )在肝细胞癌 (HCC)中的表达 ,以期找到可用于HCC免疫治疗的新靶位。方法　用逆转录 聚合酶链反应 (RT PCR)方法检测MAGE B1、B2、A1和A3在 47例HCC患者癌组织和相应癌旁组织、3 0例肝硬化和正常肝组织中的表达 ,随机选择 4例RT PCR阳性扩增产物直接进行DNA序列测定 ,并将其表达结果与临床指标之间的关系进行分析。结果　 47例HCC组织中 ,MAGE B1和MAGE B2基因阳性率分别为 44.7% (2 1/47)和61.7% (2 9/47) ,而相应癌旁组织、肝硬化组织和正常肝组织中均为阴性。测序结果证实 ,RT PCR产物确为这两种基因。MAGE A1和MAGE A3的阳性率分别为 74.5% (3 5/47)和 44.7% (2 1/47)。MAGE B和MAGE A的表达之间有显著相关性 (P <0 .0 5) ,但其中 12例不表达MAGE A1和 (或 )A3的病例中 ,有 5例表达MAGE B1和 (或 )B2。同时检测这 4种基因的表达 ,发现至少表达其中 1种、2种、3种和4种均表达者分别为 83 .0 % (3 9/47)、55.3 % (2 6/47)、48.9% (2 3 /47)和 3 8.3 % (18/47)。MAGE B基因的表达与年龄、性别、肿瘤大小、分化程度、血清甲胎球蛋白 (AFP)水平、HBV和HCV感染之间无显著相关性 (P >0 .0 5)。结论　MAGE B基因家族在HCC中呈相对高比例、高特异表达 ,可     

肝癌及癌旁组织中p53基因及AFP、CEA等表达异常及相关性研究

目的　从分子生物学及病理学角度对肝癌中抑癌基因p5 3和肿瘤标志物AFP(甲胎蛋白 )、CEA(癌胚抗原 )的表达及相关性进行研究。方法　收集 40例肝细胞肝癌 (HCC)患者癌组织连带部分癌旁组织 ,用免疫组织化学法检测p5 3基因、AFP、CEA等。结果　p5 3基因癌内表达为 80 % ,癌旁组织为 5 0 % ;AFP癌内表达为 87.5 % ,癌旁组织为80 % ;CEA癌内表达为 5 5 % ,癌旁组织表达不明显。结论　检测 p5 3基因 ,并与其它肿瘤标志物和相关基因一起 ,在基因水平诊断肝癌、估计预后及制定术后治疗方案等有着重要作用。