肝癌组织和血清中半乳糖血凝素-3的表达及其意义

目的 研究人肝癌组织及血清中半乳糖血凝素-3(galectin-3)的表达及其临床意义.方法 免疫组织化学法检测46例肝癌及其癌旁组织中galectin-3,分析galectin-3表达水平与各临床参数之间的关系; 酶联免疫吸附法检测不同肝病患者及正常人血清中galectin-3浓度,同步比较galectin-3浓度与甲胎蛋白(AFP)、γ-谷氨酰转移酶同工酶Ⅱ(GGT-Ⅱ)诊断肝癌的灵敏性及特异性,探讨3项指标对肝癌的互补诊断价值.用Stata8.0及SPSS15.0统计软件对结果进行统计分析.采用配对设计四格表假设检验、秩和检验、χ2检验、Fisher's确切概率法、Spearman等级相关方法.结果 (1)galectin-3在肝癌组织中的表达阳性率为78.2%,癌旁组织为15.2%,χ2=92.000,P<0.01,差异有统计意义.其表达水平与分化程度相关,分化程度越低表达水平越高;(2)根据ROC曲线,当确定诊断界值为0.62μg/L时,galectin-3阳性率在肝癌患者中为64.5%,肝硬化患者中为3.1%、慢性肝炎患者及正常人中均为0,P<0.05(Fisher's确切概率法);(3)肝癌患者血清中galectin-3与AFP、GGT-Ⅱ均无相关性,联合检测3项指标对肝癌诊断有互补性,可使诊断敏感性提高至94.7%.结论 galectin-3在肝癌组织中高表达,与肝癌的分化程度相关,可能与肝癌的发生和发展有关;肝癌患者联合检测血清galectin-3、AFP和GGT-Ⅱ可提高对肝癌诊断的敏感性,galectin-3有望成为新的肝癌标记物.

Abstract：

Objective To study the expression of Galectin-3 in human hepatocellular carcinoma (HCC) tissues and the clinical value of serum Galectin-3 in the diagnosis of hepatocellular carcinoma. Methods Immunohistochemistry method was used to detect the expression of Galectin-3 in the 46 pairs of HCC tissues and their paracancerous tissues. The relationship between expression levels of Galectin-3 and clinical parameters was analyzed. Serum Galectin-3 in different liver diseases were measured with ELISA. The sensitivity and specificity of galectin-3, alpha fetoprotein (AFP) and gamma-glutamyltranspeptidase Ⅱ (GGT-Ⅱ)for diagnosis of HCC were compared and the complementary diagnostic values of Galectin-3 and AFP and GGT-Ⅱ for HCC were studied. Results (1) The positive rate of Galectin-3 in the tissue of HCC was 78.2%, dramatically higher than that in paracancerous tissues (15.2%) (P < 0.01). The expression levels were correlated with differentiation and with the high expression in poor differentiation tissues; (2) Based on ROC curve, the cut-off of serum Galectin-3 for HCC diagnosis was set as 0.62 μg/L, the serum galectin-3 positive rate was 64.5% in HCC cases, which was apparently higher than that in liver cirrhosis, chronic hepatitis and healthy persons (P < 0.05); (3) Serum Galectin-3 was not correlated with AFP and GGT-Ⅱ. Combined determination of the three markers had the complementary diagnostic value for HCC and might increase the diagnostic sensitivity to 94.7%. Conclusion Galectin-3 is overexpressed in HCC tissues and is correlated with the tumor differentiation, suggesting that Galectin-3 may be associated with the carcinogenesis and development of HCC. Serum galectin-3 increases in the HCC cases and combined determination of serum Galectin-3, AFP and GGT-Ⅱ can increase the diagnostic efficiency for HCC. Galectin-3 could be a novel serum tumor marker for HCC.     

Expression and significance of new inhibitor of apoptosis protein survivin in hepatocellular carcinoma

AM: To investigate expression and significance of inhibitor of apoptosis protein survivin in hepatocellular carcinoma (HCC). METHODS: The expression of survivin and vascular endothelial growth factor (VEGF) was investigated in 38 cases of HCC tissues and 38 liver cirrhosis tissues by immunohistochemistry and Western blot. The relationship between the expression of survivin and clinicopathological factors of HCC was analyzed. RESULTS: Survivin protein was detected in 23 (60.5%) of 38 HCCs and 3 (7.9%) of 38 liver cirrhosis tissues. In 23 cases of HCC which expressed survivin, the expression of VEGF was positive in 18 cases and slight positive or negative in 5 cases. While in 15 cases of HCC which did not express survivin, 12 cases did not express or slightly expressed, and 3 cases expressed VEGF. In liver cirrhosis tissues, the expression of VEGF was as follows: 24 cases were negative, 10 cases were weak positive and 4 cases were strong positive. The expression of survivin was coincident with the expression of VEGF in HCC (P<0.01). The expression of survivin in HCC had no relationship with the patients' age, gender, tumor size and differentiation level of HCC, while it was related to the metastasis of HCC. The protein quantitative analysis by Western blot also showed that overexpression of survivin in HCC was closely correlated to the expression of VEGF (P<0.01). Furthermore, stronger expression of survivin and VEGF was also found in patients with metastasis rather than in those with no metastasis (P<0.01). CONCLUSION: Survivin plays a pivotal role in the metastasis of HCC, and it has some correlation with tumorigenesis. The expression of survivin in the primary lesion is very useful as an indicator for metastasis and prognosis of HCC. It could become a new target of gene therapy of HCC.     

Dynamic alteration of telomerase expression and its diagnostic significance in liver or peripheral blood for hepatocellular carcinoma

AIM: To investigate the dynamic alteration of telomerase expression during development of hepatocellular carcinoma (HCC) and its diagnostic implications in liver tissues or peripheral blood mononuclear cells for HCC. METHODS: Dynamic expressions of liver telomerase during malignant transformation of hepatocytes were observed in Sprague-Dawly (SD) rats fed with 0.05% of 2-fluoenyacetamide (2-FAA). Total RNA and telomerase were extracted from rat or human liver tissues. The telomerase activities in livers and in circulating blood were detected by a telomeric repeat amplification protocol-enzyme-linked immunosorbent assay (TRAP-ELISA), and its diagnostic value was investigated in patients with benign or malignant liver diseases. RESULTS: The hepatoma model displayed the dynamic expression of hepatic telomerase during HCC development. The telomerase activities were consistent with liver total RNA levels (r = 0.83, P < 0.01) at the stages of degeneration, precancerosis, and cancerization of hepatocytes. In HCC patients, the telomerase levels in HCC tissues were significantly higher than in their adjacent non-cancerous tissues, but liver total RNA levels were lower in the former than in the latter. Although the circulating telomerase of HCC patients was abnormally expressed among patients with chronic liver diseases, the telomerase activity was a non-specific marker for HCC diagnosis, because the incidence was 15.7% in normal control, 25% in chronic hepatitis, 45.9% in liver cirrhosis, and 85.2% in HCC, respectively when absorbance value of telomerase activity was more than 0.2. If the value was over 0.6, the incidence was 60% in HCC group and 0% in any of the others (P < 0.01) except in two cases with liver cirrhosis. However, the combination of circulating telomerase with serum alpha-fetoprotein level could increase the positive rate and the accuracy (92.6%, 125 of 135) of HCC diagnosis. CONCLUSION: The overexpression of telomerase is associated with HCC development, and its abnormality in liver tissues or in peripheral blood could be a useful marker for diagnosis and prognosis of HCC.     

Abnormal expression of HSP gp96 associated with HBV replication in human hepatocellular carcinoma

BACKGROUND: Heat shock protein (HSP) gp96 is a member of the HSP90 family and presumably overexpresses as a result of stimulation by mutated or abnormal proteins. Its abnormal expression correlates with carcinogenesis, progression and prognosis of hepatocellular carcinoma (HCC). In this study, we investigated the pathological characteristics of liver gp96 expression and its relationship with hepatitis B virus (HBV) replication in HCC patients. METHODS: Tumor specimens were prospectively collected from 30 HCC patients undergoing liver resection. Total RNAs were extracted from HCC or their noncancerous tissues. The distribution of gp96 expression in hepatocytes was investigated by streptavidin peroxidase (S-P) immunohistochemistry and tissue HBV-DNA was detected by the in situ molecular hybridization technique. The association of gp96 expression with HBV replication, and the histopathological characteristics of HCC were analyzed. RESULTS: The gp96 was strongly expressed in HCC (73.3%, 22 of 30) and weakly (46.7%, 14 of 30) in non-cancerous tissues. The gp96 expression in HCC tissues was correlated with degree of tumor differentiation and tumor size, but not with tumor number (P>0.05). Immunohistochemical analysis showed that 17 of 19 HCC patients with HBVDNA -positive were strongly expressed for gp96, whereas only 5 of 11 patients with HBV-DNA-negative were positive for gp96. A significant difference was found between the two groups (89.5% vs. 45.5%, P<0.05). CONCLUSIONS: The abnormal expressions of HSP gp96 in HCC tissues are associated with HBV replication. This finding indicates that HBV infection plays an important role in the development of HCC.     

Immunohistochemical assessment of angiogenesis in hepatocellular carcinoma and surrounding cirrhotic liver tissues

AIM: To investigate whether vascular endothelial growth factor (VEGF) was over-expressed in hepatocellular carcinoma (HCC) or in surrounding cirrhotic liver tissues. METHODS: Immunohistochemistry was performed to investigate the expression of VEGF proteins in HCC tissues from 105 consecutive patients undergoing curative resection for HCC. The immunostaining results and related clinicopathologic materials were analyzed with statistical methods. Kaplan-Meier method was used to calculate survival curves, and Log-rank test was performed to compare differences in survival rates of the patients with positive HCC staining and negative VEGF. RESULTS: VEGF-positive expression was found in 72 of 105 HCC patients (68.6%). Capsular infiltration (P= 0.005), vascular invasion (P= 0.035) and intrahepatic metastasis (P=0.008) were observed more frequently in patients with VEGF-positive expression than in those with VEGF-negative.expression. Kaplan-Meier curves showed that VEGF-positive expression was associated with a shorter overall survival (P= 0.014). VEGF-positive expression was found in 47 of tissues 68 HCC (69.1%), and VEGF-positive expression was found in 54 of 68 surrounding cirrhotic liver tissues (79.4%). VEGF-positive expression was significantly higher in surrounding cirrhotic liver tissues than in HCC (P=0.017). CONCLUSION: VEGF may play an important role in the angiogenesis and prognosis of HCC, as well as in the angiogenesis of liver cirrhosis.     

内皮细胞特异性分子1在原发性肝癌中的表达及其意义

Objective To study the expression and significance of endothelial cell specific molecule-1(ESM-1) in hepatocellular carcinolna(HCC)tissue and serum from HCC patients.Methods The ESM-1 expression was evaluated by immunohistochemistry staining and quantitative real-time PCR in 30 tumor tissues from HCC patients,15 surrounding non-cancerous hepatic parenchyma,and 15 normal controls.Serum levels of ESM-1 were also measured in the HCC patients and healthy controls by Enzyme-linked imrnunosorbent assay(ELISA).Results ESM-1 was expressed in endothelium of 24 HCC tissues and 6 pericarcinomatous tissues.however,it was not expressed in normal liver tissues.The mRNA level of ESM-1was 0.064±0.018,0.383±0.103,0.528±0.148 in the corresponding tissues,respectively.The mRNA level of ESM-1 in tissues was correlated with the TNM phase of HCC patients.tumor vascular invasion as well as metastasis.The serum ESM-1 was(12.643±2.280)ng/ml and(4.660±1.172)ng/ml in hepatic cell carcinoma and normal controls,respectively.Conclusion The expression of ESM-1 is significantly upregulated in HCC,suggesting that ESM-1 may play an important role in the pathoigenesis of HCC.     

Cell proliferation, apoptosis and the related regulators p27, p53 expression in hepatocellular carcinoma

AIM: To investigate the expression of cell apoptosis, proliferation and the related regulators p27,p53 in hepatocellular carcinoma (HCC). METHODS: The expression of p27, p53, proliferating cell nuclear antigen (PCNA) and apoptosis in 47 HCC specimens and 42 surrounding non-cancerous tissues were detected by the immunohistochemistry and terminal deoxy-nudeotidyl transferase-mediated nick end labeling (TUNEL) technique. Meanwhile, the clinical significance of them was analyzed combining with the clinicopathological factors and follow-up data. RESULTS: (1) The average proliferating index and apoptotic index in HCC were significantly higher than that in adjacent liver tissues. The proliferating index was associated with extrahepatic metastasis. The apoptotic index was significantly lower in TNM stage Ⅰ-Ⅱ than in stage Ⅲ-Ⅳ. The proliferating index of groups with p53-/p27+ was significantly lower than that in group with p53+/p27- (P= 0.030); (2) The level of p27 in the cytoplasmic fraction was higher in non-tumoral liver tissues and was associated with clinical stage; (3) Survival analysis showed advanced stage (P=0.031) and with extrahepatic metastasis (P = 0.045) was significantly associated with shorter survival. In addition, the prognosis of patients with p53-/p27+ was longer than that of patients with p53+/p27- (P= 0.0356). CONCLUSION: The p53 mutation and decreased p27 expression might be involved in the imbalance of proliferation and apoptosis in HCC. Cytoplasmic displacement might lead to the inactivation of p27 protein in HCC cells and acts early during carcinogenesis of HCC. The combined examination of p27, and p53 expression allows reliable estimation of prognosis for patients with primary hepatic carcinoma.     

外周血白蛋白mRNA分析在肝癌诊断和转移监测中的价值

目的     

Combined serum hepatoma-specific alpha-fetoprotein and circulating alpha-fetoprotein-mRNA in diagnosis of hepatocellular carcinoma

Introduction Hepatocellular carcinoma (HCC) is a major health problem throughout the world.[1-3] It ranks the fifth in frequency worldwideamong all malignancies and causes one million deaths annually,[4, 5] yet its incidence is increasing steadily in various countries.[6-8] The carcinogenesis of HCC is a multi-factorial, multi-step and complex process. Perhaps hepatitis B virus (HBV) infection is merely a carcinogenic factor, and is not related to the growth, infiltration and metastasis of…     

肿瘤组织中 GPC3、Hepatocyte、CD34检测对 AFP 阴性肝细胞癌的辅助诊断作用

目的评价肿瘤组织中磷脂酰肌醇蛋白聚糖3（GPC3）、Hepatocyte、CD,。蛋白检测对甲胎蛋白（AFP）阴性肝细胞癌（HCC）的辅助诊断作用。方法回顾性分析48例癌细胞AFP阴性的HCC组织及其癌旁组织中GPC3、Hepatocyte、CD。蛋白表达特点。结果48例HCC及其癌旁组织中GPC3蛋白阳性率分别为91．7％（44／48）、2．1％（1／48）,两者比较P〈0．01;Hepatocyte蛋白阳性率分别为95．8％（46／48）、100．0％（48／48）,两者比较P〉0．05;CD3。蛋白阳性率分别为100．0％（48／48）、4．2％（2／48）,两者比较P〈0．01。HCC组织中GPC3与Hepmo—cyte表达呈正相关（r=0．314,P〈0．05）。结论检测肿瘤组织中GPC3、Hepatocyte和CD。蛋白有助于AFP阴性原发性HCC的诊断。     

肝癌组织甲胎蛋白表达特点及其基因片段的扩增分析

目的探讨肝癌组织甲胎蛋白(AFP)表达的病理学特点及其基因分析的临床价值。方法以免疫组织化学法研究AFP在肝癌及癌周组织中的胞内分布及其表达;并从肝癌的癌灶、癌旁组织中制备总RNA,以巢式聚合酶链反应(nested_PCR)扩增不同癌组织AFP_mRNA片段,以探讨AFP表达的临床病理学特征及其AFP_mRNA分析的应用价值。结果肝癌细胞及癌旁肝细胞的胞浆中,均可见AFP阳性的棕黄色颗粒,癌组织中AFP阳性表达率为73.3%,癌旁组织中为40.0%,中、低分化组肝癌中AFP的表达阳性率明显高于高分化组肝癌(P<0.05),且癌旁组织中AFP的表达强度明显低于肝癌的癌灶组织。肝癌组织、癌周组织的总RNA表达水平存在差别,癌组织中AFP_mRNA片段扩增全数阳性(100%),显著高于癌周组织(P<0.01)。结论肝癌不同组织中AFP表达存在差异,AFP_mRNA分析有助于肝癌的早期发现和诊断。     

黑色素瘤抗原-A1基因多态性及其在肝癌组织中的表达

目的了解黑色素瘤抗原(MAGE)-A1基因在肝癌组织中的表达状况,探讨该基因是否存在单核苷酸多态性(SNP).方法用RT-PCR方法检测49例原发性肝细胞性肝癌(HCC)患者肝癌组织MAGE-A1基因mRNA的表达情况,同时提取其中43例HCC患者的肝癌组织、癌旁组织及外周血细胞基因组DNA,PCR扩增MAGE-A1 DNA,并对上述PCR产物进行测序.结果49例患者中MAGE-A1 mRNA阳性22例,阳性率44.9%.43例HCC患者癌组织、癌旁组织和外周血细胞中扩增的KAGE-A1 DNA序列测定显示MAGE-A1基因存在两类变异:TAG有16例,变异发生率为37.2%,包括3个位置的变异:C159T,A272G,G393A,GTG变异有7例,发生率为16.3%,亦包括3个位点的变异即:A272G,C991T,A1125G.因为C991T没有改变编码的氨基酸,A1125G不在编码区,故仅A272G引起一个氨基酸的替换(T32A).结论MAGE-A1在HCC中高表达,表达率为44.9%.MAGE-A1 mRNA的表达与AFP无关.MAGE-A1基因存在三种SNP,但不影响其mRNA的表达.     

N-glycomic changes in hepatocellular carcinoma patients with liver cirrhosis induced by hepatitis B virus

We evaluated the use of blood serum N-glycan fingerprinting as a tool for the diagnosis of hepatocellular carcinoma (HCC) in patients with cirrhosis induced by hepatitis B virus (HBV). A group of 450 HBV-infected patients with liver fibrosis or cirrhosis with or without HCC were studied. HCC was diagnosed by alpha-fetoprotein (AFP) analysis, ultrasonography, and/or computed tomography and was studied histologically. N-glycan profiles of serum proteins were determined with DNA sequencer-based carbohydrate analytical profiling technology. In this study, we found that a branch alpha(1,3)-fucosylated triantennary glycan was more abundant in patients with HCC than in patients with cirrhosis, patients with fibrosis, and healthy blood donors, whereas a bisecting core alpha(1,6)-fucosylated biantennary glycan was elevated in patients with cirrhosis. The concentration of these 2 glycans and the log ratio of peak 9 to peak 7 (renamed the GlycoHCCTest) were associated with the tumor stage. Moreover, for screening patients with HCC from patients with cirrhosis, the overall sensitivity and specificity of the GlycoHCCTest were very similar to those of AFP. CONCLUSION: This study indicates that a branch alpha(1,3)-fucosylated glycan is associated with the development of HCC. The serum N-glycan profile is a promising noninvasive method for detecting HCC in patients with cirrhosis and could be a valuable supplement to AFP in the diagnosis of HCC in HBV-infected patients with liver cirrhosis. Its use for the screening, follow-up, and management of patients with cirrhosis and HCC should be evaluated further.