肝硬化并肝细胞癌的端粒酶活性和氧化应激研究

为阐明端粒酶活性和氧化应激在肝细胞癌（HCC）合并肝硬化及肝硬化患者组织中的表达，相互关系及意义，采用TRAP－ELLISA方法测定了21例HCC合并肝硬化（下称HCC组）和23例肝硬化（下称肝硬化组）患者组织中的端粒酶活性，并用生化法测定其组织中的丙二醛（MDA），谷胱甘肽－S转移酶（GST）和总抗氧化能力（T－AOC）。结果显示，HCC组端粒酶阳性18例，阴性3例；肝硬化组阳性3例，阴性20例，两者差别有显著意义（P＜0．001）；MDA，GST和T－AOC在两组中的表达均有显著差异（P均＜0．001），端粒酶活性和MDA含量呈正相关（P＜0．05）。认为从肝硬化到肝癌的过程与机体抗氧化系统功能失调密切相关；端粒酶激活是HCC癌变的早期事件；氧化应激对端粒酶激活可能起重要作用。     

The role of telomerase activity in hepatocellular carcinoma

OBJECTIVE: The aim of this study was to clarify the role of telomerase activity in hepatocellular carcinoma (HCC). METHODS: Specimens from both HCC and noncancerous liver were obtained from 39 patients with HCC using a 14-gauge biopsy needle immediately after laparotomy. Telomerase activity was determined using a telomeric repeat amplification protocol assay. The 3+ of telomerase activity in HCC was defined as a high telomerase group, and 2+ or less of HCC telomerase activity was defined as a low telomerase group. In noncancerous liver, 2+ or more of telomerase activity was defined as an increased telomerase group, and 1+ or less of telomerase activity was defined as a nonincreased telomerase group. The correlation between telomerase activity in HCC or noncancerous liver and clinicopathological factors, including prognosis, was investigated. RESULTS: Telomerase activities in HCCs were 0 in one patient, 1+ in two, 2+ in seven, and 3+ in 29 patients. The disease-free survival rate in the high telomerase group was significantly worse than that in the low telomerase group. The des-gamma-carboxy prothrombin level in a high telomerase group (median, 330 mAU/ml) was significantly higher than that in the low telomerase group (median, 150 mAU/ml). A multivariate analysis revealed that higher TNM stage, high telomerase activity in HCC, female gender, and high alpha-fetoprotein value were independent significant factors related to be early recurrence. The incidence of multicentric HCC occurrence in the increased telomerase group (53.3%) tended to be higher than that in the nonincreased telomerase group (27.3%). CONCLUSION: A high telomerase activity in HCC correlated with the potential of HCC to be more malignant, which was expressed as both a high level of des-gamma-carboxy prothrombin and an earlier recurrence after hepatectomy than that of HCC with a low telomerase activity.     

端粒酶催化亚单位在原发性肝癌中表达的临床意义

用端粒酶催化亚单位(hTRT)原位杂交检测，探讨hTRT作为原发性肝癌早期诊断、估计预后指标的可能。     

Relationship between microvessel density and telomerase activity in hepatocellular carcinoma

AIM: To study the relationship between microvessel density (MVD), telomerase activity and biological characteristics in hepatocellular carcinoma (HCC). METHODS: S-P immunohistochemical method and telomeric repeat amplification protocol (TRAP) were respectively used to analyze the MVD and telomerase activity in 58 HCC and adjacent normal tissues. RESULTS: The MVD in HCC with metastasis, lower differentiation or without intact capsule was significantly higher than that in HCC with intact capsule, higher differentiation, or without metastasis. While MVD had no relationship with tumor size, hepatic virus infection and other clinical factors. Telomerase activity was related to differentiation degree, but not to tumor size or histological grade. MVD in HCC with telomerase activity was higher than that in HCC without telomerase activity. CONCLUSION: MVD and telomerase activity may serve as diagnostic criteria of HCC in earlier stage. Meanwhile, there may be a cooperative effect between MVD and telomerase on the growth and metastasis of HCC.     

Detection of telomerase activity in hepatocellular carcinoma by fluorescence-based TRAP method

Telomeres are important for the function and stability of eukaryotic chromosomes. Telomeric DNA is shortened every time liver cells divide. Normal human liver cells have a limited proliferative capacity, but immortalized cells prevent the shortening of the end of chromosomes by the expression of telomerase, the enzyme that elongates telomeric DNA. Although many genetic alterations have been reported in hepatocellular carcinoma, whether all hepatocellular carcinoma are immortalized cells is not known. Therefore, the telomerase activity was analyzed in 38 frozen samples from human hepatocellular carcinoma by fluorescence-based TRAP Method. Telomerase activity was detected in 34 (89.47%) of 38 hepatocellular carcinoma tissues regardless of tumor size, but telomerase activity was detected in only 12 (31.58%) of 38 nontumor liver tissues from patients with hepatocellular carcinoma. Diagnosis of hepatocellular carcinoma is sometimes difficult when the hepatomas are small and are of the differentiated type. Our results showed that the expression of telomerase may be of great value in diagnosing hepatocellular carcinoma.     

c-Myb、VEGFR2、EGFR蛋白在肝硬化、肝细胞癌组织中的表达

目的研究c-Myb蛋白在肝硬化、肝细胞癌（HCC）组织中的表达及其与VEGFR2、EGFR在HCC组织中表达的关系,探讨c-Myb基因在肝硬化、HCC的发生、发展中的作用机制。方法应用免疫组织化学法检测63例HCC组织、59例癌旁组织、12例肝硬化组织和11例正常肝组织中c-Myb蛋白表达情况,VEGFR2、EGFR分别在29、45例HCC组织中的表达情况。结果肝硬化组织和HCC组织相对于正常肝组织,癌旁组织相对于肝硬化组织,c-Myb蛋白的表达明显增高（P=0.000）;在HCC组织中,c-Myb蛋白的表达与VEGFR2和EGFR的表达无显著相关性。结论c-Myb过表达是HCC发病中的早期事件,在肝硬化、HCC的发生、发展中可能起重要作用,其作用机制值得进一步研究。     

TRA1 mRNA在肝硬化及肝癌组织中表达的研究

目的探讨肝癌(hepatocellular carcinoma,HCC)组织、肝硬化(liver cirrhosis,LC)组织及正常对照组织中肿瘤排斥抗原1(tumor rejection antigen 1,TRA1)mRNA的表达及关系。方法采用逆转录-聚合酶链反应的方法检测34例肝癌患者肝癌组织、癌旁肝硬化组织、非肝癌的肝硬化患者活检组织、肝血管瘤及肝内胆管结石等对照组织中TRA1 mRNA的表达,用捷达801分析软件对结果进行相对定量分析。结果肝癌组织、肝硬化组织、对照组织TRA1 mRNA表达率分别为95.00%、84.21%、50.00%,肝癌组与对照组间表达率的比较,差异有统计学意义(P0.05),肝硬化组织、对照组织表达率之间差异无明显统计学意义(P0.05);肝癌组织、肝硬化组织、对照组织TRA1 mRNA表达量分别为1.67±0.96、1.49±0.53、0.57±0.27;扩增产物表现出表达量的不同,呈现渐变趋势,肝癌组织、肝硬化组织与对照组之间差异有统计学意义(P0.05),肝癌组织与肝硬化组织比较,差异无统计学意义(P0.05)。结论 TRA1参与了肝硬化、肝癌的发生、发展,TRA1可能是肝硬化和肝癌潜在诊断标志物和治疗靶点。     

Unresectable hepatocellular carcinoma in cirrhosis

To evaluate the efficacy of transcatheter arterial chemoembolization in the treatment of hepatocellular carcinoma, the prognostic factors, and the side effects, 72 patients undergoing 170 chemoembolizations with lipiodol-mediated injection of adriamycin were investigated. The 1-, 2-, and 3-year survivals are 83, 61, and 56%, respectively. Significant prognostic factors for survival (by Mantael-Haenszel) are Child-Pugh and Okuda status (p=0.00001 and p=0.01 respectively), number of TACE courses (p=0.002) and of courses completed with embolization (p=0.05), stabilization or reduction of α-fetoprotein (p=0.003), and concurrent tamoxifen treatment (p=0.04). Side effects included fever, pain, increased serum amylase and transaminase levels, and one liver abscess with death of liver failure. In addition, mild hyperglycemia was observed in 19% of patients and severe in 8% (with one hyperosmolar diabetic coma), in the absence of pancreatic damage. In conclusion, transcatheter arterial chemoembolization is useful in patients with unresectable hepatocellular carcinoma. Prognostic factors are Child-Pugh and Okuda status, number of TACE courses and of embolizations, changes of α-fetoprotein levels, and association with tamoxifen treatment. The development of mild to severe changes of glucose metabolism suggests that glucose tolerance should be evaluated before and glycemia strictly monitored after each TACE course.     

Des-gamma-carboxyprothrombin in patients with hepatocellular carcinoma and liver cirrhosis

OBJECTIVES: To ascertain serum and tissue expression of des‐gamma‐carboxyprothrombin (DCP) in patients with hepatocellular carcinoma (HCC) and liver cirrhosis and clarify the relationship between DCP expression and prognosis. METHODS: Expression of DCP in tissues was evaluated with immunohistochemical staining using anti‐DCP antibody in 74 patients with a single primary HCC nodule and liver cirrhosis. Their serum DCP levels were determined using an enzyme immunoassay with a double antibody sandwich system. RESULTS: Positive DCP expression in cancerous and non‐cancerous tissues was related to a worse prognosis for patients with HCC and liver cirrhosis. The combined evaluation of tissue DCP expression and serum DCP level showed that prognosis was the worst for patients with positive tissue DCP expression and a high serum DCP level. Univariate analysis indicated that a lower 5‐year survival rate was significantly correlated with positive tissue DCP expression, a high serum DCP level and the combined factor of positive tissue DCP expression and a high serum DCP level. Multivariate analysis indicated that the combined factor of positive tissue DCP expression and a high serum DCP level was a significant prognostic factor. CONCLUSION: The combined evaluation of tissue DCP expression and serum DCP level is more useful than either factor alone in predicting prognosis for patients with HCC and liver cirrhosis.     

Juvenile hepatocellular carcinoma with congestive liver cirrhosis

A case of juvenile hepatocellular carcinoma (HCC) with congestive liver cirrhosis is reported. The patient was a 21-year-old woman. She had been diagnosed as having transposition of the great arteries, type 2, in 1978. She underwent the Mustard operation, but suffered from chronic heart failure. In 1995, she experienced abdominal pain and underwent examination. The laboratory data were normal, except for elevated total bilirubin (5.2mg/dl). Blood examinations were performed at frequent intervals, and the total bilirubin level fluctuated between 0.9 and 8.1mg/dl over the next 4 years, but the transaminase level remained normal. In 1999, she experienced abdominal pain again and was admitted to our hospital. Computed tomography showed four space-occupying lesions in the liver; 45mm, 20mm, 12mm, and 10mm in size. She was diagnosed as having HCC, and transcatheter arterial chemoembolization and percutaneous ethanol injection therapy were performed. Histology of the cancerous and the noncancerous liver tissue revealed HCC, moderately differentiated type, in cirrhotic liver with congestion. This patient had no background factors of liver disease, except for liver congestion, associated with the chronic heart failure. Because most patients with cardiac cirrhosis die of cardiac disease, only a small number of these patients develop liver failure. However, the incidence of HCC in patients with congestive liver disease is likely to increase in the future, as survival time is prolonged with the advances in treatment for chronic heart failure. Therefore, patients with congestive liver disease should be followed, taking into account the possibility of HCC.     

Deficiency in calcineurin activity in liver transplantation candidates with alcoholic cirrhosis or hepatocellular carcinoma

Background: Tacrolimus and cyclosporin inhibits the activity of calcineurin, a serine/threonine phosphatase that is involved in many physiological and pathological pathways. However, the baseline calcineurin phosphatase activity (CPA) measured before the transplant is unknown. In this study, we determine baseline CPA in liver transplant (LT) candidates and explore some factors that might modify it. Patients and methods: Thirty‐two consecutive LT candidates (25 men, seven women, average age 53.4 years) were included. Seven millilitres of whole blood was collected from each patient. CPA was determined in lymphocytes quantifying a dephosphorylated peptide phosphorylated previously (D‐L‐D‐V‐P‐I‐P‐G‐R‐F‐D‐R‐R‐V‐S‐V‐A‐A‐E) by high‐performance liquid chromatography. The relationship between CPA and the quantitative variables was tested according to Pearson's correlation. A two‐way analysis of variance was performed to test the independent role of categorical parameters in CPA. Results: The median CPA was significantly lower in LT candidates than in healthy volunteers [179.2 (146.9–226.3) vs 247.8 (220.9–292.5) pmol/min/10⁶ peripheral blood mononuclear cell (PBMC), respectively, P=0.0002]. CPA was also significantly lower in alcoholic cirrhosis (152.2 vs 211.1 pmol/min/10⁶ PBMC, P=0.04) and in the presence of hepatocellular carcinoma (HCC) (152.0 vs 213.5 pmol/min/10⁶ PBMC, P=0.0074) compared with other liver diseases. A two‐way analysis of variance showed that these parameters were independently associated with lower CPA (P=0.05 for alcohol and P=0.0056 for HCC respectively). Conclusion: This pilot study showed a lower CPA in patients with AC and HCC. This phenomenon may contribute towards lowering the risk of acute rejection in these patients after LT and, on the other hand, may increase the risk of de novo cancers.