Quantitative analysis of hepatoma-specific α-fetoprotein(HS-AFP) by a new mini-column affinity chromatography and its clinical value in diagnosis of hepatocellular carcinoma

Objective:To establish a convenient and economic method to determine hepatoma-specific α-fetoprotein(HS-AFP) for diagnosis of hepatocellular carcinoma(HCC). Methods:HS-AFP from serum of HCC patients was separated by a mini-column Lens culinaris agglutinin(LCA)-affinity chromatography. The levels of serum total AFP and separated HS-AFP were detected by radioimmunoassay(RIA). Results:Circulating AFP was separated into three peaks(AFP-1,AFP-2,and AFP-3) by LCA-affinity chromatography. During the elution course,the AFP-1 and AFP-2 could be eluted with TE buffer. HS-AFP(AFP-3) from sera of HCC patients was eluted clearly on the LCA-sepharose gel mini-column with a solution containing α-methyl-D-mannoside. It was a part of total AFP and only found in sera of HCC patients. A ratio of more than 15% for HS-AFP to total AFP in serum was considered as a specific marker for HCC diagnosis with higher sensitivity(92.7%) and specificity(88.2%). Conclusion:The new assay for circulating HS-AFP analysis is more sensitive,repeatable,and convenient. Its clini-cal application would be useful to early diagnosis of HCC.

Abnormal expression of vascular endothelial growth factor (VEGF) and its clinical features in tissues of human lung cancer

Objective： Angiogennesis, the formation of new blood vessels from the existing vascular bed, is essential step for growth and invasion of primary rumor. Vascular endothelial growth factor （VEGF） is known to play crucial role in tumor angiogenesis. In the present study, we investigate the expression of VEGF and VEGF-mRNA in the angiogennesis, metastasis and prognosis of lung cancer. Methods： The VEGF cellular distributions and expression in 38 specimens of patients with lung cancer were investigated with immunohistochemistry stain technology. The total RNAs in 38 tissues of lung cancer was measured, then the levels of VEGF-mRNA expression were analyzed by a reverse-transcription polymerase chain reaction （RT-PCR） assay. The levels of VEGF in sera of patients with lung cancer, benign lung diseases and healthy controls were detected through Enzyme linked immunosorbent assay （ELISA） method. Results： The VEGF positive stain was 76% in 38 cases of lung cancer specimens. The 89% rate of VEGF stain was found for clinical stage Ⅲ cases and 92% for stage Ⅳ lung cancers. The significantly higher expression of VEGF was evidenced in patients with lymph node metastasis （84%）, distant metastasis （90%）, and lung cancers with lower histological differentiation （89%）, respectively. The expression level of total RNA was significantly higher in patients with lung cancers than that in their paracancerons or distant lung tissues. The VEGF expressions were tightly correlated with total RNA concentration of lung carcinoma （ P 〈 0.01 ）. The predominant expressions of VEGF121 and VEGF165 gene fragments were found in lung cancer specimens by RT-PCR analysis. No significant difference of serum VEGF levels was detected between cases with lung cancer and patients with benign diseases. However, the VEGF level of cases with benign diseases was decreased significantly after patients with anti-inflammation medication. Conclusion： The present data suggested that the rumor tissue VEGF expression and VEGF-mRNA analysis in patients with lung cancer be a useful indicator for angiogenesis and metastasis of lung cancer.

Abnormal expression of VEGF and its gene transcription status as diagnostic indicators in patients with non-small cell lung cancer

Objective Angiogenesis is known to be essential for the survival,growth,invasion,and metastasis of lung cancer cells. Vascular endothelial growth factor(VEGF) is an important factor regulating angiogenesis of non-small cell lung cancer(NSCLC); however,its pathologic features and significance are unclear. In this study,the tissue VEGF expression levels and its gene transcriptional status,as well as circulating VEGF levels,were investigated in patients with lung disease. Methods VEGF protein and m RNA expression levels in 38 lung tissue samples were analyzed by immunohistochemistry and reverse transcription-polymerase chain reaction(RT-PCR),respectively. Circulating VEGF levels were detected quantitatively by an enzyme linked immuno-sorbent assay. Results The level of VEGF expression was significantly higher in lung cancer tissue than in the corresponding paracancerous or non-cancerous tissues. The average level of VEGF-positive staining was 76% in tissue samples from NSCLC patients; the levels were 89% in tissue samples from stage III patients and 92% in stage IV patients. High VEGF expression was also evident in cases with lymph node metastasis(84%),distant metastasis(90%),and lower differentiation degree(89%). VEGF m RNA in cancerous tissues was represented predominantly by the VEGF121 and VEGF165 isoforms. Circulating VEGF levels were significantly higher in NSCLC patients [(840 ± 324) pg/m L] than in patients with benign lung diseases [(308 ± 96) pg/m L] or in healthy individuals serving as controls [(252 ± 108) pg/m L]. Conclusion The over-expression of lung VEGF and its gene transcription status should be useful molecular indicators for NSCLC diagnosis.

Clinicopathological features of hypoxia-inducible factor-1α and vascular endothelial growth factor expression in patients with lung cancer

Objective The aim of the study was to investigate the clinicopathological characteristics of hypoxiainducible factor-1α(HIF-1α) and vascular endothelial growth factor(VEGF) expression in patients with lung cancer.Methods Cancerous and noncancerous tissues were collected post-operation from 115 patients with lung cancers by the self-control method. Total RNA was extracted from the lung tissues. The status of tissue HIF-1α expression and intercellular distribution was observed by immunochemistry using a tissue microarray. The expression levels of circulating HIF-1α and VEGF were detected by enzyme-linked immunosorbent assay(ELISA).Results The expression of serum HIF-1α [(138.3 ± 28.8) μg/L] in the group of patients with lung cancer was significantly higher(P < 0.01) than that in the group of patients with pneumonia [(58.8 ± 14.5) μg/L] and the control group of patients ((24.1 ± 3.3) μg/L)There was a strong positive correlation of serum HIF-1α levels(r = 0.937, P < 0.01) with serum VEGF levels. The specific concentration of total RNA [(1.52 ± 1.14) μg/mg wet lung tissues] in the cancerous tissues was significantly higher(t = 8.494, P < 0.001) than that in the noncancerous tissues ((0.58 ± 0.33) μg/mg)The clinicopathological features of HIF-1α expression in lung cancer tissues revealed a significant relationship between positive HIF-1α expression and patient sex(χ~2 = 4.494, P = 0.034), tumor size(χ~2 = 4.679, P = 0.031), differentiation degree(χ~2= 8.846, P = 0.012), and presence of lymphatic node metastasis(χ~2= 6.604, P = 0.037).Conclusion Abnormal HIF-1α expression in lung cancer is closely related with nucleic acid metabolism and angiogenesis, and it may be helpful in the diagnosis and identification of lung cancer.

循环血AFP-mRNA与HS-GGT检测对肝癌的诊断与监测价值(英文)

Objective: Early finding of hepatocellular carcinoma(HCC) and monitoring of its metastasis are of the utmost importance. The objectives of this study were to investigate the values of circulating hepatoma-specific gamma-glutamyl transferase(HS-GGT) fraction and α-fetoprotein mRNA(AFP-mRNA) from peripheral blood mononuclear cells(PBMCs) in diagnosis or monitoring metastasis of HCC. Methods: Total RNA was extracted from hepatomas or PBMCs of patients, synthesized to AFP-cDNA through random primers and reverse transcriptase, amplified by using a nested PCR assay, and confirmed by sequencing. Simultaneity, the HS-GGT activities were quantitatively determined in sera of patients. The comprehensive assessments of two markers were investigated in HCC patients. Results: The amplified fragments of AFP-mRNA were identical to original designed ones(159 bp) and confirmed by sequencing. The lowest sensitivity was 2 ng/L of total RNA. The incidences of AFP-mRNA and HS-GGT were 60.4%(113 of 187) and 84.5%(158 of 187) in the HCC group, and their significantly higher(P < 0.01) than that in any of other groups. The high frequencies of HS-GGT and AFP-mRNA were 78.2% and 56.4% in the HCC patients with AFP < 50 ng/mL. All positive of AFP-mRNA was found in HCC patients with extrahepatic metastasis. No significant correlation was found between tumor size and AFP-mRNA or HS-GGT. The total incidence of combined AFP-mRNA and HS-GGT detection was 93.6 % for HCC diagnosis. Conclusion: HS-GGT and AFP-mRNA are specific markers for HCC diagnosis or differentiation, especially the former for early finding and the later for monitoring metastasis of HCC.

肝癌缺氧诱导因子-lα异常表达与靶向干预的临床价值研究(英文)

Objective:The aim of this study was to analyze the expression features of hypoxia inducible factor-1α (HIF-1α) in hepatocellular carcinoma (HCC) and effects of HIF-1α silencing on HepG2 cells.Methods:HIF-1α expression was analyzed in the self-control HCC specimens by immunohistochemistry.After HepG2 cells with miRNA transfection,the expression of HIF-1α was determined at mRNA or protein level by real-time polymerase chain reaction (PCR) or Western blotting.Vascular endothelial growth factor (VEGF) and angiopoietin-2 (ANG-2) were determined by ELISA.Alterations of cell cycles and apoptosis of HepG2 cells were measured using a flow cytometer.Results:Positive HIF-1α was brown and granule-like in the cytoplasm or nucleus.Significant difference was found between HCC (80%) and its surrounding tissues (100%,χ2=22.35,P < 0.001) and HIF-1α expression related to tumor size.At 72 h after miRNA transfection,the expression of HIF-1α in HepG2 cells was down-regulated by 87% at mRNA or 65% at protein level,with VEGF and ANG-2 decreased to 54% and 36%,respectively.After RNA interference combined with anti-cancer drug,the apoptotic rate of HepG2 cells was increasing from 22.46% ± 0.61% to 36.99% ± 0.88%,with up-regulation of G1 phase (65.68% ± 0.91%) and down-regulation of S phase (19.47 ± 1.34 %).Conclusion:Abnormal expression of HIF-1α is associated with development of HCC,and HIF-1α gene silencing can effectively inhibit HepG2 cell proliferation.

Nonalcoholic Lipid Accumulation and Hepatocyte Malignant Transformation

Worldwide incidence of hepatocellular carcinoma (HCC) is steadily increasing,highlighting its status as a public health concern,particularly due to its significant association with other comorbidities,such as diabetes.However,nonalcoholic fatty liver disease (NAFLD) has emerged as a primary risk factor,with its own prevalence increasing in recent years,and it has gradually caught up with the historical primary etiological factors of infection with hepatitis B virus and hepatitis C virus,exposure to aflatoxin,or alcohol liver disease.The deeply worrisome aspects of all of these high risk factors,however,are their remarkable presence within populations.Systemic and genetic mechanisms involved in the malignant transformation of liver cells,as well as useful biomarkers of early stage HCC are being investigated.However,the exact mechanisms underlying the interrelation of NAFLD and HCC remain largely unknown.In this review,some of the recent advances in our understanding of liver lipid accumulation are summarized and discussed to provide insights into the relationship between NAFLD and hepatocyte malignant transformation.(C) 2016 The Second Affiliated Hospital of Chongqing Medical University.Published by XIA & HE Publishing Inc.All rights reserved.