Establishment of a sandwich ELISA method for detection of vascular endothelial growth factor in serum samples of hepatocellular carcinoma patients.

OBJECTIVE: To establish a sandwich ELISA method for detecting vascular endothelial growth factor (VEGF) in sera of population and the patients with hepatocellular carcinoma (HCC). METHODS: Full length and two truncated human VEGF cDNA sequences were amplified from a commercial plasmid pBLAST49-hVEGF by PCR and inserted into the prokaryotic-expression plasmid pET-32a or pGEX-2T. Various VEGF proteins were expressed and purified from E. coli in His-Trx or GST fusion forms. The specific VEGF antibodies were elicited in experimental rabbits and mice by immunization of the full length VEGF fusion protein His-Trx-VEGF1-165. After purification of antibodies with chromatograph of Protein G, a sandwich ELISA technique was established. Serum VEGF levels were evaluated in 229 adults and 291 HCC patients. RESULTS: SDS-PAGE displayed that the molecular weights of the expressed full length (His-Trx-VEGF1-165), N-terminal (His-Trx-VEGF1-100) and C-terminal (GST-VEGF100-165) human VEGF fusion proteins were about 38KD, 31KD, and 33KD, respectively. Western blots confirmed that the prepared antisera were able to recognize both prokaryoticly and eukaryoticly expressed recombinant VEGF proteins. Assays of serially diluted His-Trx-VEGF1-100 by the established sandwich ELISA method showed that the linear range of the standard curve was 0.625-320 ng/mL, with the squared correlation coefficient R2 = 0.991. Screening of a serum panel containing 291 serum samples of HCC patients and 229 health adults revealed that the average VEGF level in HCC patients was higher than that in healthy controls, with a statically significant difference. CONCLUSION: The established sandwich ELISA reflects the level of serum VEGF and provide scientific basis for screening metastasis and recurrence of HCC using serum VEGF as an index.

[Long-term effect of united hepatectomy and splenectomy on treatment of hepatocellular carcinoma complicated with cirrhosis and hypersplenism].

OBJECTIVE: To discuss the long-term effect of united liver and spleen resection on treatment of hepatocellular carcinoma (HCC) complicated with cirrhosis and hypersplenism. METHODS: The clinical data of 102 patients of HCC complicated with cirrhosis and hypersplenism, 57 receiving united hepatectomy and splenectomy (group A), and 45 receiving hepatectomy only (group B), were retrospectively examined. RESULTS: The volume of operative blood loss of group A was 765 ml (100 - 2,400 ml), not significantly different from that of group B (720 ml, 200 - 2,000 ml). The operative morbidity was 1.7% (1/57) of group A, significantly higher lower than that of group B (4.4%, 2/45). After the operation, the platelet count increased to 275.8 x 10(9)/L in group A, significantly higher than that in group B (83 x 10(9)/L, P < 0.05); and the white blood cell count of group A increased to 10.4 x 10(9)/L, significantly higher than that of group B (4.19 x 10(9)/L, P < 0.05). Post-operative gamma glutamyl transferase, a prognostic factor for liver cirrhosis, of group A was 68 U/L, significantly lower than that of group B (132 U/L, P < 0.05). The incidence of complication was 29.8% (17/57) in group A, not significantly different from that of group B (33.3%, 15/45). The 1, 3, and 5-year recurrence rates were 27.1%, 48.6%, and 69.3% in group A, versus 41.2%, 50.0%, and 77.9% in group B, whereas the 1, 3, and 5-year survival rates were 92.6%, 59.1%, and 41.8% in group A, versus 80.3%, 43.9%, and 29.1% in the group B. The longest survival time in group A was 12.9 months, significantly longer than that in group B (6.3 months, P < 0.01). CONCLUSION: United liver and spleen resection is a safe and effective modality for the treatment of HCC complicated with cirrhosis and hypersplenism.