Isolation ,identification and comparison of tumor-derived endothelial cells from human hepatocellular carcinoma

Abstract

Objective To isolate, identify and compare vascular endothelial cells derived from human hepatocellular carcinoma (HCC). Methods Modified immunomagnefic methods using magnetic beads conjugated with anti-CD105 and anti-CD31 antibody were used to isolate tumor-derived endothelial cells (TEC), namely CD31 +TEC and CD105 +TEC. CD31 +TEC and CD105 +TEC were cultured in vitro,identified and compared by morphological appearance, functional characterization, flow cytometry and realtime polymerase chain reaction (PCR) analyses. Results The isolated CD105 + TEC presented fibrous bands appearance while CD31 + TEC presented fusiformis appearance. Macrophages, fibrocytes, and tumor cells contamination was excluded. Surface CD68 and α-SMA expression was less than 5%. Alpha fetoprotein expression was significantly lower in CD31 + TEC and CD105 + TEC than that in HepG2 cells ( P ＜0. 01 ), and the expression level was less than 1/8 folds of HepG2 cells. Internalization of acetylated lowdensity lipoprotein was positive in more than 95% of isolated cells. The absorbance (A) values of CD105 +TEC and CD31 + TEC grown for 48 h were 0. 45 ± 0. 04 and 0. 35 ± 0. 02 respectively ( P ＜ 0. 05 ). Proliferation of CD105 + TEC was significantly more active than CD31 + TEC when cultured in the serum supplemented with medium for 24, 48, and 72 h. The isolated cells could form capillary-like tubes on Matrigel matrix, stronger capability of CD31 + TEC than CD105 + TEC. Conclusion CD31 + TEC and CD105 + TEC can be conveniently purified by modified immunomagnetic methods. Key words: Carcinoma,hepatocellular; Endothelial cells; Angiogenesis