Isolation, culture and identification of hepatocellular carcinoma-associated fibroblast and its effect on the biological function of hepatocellular carcinoma cells / 肿瘤研究与临床

ABSTRACT

Objective To establish an in vitro isolation and culture system for hepatocellular carcinoma (HCC)-associated fibroblast (CAF) and identify based on its specific markers of CAF. Methods CAF was isolated from the tumor tissue of the HCC patients after hepatectomy, by digesting with collagenase enzyme, centrifugation and resuspension. The morphology of CAF was observed by electron microscopy. Immunofluorescence staining was performed for detecting α-SMA and fibronectin as well as other specific markers such as AFP on the surface of HCC cells and CD31 on the surface of vascular endothelial cells. On the basis of the study, the function work between CAF and HCC cell line Huh7, HepG2 was detected under co-culture system. Meanwhile, CCK-8 was used to observe the effect of CAF on the proliferation of Huh7 and HepG2 cells, and Transwell assay was used to analyze CAF effect on the invasion of Huh7 and HepG2 cells. Results Compared with the other cells, the morphological analysis showed that CAF was more elongated or spindle-shaped. Moreover, the cell size and the nucleus were larger than normal epithelial cells. Immunofluorescence staining revealed that the CAF surface specific markers including α-SMA and fibronectin were positive, and mainly were the cell membrane staining. The proliferation and invasion of Huh7 and HepG2 were significantly increased by CAF. The results show that the increasing percentage of cells in 24 hours between blank group and the experimental group was (63 ± 4) %, (78 ± 5) % and (69 ± 5) %, (81 ± 3) %respectively after co-culture with CAF, the difference was statistically significant (P<0.05). In transwell model, the number of cells in the blank group and the experimental group was (59.4 ± 3.1), (162.9 ± 3.9) and (104.8 ± 2.6), (166.4 ± 4.2), and the difference was also statistically significant (P< 0.05). Conclusion The isolated CAF from HCC enhances the ability of tumor's proliferation and invasion.