To investigate the effect of activated hepatic stellate cells(HSC) to angiogenesis, proliferation and metastasis of hepatocellular carcinoma (HCC).

Thirty BALB/c-nu female nude mice were divided into 3 groups according to randomly digital table: normal control group, low consistency group and high consistency group with 10 mice in each group. Mice in the normal control group received an intrahepatic injection of 1×10⁶ hepatoma cells(H22), low consistency group 1×10⁶ H22 and 1×10⁵ HSC-T6 cells labeled by (5, 6-carboxyfluorescein diacetate, succinimidyl ester, CFSE) , and high consistency group 1×10⁶ H22 and 4×10⁵ HSC-T6 cells labeled by CFSE. The mice in 3 groups were killed after 50 days and the number and size of tumors in situ, hepatic and pulmonary metastases were observed. The expression level of α-smooth muscle actin(α-SMA), glial fibrillary acidic protein (GFAP) , cluster of differentiation(CD) 34, vascular endothelial growth factor (VEGF) , proliferating cell nuclear antigen (PCNA) in tissues of primary tumors, hepatic and pulmonary metastases were tested by immunohistochemistry. Western blot were used to examine the protein level of VEGF. The effect of HSC to the angiogenesis, proliferation of HCC was observed. The experimental data between two groups was compared using t test. And the data of three groups were compared using one-way analysis of variance, and the comparison between two groups was performed using LSD-t test.

The cell purity of HSC>95% in the cell line of HSC-T6. The HSC labeled by CFSE were green fluorescent cells. The fluorescent intensity of CFSE staining of was stable after 8 weeks of cell culture. The tumor formation rate in the normal control group was 70%, low consistency group was 70%, and the high consistency group was 80%. The size of primary tumors in the high consistency group was obviously larger than the other 2 groups, and there was significant difference (LSD-t=0.04, 0.04; P<0.05). The number of intrahepatic and pulmonary metastases of mice in the high consistency group was significantly higher than the low consistency group and normal control group (LSD-t=0.33, 0.57; P<0.05). The number density of α-SMA of primary tumor tissue in the normal control group, low consistency group and high consistency group were (19±4) , (42±4) and (62±6) /high power field. The number density of α-SMA in the high consistency group was significantly larger compared with the other 2 groups (LSD-t=2.12, 2.12; P<0.05). The number density of GFAP in the normal control group, low consistency group and high consistency group were (15±4), (36±5), (57±5)/high power field respectively. The number density of GFAP in the high consistency group was obviously larger compared with the other 2 groups, and significant difference was observed (LSD-t=2.00, 2.00; P<0.05). The number density of CD34 of primary tumor in the normal control group, the low consistency group and high consistency group were (19.0±2.6), (31.0±6.2), (43.0±8.3)/high power field, of which the high consistency group was significantly larger than the other 2 groups (LSD-t=2.75, 2.75; P<0.05). The number density of CD34 in the liver metastases with the tumor diameter less than 1 mm was (2.1±0.6)/high power field, and in the metastases with diameter between 1 to 3 mm was (11.2±3.3)/high power field, which was significantly higher than the former(t=8.69, P<0.05). The expression (optical density) of VEGF from primary tumors in the normal control group, the low consistency group and high consistency group were 0.21±0.05, 0.31±0.06, 0.42±0.06 respectively and the optical density of VEGF in the high consistency group was higher compared with the other 2 groups (LSD-t=0.07, 0.07; P<0.05). The protein level of VEGF of the hepatic primary tumor tissue in the normal control group, the low consistency group and high consistency group were 0.19±0.02, 0.36±0.05, 0.45±0.06 respectively, of which the high consistency group was obviously higher than the other 2 groups (LSD-t=0.02, 0.02; P<0.05). The proliferating cell nuclear antigen-labeling index(PCNA-LI) in the normal control group, low consistency group and high consistency group were (46±7)%,(68±10)%,(87±9)% respectvely, of which the high consistency group was significantly higher than the other 2 groups (LSD-t=3.89, 3.89; P<0.05).

HSC may promote HCC proliferation and metastasis through regulation of angiogenesis.