探讨活化态的肝星状细胞（HSC）对小鼠肝细胞肝癌（肝癌）血管形成、肿瘤生长及转移的作用。

将30只BALB/c-nu雌性裸鼠按随机数字表法分成3组，每组各10只，单纯组将1×10⁶个H22肝癌细胞注射于裸鼠肝脏，低浓度组将1×10⁶个H22肝癌细胞及1×10⁵个羟基荧光素二醋酸盐琥珀酰亚胺酯（CFSE）标记的HSC-T6细胞注射于裸鼠肝脏，高浓度组将1×10⁶个H22肝癌细胞及4×10⁵个CFSE标记的HSC-T6细胞注射于裸鼠肝脏；各组裸鼠生长50 d后处死，观察肝原位肿瘤、肝转移瘤、肺转移瘤的数量和体积。应用免疫组织化学方法（免疫组化法）检测肝原发肿瘤组织、肝转移瘤组织及肺转移瘤α-平滑肌肌动蛋白（α-SMA)、胶质纤维酸性蛋白（GFAP）、分化群抗原（CD）34、血管内皮生长因子（VEGF）及增殖细胞核抗原（PCNA）的表达水平；用蛋白质印迹法检测VEGF蛋白含量。观察HSC对肝癌血管形成、增殖作用影响。两组实验数据比较采用t检验，3组实验数据比较采用单因素方差分析，两两比较采用LSD-t检验。

免疫荧光法检测显示HSC-T6细胞株中HSC细胞纯度>95%，CFSE标记的HSC为绿色荧光细胞，细胞培养8周，CFSE染色的荧光强度稳定。单纯组肝癌成瘤率70%，低浓度组成瘤率70%，高浓度组成瘤率为80%。高浓度组肝内原位肿瘤体积明显大于低浓度组及单纯肝癌细胞组，差异有统计学意义（LSD-t=0.04，0.04；P<0.05）。高浓度组裸鼠肝内转移瘤及肺转移瘤数目明显多于低浓度组及单纯组（LSD-t=0.33，0.57；P<0.05）。单纯组、低浓度组及高浓度组原发肿瘤组织中α-SMA数密度分别为（19±4）、（42±4）及（62±6）个/高倍镜视野，高浓度组α-SMA数密度明显多于低浓度组及单纯肝癌细胞组，差异有统计学意义（LSD-t=2.12，2.12；P<0.05）；单纯组、低浓度组及高浓度组原发肿瘤组织中GFAP数密度分别为（15±4）、（36±5）、（57±5）个/高倍镜视野，高浓度组GFAP数密度明显多于低浓度组及单纯肝癌细胞组，差异有统计学意义（LSD-t=2.00，2.00；P<0.05）。单纯组、低浓度组及高浓度组原发肿瘤组织中CD34数密度分别为（19.0±2.6）、（31.0±6.2）、（43.0±8.3）个/高倍镜视野，高浓度组CD34数密度明显多于低浓度组及单纯组（LSD-t=2.75，2.75；P<0.05）。肝转移瘤组织标本中，肿瘤直径<1 mm的肝转移瘤中见微量CD34表达，数密度为（2.1±0.6）个/高倍镜视野；在肿瘤直径为1~3 mm的肝转移瘤中，CD34表达的数密度为（11.2±3.3）个/高倍镜视野，明显多于在直径<1 mm的肝转移瘤组织（t=8.69，P<0.05)。单纯组、低浓度组及高浓度组原位肿瘤标本中VEGF吸光度值分别为0.21±0.05、0.31±0.06、0.42±0.06，高浓度组VEGF吸光度值明显高于低密度组及单纯组（LSD-t=0.07，0.07；P<0.05）。单纯组、低浓度组及高浓度组肝原位肿瘤VEGF蛋白相对含量分别为0.19±0.02、0.36±0.05、0.45±0.06，高浓度组VEGF蛋白相对含量明显高于低密度组及单纯组（LSD-t=0.02，0.02；P<0.05）。单纯组、低浓度组及高浓度组原位肿瘤标本PCNA增殖指数（PCNA-LI）分别为（46±7）%、（68±10）%、（87±9）%，高浓度组明显多于低浓度组和单纯组，差异有统计学意义（LSD-t=3.89，3.89；P<0.05）。

HSC可能通过调节肝癌血管形成而影响肝癌生长及转移。

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.