肝癌相关成纤维细胞的培养和鉴定及其对肝癌细胞增殖的影响

目的：分离、培养及鉴定原代肝癌相关成纤维细胞(hCAF)，阐明其在肝癌发生发展中的作用。方法：采用酶消化法分离、纯化人hCAF, Western blotting法鉴定其相关蛋白标记物的表达，流式细胞术检测肝癌细胞Hep3B经hCAF上清处理后的增殖速度及细胞周期的变化，建立裸鼠移植瘤模型，观察hCAF对肝癌细胞生长的影响。结果：在肝癌组织标本中成功分离出hCAF，Western blotting结果显示hCAF特异性地高表达α-平滑肌肌动蛋白（α-SMA）。CCK8细胞增殖检测试剂盒检测证实，经hCAF上清处理后的Hep3B细胞增殖量为完全培养基处理48 h细胞增殖量的126.0%、125.0%及120.5%，细胞增殖量差异有统计学意义（P<0.05）。流式细胞术结果显示，经hCAF上清处理后的Hep3B细胞增殖速度及细胞周期中处于S期的细胞数目均高于完全培养基处理的Hep3B细胞（P<0.05）。hCAF与Hep3B共同接种至裸鼠皮下的成瘤体积［（1.34±0.52） cm3］明显大于单独接种Hep3B组［(0.51±0.09)　cm3］，差异有统计学意义（P<0.05）。结论：hCAF可以明显促进肝癌细胞系Hep3B的生长，其可能在肝癌生长过程中发挥重要作用。

Culture and identification of hepatocellular carcinoma associated fibroblasts and their effects on proliferation of hepatocellular carcinoma cells

Objective To isolate,cultivate and identify the hepatocellular carcinoma associated fibroblasts(hCAF) and to investigate their possible role in the occurrence and development of hepatocellular carcinoma.Methods The primary hCAF were isolated and purified by enzyme digestion method.The expressions of the specific proteins associated with hCAF were examined by Western blotting method.The changes of cell cycles of Hep3B cells after hCAF supernatant treatment were evaluated by flow cytometry.The effect of hCAF on hepatocellular carcinoma cells was observed though building a xenograft tumor model in nude mice.Results The hCAF were successfully isolated and confirmed and highly specificly expressed α-smooth muscle actin(α-SMA) detected by Western blotting method.The proliferation capacity of Hep3B was increased significantly after treated with hCAF conditioned media.The proliferation capacities of Hep3B were 126.0%,125.0% and 120.5% of those treated with complete medium for 48 h(P<0.05).The number of carcinoma cells and the percentages of carcinoma cells in S phase were higher than those treated with complete medium for 48 h(P<0.05).The tumor volume in Hep3B cells and hCAF co-injection group (1.34±0.52) cm2] was significantly larger than that in Hep3B cells inoculation group (0.51±0.09)cm2] in nude mice(P<0.05).Conclusion hCAF could promote the proliferation of hepatocellular carcinoma cell line Hep3B,and hCAF might play an important role in the progression of hepatocellular carcinoma.