HepG2及其克隆形成细胞端粒酶活性的异质性研究

Heterogeneity of telomerase activation in HepG2 and its colony-forming cells

目的研究端粒酶在肝癌细胞株HepG2及其克隆形成细胞中的表达,探讨不同增殖能力的肝癌细胞中端粒酶活性的异质性,为肝癌的诊断以及治疗提供新的思路。方法利用软琼脂克隆形成实验富集分离人肝癌HepG2细胞的克隆形成细胞;常规培养HepG2及其克隆形成细胞,利用免疫细胞化学、Western blotting和RT-PCR检测hTERT蛋白和mRNA在HepG2细胞及其克隆形成细胞中表达的异质性。结果①HE染色显示,克隆形成细胞的胞核较HepG2细胞大,核仁明显;细胞伸出较多的细长突起,并连接形成网状。②免疫细胞化学染色显示,hTERT在HepG2细胞的表达以细胞质为主,克隆形成细胞则以细胞核为主。③Western blotting和RT-PCR结果显示,克隆形成细胞中hTERT蛋白质和mRNA的表达均高于HepG2细胞。结论①hTERT在HepG2细胞及其克隆形成细胞中的表达存在异质性,且在克隆形成细胞中表达较高。②hTERT在HepG2细胞及其克隆形成细胞中的表达模式提示克隆形成细胞具有肝癌干细胞的特征。

Objective To illustrate the heterogeneity of telomerase in cells with different reproductive activity and to provide a new target for diagnosis and treatment of hepatoma, we studied the expression of telomerase in HepG2 and its colony-forming cells. Methods The soft agar clone formation experiment was adopted to separate the colony-forming cells from HepG2 cells. HepG2 ceils and its colony-forming cells were cultured. Then immunocytochemistry ,western blotting and RT PCR were used to examine the ex- pressions of hTERT in HepG2 and its colony forming cells, respectively. Results （1） Compared with HepG2 ceils, the colony-forming cells had a characteristic of larger nuclei, prominent nucleoli and more cell processes. （~） Immunocytochemistry showed that the expression of hTERT protein was mainly located in the cytoplasm of HepG2 and in the nuclei of colony-forming cells. （3） Western blotting and RT-PCR showed that the expressions of hTERT protein and mRNA in the clone-forming cells were higher than those in HepG2 cells, respectively. Conclusion （1） The expression of hTERT in HepG2 cells and its colony forming cells is heterogeneous, and the expression of hTERT in the colony forming cells is higher than that in HepG2 cells. （2） The expression patterns of hTERT in HepG2 and its colony-forming cells imply that the colony-forming cells have the characteristics of liver cancer stem cells.