A study for replicative senescence in hepatocytes in hepatic fibrosis

肝细胞的显著端粒缩短与慢性肝病中的复制性衰老和非分裂状态相关，导致终末期肝衰竭和/或肝细胞癌的发展。为了防止肝细胞端粒缩短，我们正在关注雌激素依赖的人端粒酶逆转录酶（hTERT）基因的反式激活作为慢性肝病的端粒酶治疗。我们检测了17β-雌二醇（E2）处理前后人正常肝细胞株（HC-细胞、h-Nheps和WRL-68）hTERTmRNA和蛋白表达及端粒酶活性（TA）。Hc-细胞和h-Nheps经E_2处理后hTERT基因mRNA和蛋白表达均上调，而WRL-68细胞在E_2处理前hTERT基因就已过表达。Hc-细胞和h-Nheps的端粒长度随传代次数的增加而减少，而长期E_2暴露的端粒长度比无E_2暴露的端粒长度长。E_2处理组细胞中β-半乳糖苷酶阳性细胞数显著低于未处理组（P<0.05）。采用四氯化碳（CCl_4）诱导的大鼠肝纤维化模型，观察外源性E_2对肝纤维化大鼠肝纤维化组织中TA和端粒长度的影响。雌、雄CCl_4肝纤维化大鼠给予E_2后TA均显著高于未给予E_2组（P<0.05）。未观察到TA的性别相关差异。长期给予E_2可显著地挽救雄性和雌性大鼠肝脏端粒的广泛缩短。这些结果表明，雌二醇作为一个积极的调节剂的hTERT基因在肝脏中，并可能延长寿命的肝细胞通过防止广泛的端粒缩短。雌激素依赖性hTERT基因的反式激活是减缓慢性肝病进展的新策略。

Significant telomere shortening of hepatocytes is associated with replicative senescence and a non-dividing state in chronic liver disease, resulting in end-stage liver failure and/or development of hepatocellular carcinoma. To prevent hepatocytes from critical telomere shortening, we are focusing on an estrogen-dependent transactivation of human telomerase reverse transcriptase (hTERT) gene as a telomerase therapy in chronic liver disease. We examined hTERTmRNA and protein expressions, and telomerase activity (TA) in 3 human normal hepatic cell lines (Hc-cells, h-Nheps and WRL-68) before and after treatment with 17β-estradiol (E2). The hTERT mRNA and protein expressions were up-regulated in Hc-cells and h-Nheps by E_2-treatment, while overexpression of hTERT gene was observed in WRL-68 even before the treatment. Telomere length decreased with accumulated passages in Hc-cells and h-Nheps, whereas that with long-term E_2 exposure was longer than that without E_2. Incidence of β-galactosidase positive cells, indicating senescence state, significantly decreased in E_2-treated cells in comparison with non-treated cells (P<.05). Effects of exogenous E_2 administration on TA and telomere length were examined in CCl4-induced liver fibrosis model of rats. TA of both male and female rats of CCl4-induced liver fibrosis with E_2 administration significantly higher than those without E_2 administration (P<.05). Gender-related difference in TA was not observed. Long-term E_2 administration could drastically rescue the hepatic telomere from extensive shortening in both male and female rats. These results suggest that estradiol acts as a positive-modulator of hTERT gene in the liver and may prolong the lifespan of hepatocytes by the prevention of extensive telomere shortening. The estrogen-dependent transactivation of the hTERT gene is a new strategy for slowing the progression of chronic liver disease.