A study for replicative senescence in hepatocytes in hepatic fibrosis

肝纤维化过程中肝细胞复制性衰老的研究

• 批准号: 14570156
• 负责人: MASUDA Tomoyuki
• 金额: $ 2.24万
• 依托单位: Iwate Medical University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570156/
• 关键词: stem cell; hTERT; TRE assay; replicative senescence; estradiol; biomolecular ineraction; telomere; telomerase; 肝臓; 肝不全

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.

在慢性肝病中，肝细胞显著的端粒缩短与复制性衰老和非分裂状态有关，从而导致终末期肝功能衰竭和/或肝细胞癌的发生。为了防止肝细胞发生严重的端粒缩短，我们将重点放在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上。用17-雌二醇(17β-estadiol，E_2)处理人正常肝细胞系(HC-Cells、h-Nheps和WRL-68)，检测其端粒酶活性(TA)和端粒酶活性(TA)。经E_2处理后，hTERT基因在HC-细胞和h-Nhep细胞中的表达上调，而在WRL-68细胞中hTERT基因的表达甚至在治疗前就已经过表达。在HC细胞和h-NHEP中，随着传代次数的增加，端粒长度逐渐缩短，但长期暴露于E_2的细胞端粒长度明显长于未处理的细胞。提示衰老状态的β-半乳糖苷酶阳性细胞的发生率在E_2处理的细胞中显著低于未处理的细胞(P&lt；0.05)。在四氯化碳诱导的大鼠肝纤维化模型上，观察外源性E_2对TA和端粒长度的影响。CCl4诱导的肝纤维化雌雄大鼠给予E_2后，TA均显著高于未给予E_2者(P&lt；0.05)。在TA上未发现性别差异。雌雄大鼠长期给予E_2，均可显著减轻大鼠肝细胞端粒的广泛缩短。这些结果表明，雌二醇是肝脏hTERT基因的正向调节剂，可能通过阻止广泛的端粒缩短而延长肝细胞的寿命。雌激素依赖的hTERT基因反式激活是减缓慢性肝病进展的新策略。

项目成果

Uchiyama M, Maesawa C, Yashima A, Itabashi T, Ishida Y Masuda T.: "Consensus primers for detecting monoclonal immunoglobulin heavy chain rearrangement in B-cell lymphomas."J Chin Pathol.. 56(10). 778-779 (2003)

Uchiyama M、Maesawa C、Yashima A、Itabashi T、Ishida Y Masuda T.：“用于检测 B 细胞淋巴瘤中单克隆免疫球蛋白重链重排的共有引物。”J Chin Pathol.. 56(10)。

Sato R, Maesawa C, Fujisawa K, Wada K, Oikawa K, Takikawa Y, Suzuki K, Oikawa H, Ishikawa K, Masuda T: "Prevention of critical telomere shortening by estradiol in human normal hepatic cultured cells and CCl4-induced rat liver fibrosis."Gut. 53(in press).

Sato R、Maesawa C、Fujisawa K、Wada K、Oikawa K、Takikawa Y、Suzuki K、Oikawa H、Ishikawa K、Masuda T：“雌二醇在人正常肝培养细胞和 CCl4 诱导的大鼠肝脏中预防关键端粒缩短

Tarusawa M, Yoshima A, endo M, Maesawa C.: "Quantitative assessment of minimal residual disease in childhood lymphold malignancies using an allele-specific oligonucleotide real-time quantitative polymerase chain reaction"Int J Hematol. 75. 166-173 (2002)

Tarusawa M、Yoshima A、endo M、Maesawa C.：“使用等位基因特异性寡核苷酸实时定量聚合酶链反应对儿童淋巴恶性肿瘤中微小残留病进行定量评估”Int J Hematol。

Maesawa C, Inaba T, Sato H, Iijima S, Ishida K, Trerashima M, Sato R, et al.: "A rapid biosensor chip assay for measuring of telomerase activity using surface plasmon resonance"Nucleic Acids Res. 31. E4-4 (2003)

Maesawa C、Inaba T、Sato H、Iijima S、Ishida K、Trerashima M、Sato R 等人：“使用表面等离子体共振测量端粒酶活性的快速生物传感器芯片测定”核酸研究。

Oikawa H, Masuda T, Kawaguchi J, Sato R.: "Three-dimensional examination of hepatic stellate cells in rat liver and response to endothelin-1 using confocal laser scanning microscopy"J Gastroenterol Hepatol. 17. 861-872 (2002)

Oikawa H、Masuda T、Kawaguchi J、Sato R.：“使用共聚焦激光扫描显微镜对大鼠肝脏中的肝星状细胞进行三维检查以及对内皮素-1 的反应”J Gastroenterol Hepatol。