Molecular Analysis of Stem Cell in Hepatocarcinogenesis

干细胞在肝癌发生中的分子分析

• 批准号: 13670528
• 负责人: OHTSURU Akira
• 金额: $ 2.56万
• 依托单位: Nagasaki 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: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670528/
• 关键词: Hepatoma; Stem Cell; Carcinogenesis; Apoptosis; MEKK; Radiation; 肝細胞; 肝癌; 前癌病変; HUMARA assay

We compared the expression profile of RTK genes in rat normal liver and diethylnitrosamine-induced hepatoma tissues using a homology cloning method with degenerated primers. The Tie-2, c-Met, and Flk-1 genes were the most abundant RTK genes cloned in rat hepatoma compared to normal liver. In situ hybridization and immunohistochemical studies showed overexpression of c-Met and Flk-1 in GST-P positive preneoplastic lesions as well as neoplastic lesions. Tie-2 was expressed not only in endothelial cells but also in so-called oval cells, which are thought to be liver stem cells. Tie-2 ligand, angiopointin-1, mRNA was detected in both normal livers and hepatoma cells/tissues. In contrast, angiopoietin-2 mRNA was detected only in hepatoma tissues.We next aimed to examine the involvement of caspases and calpains in H2O2-induced hepatic cell apoptosis. TUNEL-positive apoptotic cells appeared in parallel with poly(ADP-ribose) polymerase (PARP) cleavage and procaspase-3 proteolysis by H2O2 treatment in a dose-dependent manner (250-1000μM). Bcl-xL and intact Bax expression levels decreased when H2O2 was >250μM. The cleaved form of Bax appeared prior to caspase-3 activation, increasing in a dose-dependent manner. These results indicate that Bax cleavage is upstream signal of caspase-dependent apoptosis in hepatocytes. Molecular analysis of Bax cleavage may allow the mechanism of development of hepatoma cell.Finally, we acknowledge a support from this grant and a valuable contribution of our post doc fellows and staffs.

我们用简并的同源克隆方法比较了RTK基因在大鼠正常肝脏和二乙基亚硝胺诱发的肝癌组织中的表达谱。与正常肝相比，Tie-2、c-Met和Flk-1基因是在大鼠肝癌中克隆的最丰富的RTK基因。原位杂交和免疫组织化学研究显示，c-Met和Flk-1在GST-P阳性癌前病变和癌前病变中均有高表达。Tie-2不仅在内皮细胞中表达，在所谓的卵圆形细胞中也有表达，椭圆形细胞被认为是肝脏干细胞。Tie-2配体Angiopointin-1在正常肝脏和肝癌细胞/组织中均有表达。相反，血管生成素-2mRNA仅在肝癌组织中检测到。接下来，我们的目标是研究caspase和calain在过氧化氢诱导的肝细胞凋亡中的作用。在H_2O_2作用下(250~1000μM)，TUNEL阳性的细胞出现与多聚腺苷二磷酸-核糖聚合酶(PARP)裂解和原天冬氨酸氨基转移酶-3蛋白降解平行的细胞形态。当H_2O_2浓度为250μ时，bcl-xl和全长bax的表达水平下降，bax的断裂形式出现在caspase-3激活之前，且呈剂量依赖性增加。这些结果表明，Bax裂解是caspase依赖的肝细胞凋亡的上游信号。BAX裂解的分子分析可能有助于肝癌细胞的发展机制。最后，我们感谢这笔资金的支持以及我们博士后研究员和工作人员的宝贵贡献。

项目成果

S.S.Shklyaev, et al.: "Transient activation of c-Jun NH2-terminal kinase by gowth factors closely likes to human thyroid cell survival."Thyroid. 11. 629-636 (2001)

S.S.Shklyaev 等人：“生长因子对 c-Jun NH2 末端激酶的瞬时激活与人类甲状腺细胞的存活密切相关。” 甲状腺。

K.Hamasaki, et al.: "The sympathetic nervous system promotes carbon tetrachloride-induced liver cirrhosis in rats by suppressing apoptosis and enhancing the growth kinetics of regenerating hepatocytes."J.Gastroenterology. 36. 111-120 (2001)

K.Hamasaki 等人：“交感神经系统通过抑制细胞凋亡和增强再生肝细胞的生长动力学，促进四氯化碳诱导的大鼠肝硬化。”J.Gastroenterology。

N.Mitsutake, et al.: "PKC σ mediates ionzing radiation-induced activation of c-Jnu NH2-terminal kinase through MKK7 in human thyroid cells."Oncogene. 20. 989-996 (2001)

N. Mitsutake 等人：“PKC σ 通过人甲状腺细胞中的 MKK7 介导电离辐射诱导的 c-Jnu NH2 末端激酶的激活。”Oncogene。 20. 989-996 (2001)

K.Hamasaki, et al.: "The sympathetic nervous system promotes carbon tetrachloride-induced liver cirrhosis by suppressing apoptosis and enhancing the growth kinetics of regenerating rat hepatocytes"J Gastroenterology. 36. 111-120 (2001)

K.Hamasaki 等人：“交感神经系统通过抑制细胞凋亡和增强再生大鼠肝细胞的生长动力学来促进四氯化碳诱导的肝硬化”J Gastroenterology。

N.Mitsutake, A.Ohtsuru, et al.: "PKC δ mediates ionizing radiation-induced activation of c-Jun NH2-terminal kinase through MKK7 in human thyroid cells"Oncogene. 20. 989-996 (2001)

N.Mitsutake、A.Ohtsuru 等人：“PKC δ 在人甲状腺细胞中通过 MKK7 介导电离辐射诱导的 c-Jun NH2 末端激酶的激活”Oncogene 20. 989-996 (2001)。