Establishment of immotalized cell lines from transgenic mouse nephron segments

从转基因小鼠肾单位片段建立永生化细胞系

• 批准号: 04557122
• 负责人: ENDOU Hitoshi
• 金额: $ 11.14万
• 依托单位: Department of Pharmacology and Toxicology, Kyorin University School of Medicine (1993-1994)The University of Tokyo (1992)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-04557122/
• 关键词: transgenic mouse; SV40 large T antigen gene; single nephron; immortalized cell lines; nephron segment-specific enzyme; nephron segment-specific metabolism; ATP receptor; cisplatin; トランスジエニックマウス; 温度感受性因子; 近位尿細管; 集合尿細管; サイクリツクAMP; アルカリフォスファターゼ

The results that we could obtain according to our plan are as follows.1)Establishment of immortalized cultured cell lines from transgenic mouse kidneys.A ts-SV40 large T-antigen gene was injected into pronuclei of fertilized eggs of C57BL/6 mice, and mice of first generation were abtained. Various nephron segments were microdissected from collagenase-treated kidneys and cultivated. Cells thus made can be maintained over 100 passages.2)Characterization of established cell lines.Several characters like nephron segment-specific enzymes, metabolisms, and hormone recepters in the cell lines were compared with those in freshly isolated respective nephron segments. These cell lines maintained most site-specific characters except gamma-GTP activity, gluconeogenesis and porathyroid hormone receptor.3)Application of these cell lines for pharmacological and toxicological studies.Cells originated from the terminal proximal tubule could be successfully applied for the evaluation of cisplatin-induced nephrotoxicity. Since ATP-purinoceptor was highly expressed in cells form outer medullary collecting duct, these cells might be suitable sample for cloning cDNA of ATP receptr protein (s) . This trial is now under investigation.

1）建立转基因小鼠肾脏永生化培养细胞系将ts-SV 40大T抗原基因注入C57 BL/6小鼠受精卵原核，获得第一代小鼠。从胶原酶处理的肾脏显微切割各种肾单位片段并培养。2）建立的细胞系的特性：将该细胞系中的肾单位节段特异性酶、代谢和激素受体等特性与新鲜分离的相应肾单位节段进行比较。这些细胞系除γ-GTP活性、促性腺激素生成和甲状腺激素受体外，其余均保持了大部分位点特异性特征。3）这些细胞系在药理学和毒理学研究中的应用：来源于终末近曲小管的细胞可成功用于评价顺铂的肾毒性。由于ATP-嘌呤受体在外髓集合管细胞中高表达，这些细胞可能是克隆ATP受体蛋白cDNA的合适样本。此案正在调查中。

项目成果

Takeda,M.: "Intranephron distribution of creatine content in rats" Biochemistry and Molecular Biology International. 32. 435-440 (1994)

Takeda，M.：“大鼠肌酸含量的肾单位分布”国际生物化学和分子生物学。

Takeda, M. , Hosoyamada, M. , Shirato, I. , Obinata, M. , Suzuki, M. and Endou, H.: "Establishment of vasopressin-responsive early proximal tubular cell lines derived from transgenic mice harboing temperature-sensitive simian virus 40 large T-antigen gene

Takeda, M.、Hosoyamada, M.、Shirato, I.、Obinata, M.、Suzuki, M. 和 Endou, H.：“建立源自温度敏感猿猴的转基因小鼠的加压素响应性早期近端肾小管细胞系

遠藤 仁(丸茂文昭、黒川清、越川昭三、藪田敬次郎、小川龍、北島政樹 編集): "輸液ハンドブック" 中外医学社, 479 (1994)

远藤仁（丸茂文明、黑川清、越川正三、薮田敬二郎、小川龙、北岛正树编）：《输液手册》中外医学社，479（1994）

Kanai, Y,and Endou, H: "Roles of glutamate transporters in neuronal cell death" J.Toxicol.Sci. 19. 295-297 (1994)

Kanai, Y 和 Endou, H：“谷氨酸转运蛋白在神经元细胞死亡中的作用”J.Toxicol.Sci。

Takeda, M., Hosoyamada, M., Shirato, I., Obinata, M., Suzuki, M.and Endou, H: "Establishment of vasopressin-responsive early proximal tubular cell lines derived from transgenic mice harboing temperature-sensitive simian virus 40large T-antigen gene." Bioc

Takeda, M.、Hosoyamada, M.、Shirato, I.、Obinata, M.、Suzuki, M.和 Endou, H：“建立源自携带温度敏感猿病毒的转基因小鼠的加压素反应性早期近端肾小管细胞系