The Investigation of the protective effect of hoat shock protein on neuronal death

热休克蛋白对神经元死亡保护作用的研究

• 批准号: 12671339
• 负责人: KINOUCHI Hiroyuki
• 金额: $ 2.18万
• 依托单位: Akita University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671339/
• 关键词: PC12; electroporation; heat shock protein; cerehral ischemia; fransfection; DNA; HSP; apoptosis; heat shock protein; HSP70; transfection; 熱ショック蛋白; hsp; トランスフェクション; 遺伝子組み換え

Gene transfer into rat brain: Rats (4, 8, or 12 weeks old) were anesthetized and fixed onto a stereotaxic operating table. A burr hole was opened and 20μg of plasmid DNA (pBK-CMV-HSP70) was injected into the hippocampus or striatum using a cannula. The injection time was changed from 10 sec to 10 min. Subsequently, a pair of stainless steel electrodes was inserted the region. Electric pulses were generated with a Square Electro Porator (CUY21) and were administered at 10 Hz. Electric pulses were 2ms in duration at 50, 100, or 150 V. However, at any condition, the sufficient gene transfer was not obtained. Only a couple of neurons showed β-galactosidase positive coincidentally.Transfection of HSP70 gene into PC12 cells: Human HSP75 cDNA was transfected using the pBK-CMV vector. Production of HSP70 protein was confirmed by Western blot analysis. The PC12 cells overexpressing HSP70 were exposed to hydrogen peroxide (50, 100, 150μM). MTT assay after 24h of exposure revealed the significant viability of HSP70-transfected PC12. The protective effect of HSP70 against oxidative injury was revealed.

基因转移到大鼠脑中：将大鼠（4、8或12周龄）麻醉并固定在立体定位手术台上，打开钻孔并将20μg质粒DNA（pBK-CMV-HSP70）注射到海马或脑中。使用套管将注射时间从 10 秒改为 10 分钟。使用方形电穿孔器 (CUY21) 产生电脉冲，并以 10 Hz 的频率施加持续时间为 2 毫秒、电压为 50、100 或 150 V 的电脉冲。然而，在任何条件下，都没有足够的基因转移。仅少数神经元同时显示β-半乳糖苷酶阳性。 将HSP70基因转染至PC12细胞：使用人HSP75 cDNA转染。 pBK-CMV 载体通过蛋白质印迹分析证实了 HSP70 的产生，将过氧化氢（50、100、150μM）暴露 24 小时后，MTT 测定显示了 HSP70 转染的 PC12 的显着活力。揭示了 HSP70 对氧化损伤的保护作用。

项目成果

Atsuya Okubo: "Simultaneous induction of mitochondrial heat shock protein mRNAs in rat forebrain ischemia"Molecular Brain Research. 84・1-2. 127-134 (2000)

Atsuya Okubo：“大鼠前脑缺血中线粒体热休克蛋白mRNA的同时诱导”分子脑研究84・1-134（2000）。

Okubo Atsuya: "Simultaneous induction of mitochordrial heat shock protein mRNAs in cat forebrain ischemia"Mdecular Brain Research. 84. 127-134 (2000)

Okubo Atsuya：“猫前脑缺血中线粒体热休克蛋白 mRNA 的同时诱导”医学脑研究。

Izaki K, Kinouchi H, Watanabe K, Owada Y, Okubo A, Itoh H, Kondo H, Tashima Y, Tamura S, Yoshimoto T, Mizoi K: "Induction of mitochondrial hear shock protein 60 and 10 mRNAs follorving transient focal cerebral ischemia in the rat"Molecular Brain Research.

Izaki K、Kinouchi H、Watanabe K、Owada Y、Okubo A、Itoh H、Kondo H、Tashima Y、Tamura S、Yoshimoto T、Mizoi K：“在短暂性局灶性脑缺血后诱导线粒体休克蛋白 60 和 10 mRNA

Ckubo A, Kinouchi H, Owada Y, Kunizuka H, Itoh H, Izaki K, Kondo H, Yoshimoto T, Mizoi K: "Simultaneous induction of mitochondrial heat shock protein mRNAs in rat forebrain ischemia"Molecular Brain Research. 84. 127-134 (2000)

Ckubo A、Kinouchi H、Owada Y、Kunizuka H、Itoh H、Izaki K、Kondo H、Yoshimoto T、Mizoi K：“大鼠前脑缺血中线粒体热休克蛋白 mRNA 的同时诱导”分子脑研究。

Izaki Kenji: "Induction of mitochordrial heat schock protein 60 and 10 mRNAs following fransient focal cerehral ishemia in the rat"Molecular Brain Research. 88. 14-25 (2001)

Izaki Kenji：“大鼠瞬时局灶性脑缺血后线粒体热休克蛋白 60 和 10 mRNA 的诱导”分子脑研究。