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

将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。打开钻孔并使用套管将20μg质粒DNA（pBK-CMV-HSP 70）注射到海马或纹状体中。注射时间从10秒变为10分钟。随后，将一对不锈钢电极插入该区域。用方形电穿孔器（CUY 21）产生电脉冲，并以10 Hz施用。电脉冲在50、100或150 V下持续2 ms。然而，在任何条件下，都没有获得足够的基因转移。HSP 70基因转染PC 12细胞：用pBK-CMV载体转染人HSP 75 cDNA，用流式细胞仪检测HSP 75基因的表达。Western blot分析证实HSP 70蛋白的产生。将过表达HSP 70的PC 12细胞暴露于过氧化氢（50、100、150μM）。MTT法显示HSP 70转染的PC 12细胞在24小时后具有明显的存活能力。热休克蛋白70对氧化损伤有保护作用。

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.