【摘要】 目的观察腺病毒介导的人诱导型HSP70过表达对低钾诱导的原代培养的大鼠小脑颗粒神经元(cerebellargranuleneurons,CGN)凋亡的影响。方法原代培养5d的CGN共感染含人诱导型HSP70和绿色荧光蛋白(GFP)的腺病毒(AdTR5/HSP70-GFP)和四环素调控的启动子(AdCMV/tTA),或共感染含GFP的腺病毒(AdTR5/GFP)和AdCMV/tTA(对照组)。48h后,采用细胞荧光免疫组织化学法、Westernblot法检测HSP70的表达,或者换成无血清含5mmol·L-1KCl的培养基以诱导神经元凋亡。24h后,采用相差显微镜观察细胞形态学变化,MTT法检测神经元存活率,Hoechst33258核染色和DNA琼脂糖凝胶电泳分析神经元凋亡,以观察HSP70过表达对低钾诱导的CGN凋亡的影响。结果共感染了AdCMV/tTA和AdTR5/HSP70-GFP的CGN过表达了HSP70,抑制了低钾诱导的CGN的凋亡:使神经元存活率由45·5%±5·2%提高至82·3%±5·2%(P<0·01),核固缩减少,DNA的片段化减轻。结论腺病毒介导的人诱导型HSP70的过表达抑制了低钾诱导的CGN凋亡。更多还原

【Abstract】 Aim To observe effects of adenovirus-mediated human inducible HSP70 overexpression on apoptosis of primary cultured rat cerebellar granule neurons (CGN) induced by low potassium. Methods CGN in vitro at 5 d were coinfected with adenoviruses containing human inducible HSP70 and green fluorescent protein (GFP) (AdTR5/HSP70-GFP) on separate cistrons, as well as a tetracycline-regulated promoter (AdCMV/tTA). Control cells were coinfected only with AdTR5/GFP and AdCMV/tTA. After 48 h, the expression of HSP70 was measured by immunofluorescence analysis and Western blot analysis. Apoptosis was induced by switching CGN to serum-free medium containing 5 mmol·L~ -1 KCl. After 24 h, the neuronal viability was measured by MTT methods, the morphology of neurons was observed by phase-contrast microscopy, Hoechst 33258 nucleus staining and DNA agarose gel electrophoresis fragmentation were used to analyzeneuronal apoptosis. Results HSP70 was overexpressed in CGN coinfected with AdCMV/tTA and AdTR5/HSP70-GFP. HSP70 overexpression protected CGN from apoptosis induced by low potassium. The neuronal viability was elevated from 45.5%±5.2% to 82.3%±5.2%(P<0.01), nuclear condensation/aggregation was reduced and a typical apoptotic DNA ladder almost disappeared. Conclusion Adenovirus-mediated human inducible HSP70 overexpression inhibits apoptosis of CGN induced by low potassium.更多还原