摘要
目的：探讨慢病毒介导的热休克蛋白70（HSP70）基因表达对缺血/缺氧嗜铬细胞瘤（PC12）细胞内质网钙稳态的影响及钙通道的调节机制。方法取对数生长期的PC12细胞，分为重组慢病毒感染组〔感染含HSP70及绿色荧光蛋白（GFP）基因的慢病毒〕、慢病毒对照组（感染含GFP但不含HSP70基因的慢病毒）及未感染组。采用四甲基偶氮唑盐比色法（MTT）检测PC12细胞经缺血/缺氧处理4、8、12、24h后的细胞活性，以选取最佳缺血/缺氧时间。采用实时定量反转录-聚合酶链反应（RT-qPCR）检测缺血/缺氧8h时HSP70、肌质/内质网Ca2＋-ATP酶亚型（SERCA2a、SERCA2b）、兰尼碱受体2（RyR2）、三磷酸肌醇受体1（IP3R1）的mRNA转录水平；采用蛋白质免疫印迹试验（WesternBlot）检测缺血/缺氧8h时HSP70、SERCA、IP3R的蛋白表达水平；采用流式细胞仪检测细胞内活性氧（ROS）及游离钙离子（[Ca2＋]i）水平。结果随着缺血/缺氧时间延长，各组细胞活性呈先增强后减弱的趋势，均于8h时达峰值。缺血/缺氧8h时，重组慢病毒感染组细胞活性较未感染组和慢病毒对照组均明显升高〔A值（×10-2）：20.3±2.2比14.1±2.1、15.0±1.6，均P＜0.01〕，HSP70和SERCA的mRNA及蛋白表达均明显上调〔HSP70mRNA（2-ΔΔCt）为0.785±0.018比0.428±0.019、0.423±0.023，HSP70蛋白（灰度值）为2.72±0.20比1.56±0.36、1.63±0.41，SERCA2amRNA（2-ΔΔCt）为0.971±0.037比0.367±0.014、0.347±0.012，SERCA2bmRNA（2-ΔΔCt）为8.869±0.162比3.015±0.091、2.941±0.091，SERCA蛋白（灰度值）为2.84±0.18比1.48±0.26、1.52±0.29〕，IP3R2的mRNA及蛋白表达均明显下调〔IP3R2mRNA（2-ΔΔCt）为0.183±0.020比0.439±0.020、0.433±0.040，IP3R2蛋白（灰度值）为1.15±0.12比1.91±0.20、1.83±0.19〕，差异均有统计学意义（均P＜0.01），而RyR1mRNA表达差异无统计学意义〔2-ΔΔCt（×10-3）：1.97±0.63比2.02±0.22、2.01
Objective To investigate the effects of lentivirus-mediated heat shock protein 70 （HSP70） gene on calcium homeostasis and calcium channels of PC12 cells induced by ischemic and hypoxia and its mechanisms. Methods PC12 cells at logarithmic phase were collected, and they were divided into recombined lentiviral infection group [infected by lentivirus containing HSP70 and green fluorescent protein （GFP） fluorescin gene], lentivirus control group （infected by lentivirus containing GFP without HSP70 gene） and non-infection group. PC12 cells were subjected ischemia/hypoxia for 4, 8, 12, 24 hours, and the cell activity was determined by methylthiazolyl tetrazolium （MTT） assay test inorder to determine the best time for ischemia/hypoxia. The mRNA expressions of HSP70, muscle/endoplasmic reticulum Ca2＋-ATP isoforms （SERCA2a, SERCA2b）, ryanodine receptor 2 （RyR2）, and inositol 1,4,5-triphosphate receptor 1 （IP3R1） were determined by real-time quantitative reverse transcription-polymerase chain reaction （RT-qPCR）, and the protein expressions of HSP70, SERCA, and IP3R were determined by Western Blot at 8 hours after ischemic/hypoxia. Flow cytometry was used to determine the levels of intracellular reactive oxygen （ROS） and intracellular Ca2＋ （[Ca2＋]i）. Results With the prolongation of time of ischemia/hypoxia, the cell viability in all groups showed an increase followed by a weakening, and peaked at 8 hours. The cell viability at 8 hours in lentiviral infection group was significantly higher than that of the non-infection group and lentivirus control group [A value （×10-2）： 20.3±2.2 vs. 14.1±2.1, 15.0±1.6, both P 〈 0.01], the mRNA and protein expressions of HSP70 and SERCA in lentiviral infection group were significantly increased [HSP70 mRNA （2-ΔΔCt ）： 0.785±0.018 vs. 0.428±0.019, 0.423±0.023; HSP70 protein （gray value）： 2.72±0.20 vs. 1.56±0.36, 1.63±0.41; SERCA2a mRNA （2-ΔΔCt ）： 0.971±0.037 vs. 0.367±0.014, 0.347±0.012; SERCA2b mRNA （
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2016年第3期205-210,共6页
Chinese Critical Care Medicine
基金
国家自然科学基金（81571938,81501706）
山东省自然科学基金（Y2007C133）
关键词
热休克蛋白70
缺血/缺氧
PC12细胞
内质网钙通道
Heat shock protein 70
Ischemia and hypoxia
PC12 cell
Endoplasmic reticulum calciumchannel
