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）细胞钙稳态的影响及机制。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及荧光蛋白基因的慢病毒）、慢病毒对照组（感染含荧光蛋白而不含HSP70基因的慢病毒）及未感染组3组。用反转录-聚合酶链反应（RT-PCR）检测转染细胞HSP70 mRNA表达，蛋白质免疫印迹试验（Western Blot）检测HSP70蛋白表达。PC12细胞缺血/缺氧处理4 h后，用四甲基偶氮唑盐比色法（MTT）检测细胞活性，乳酸脱氢酶（LDH）检测试剂盒测定细胞上清液LDH水平，流式细胞仪检测细胞内游离钙离子（[Ca2＋]i）浓度，ATP酶试剂盒测定Na＋-K＋-ATP酶、 Ca2＋-Mg2＋-ATP酶、总ATP酶活性。结果重组慢病毒感染组PC12细胞HSP70 mRNA和HSP70蛋白表达呈阳性。经缺血/缺氧处理后，与慢病毒对照组和未感染组比较，重组慢病毒感染组细胞活性明显增强（A值：0.575±0.020比0.395±0.014、0.363±0.045，t1＝17.996，t2＝10.600，均P＜0.001），细胞上清液中LDH水平明显降低（U/L：743.46±23.68比935.43±34.77、962.89±26.68，t1＝11.179，t2＝15.044，均P＜0.001），[Ca2＋]i浓度显著降低〔相对荧光强度：（31.60±2.43）%比（41.48±3.33）%、（40.40±3.05）%， t1＝5.853，t2＝5.502，均P＜0.001〕，Na＋-K＋-ATP酶、Ca2＋-Mg2＋-ATP酶、总ATP酶活性显著升高〔Na＋-K＋-ATP酶（μmol·mg-1·h-1）：8.608±0.307比6.728±0.173、6.450±0.091，t1＝9.237、P1＝0.001，t2＝11.675、P2＜0.001；Ca2＋-Mg2＋-ATP酶（μmol·mg-1·h-1）：10.523±0.036比7.910±0.209、8.064±0.195，t1＝9.718、P1＝0.001，t2＝11.535、P2＜0.001；总ATP酶（μmol·mg-1·h-1）：17.041±0.324比14.150±0.182、13.983±0.085，t1＝16.113，t2＝17.602，均P＜0.001〕。慢病毒对照组与未感染组各指标差异均无统计学意义。结论 HSP70能维持缺血/缺氧P
ObjectiveTo investigate the effects of lentivirus-mediated heat shock protein 70 （HSP70） gene on calcium homeostasis in PC12 cells undergone ischemia and hypoxia, and the mechanism involved.Methods PC12 cells at logarithmic phase were collected, and were divided into recombination lentivirus infection group （infected by lentivirus containing HSP70 and fluorescent gene）, lentivirus control group （infected by lentivirus containing fluorescin without HSP70 gene） and non-infection group. HSP70 gene and protein expressions in PC12 cells were determined by reverse transcription-polymerase chain reaction （RT-PCR） and Western Blot. After being challenged with ischemia and hypoxia for 4 hours, the viability of cells was detected by methyl thiazolyl tetrazolium （MTT）, the levels of lactic acid dehydrogenase （LDH） in cell supernatant were determined by LDH measurement test kit. The concentration of intracelluer calcium （[Ca2＋]i） was assayed by flow cytometer. The activities of Na＋-K＋-ATPase, Ca2＋-Mg2＋-ATPase and total ATPase were measured by ATPase test kits.Results The expressions of exogenous HSP70 gene and protein were found by RT-PCR and Western Blot in the recombination lentivirus infection group. After being challenged with ischemia and hypoxia, the viability of cells in the recombination lentivirus infection group was increased significantly as compared with the lentivirus control group and non-infection group （A value： 0.575±0.020 vs. 0.395±0.014,0.363±0.045,t1= 17.996,t2= 10.600, bothP〈 0.001）, the levels of LDH in culture medium and the concentration of [Ca2＋]i were decreased significantly [LDH （U/L）： 743.46±23.68 vs. 935.43±34.77, 962.89±26.68,t1= 11.179, t2= 15.044, bothP〈 0.001; [Ca2＋]i relative fluorescence： （31.60±2.43）% vs. （41.48±3.33）%, （40.40±3.05）%, t1= 5.853,t2= 5.502, bothP〈 0.001], and the activities of Na＋-K＋-ATPase, Ca2＋-Mg2＋-ATPase and total ATPase were increased significantly [Na＋-K＋-ATPase （μmol·mg-
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2015年第4期295-299,共5页
Chinese Critical Care Medicine
基金
山东省自然科学基金（Y2007C133）
山东省青岛市市南区科技局项目（2014-14-041-YY）
