Calcium influx into neurons triggers neuronal death during cerebral ischemia/reperfusion injury.Various calcium channels are involved in cerebral ischemia/reperfusion injury.Cav3.2 channel is a main subtype of T-type ...Calcium influx into neurons triggers neuronal death during cerebral ischemia/reperfusion injury.Various calcium channels are involved in cerebral ischemia/reperfusion injury.Cav3.2 channel is a main subtype of T-type calcium channels.T-type calcium channel blockers,such as pimozide and mibefradil,have been shown to prevent cerebral ischemia/reperfusion injury-induced brain injury.However,the role of Cav3.2 channels in cerebral ischemia/reperfusion injury remains unclear.Here,in vitro and in vivo models of cerebral ischemia/reperfusion injury were established using middle cerebral artery occlusion in mice and high glucose hypoxia/reoxygenation exposure in primary hippocampal neurons.The results showed that Cav3.2 expression was significantly upregulated in injured hippocampal tissue and primary hippocampal neurons.We further established a Cav3.2 gene-knockout mouse model of cerebral ischemia/reperfusion injury.Cav3.2 knockout markedly reduced infarct volume and brain water content,and alleviated neurological dysfunction after cerebral ischemia/reperfusion injury.Additionally,Cav3.2 knockout attenuated cerebral ischemia/reperfusion injury-induced oxidative stress,inflammatory response,and neuronal apoptosis.In the hippocampus of Cav3.2-knockout mice,calcineurin overexpression offset the beneficial effect of Cav3.2 knockout after cerebral ischemia/reperfusion injury.These findings suggest that the neuroprotective function of Cav3.2 knockout is mediated by calcineurin/nuclear factor of activated T cells 3 signaling.Findings from this study suggest that Cav3.2 could be a promising target for treatment of cerebral ischemia/reperfusion injury.展开更多
本研究旨在探讨慢病毒介导的CAV1过表达对HL-60细胞增殖与凋亡的影响。构建慢病毒重组表达载体pcDNA-EF1-CAV1,并与pPACK包装质粒混合物共转染至293TN细胞后,收集病毒液感染HL-60细胞,使CAV1基因在细胞中稳定转染并高表达。采用Western ...本研究旨在探讨慢病毒介导的CAV1过表达对HL-60细胞增殖与凋亡的影响。构建慢病毒重组表达载体pcDNA-EF1-CAV1,并与pPACK包装质粒混合物共转染至293TN细胞后,收集病毒液感染HL-60细胞,使CAV1基因在细胞中稳定转染并高表达。采用Western blot方法评价转染后HL-60 CAV1蛋白表达情况。采用CCK-8法、流式细胞术分别检测转染前后HL-60细胞的增殖活性和凋亡情况。结果表明:PCR阳性克隆筛选及核苷酸测序结果证实CAV1基因正确插入表达载体pcDNA-EF1-GFP中。重组慢病毒颗粒Lv-CAV1成功转染HL-60细胞,转染率达90%。Western blot检测结果显示,转染后48 h HL-60细胞中CAV1蛋白表达明显增高。CCK-8检测结果显示转染后48 h HL-60细胞增殖活性降低(P<0.05),流式细胞仪检测结果显示转染后HL-60细胞凋亡率明显增加(P<0.01)。结论:CAV1过表达对HL-60细胞具有抑制细胞的增殖活性、促进细胞凋亡的作用。展开更多
基金supported by the Natural Science Foundation of Anhui Province of China,No.2208085Y32Scientific Research Plan Project of Anhui Province of China,No.2022AH020076the Chen Xiao-Ping Foundation for the Development of Science and Technology of Hubei Province,No.CXPJJH12000005-07-115(all to CT).
文摘Calcium influx into neurons triggers neuronal death during cerebral ischemia/reperfusion injury.Various calcium channels are involved in cerebral ischemia/reperfusion injury.Cav3.2 channel is a main subtype of T-type calcium channels.T-type calcium channel blockers,such as pimozide and mibefradil,have been shown to prevent cerebral ischemia/reperfusion injury-induced brain injury.However,the role of Cav3.2 channels in cerebral ischemia/reperfusion injury remains unclear.Here,in vitro and in vivo models of cerebral ischemia/reperfusion injury were established using middle cerebral artery occlusion in mice and high glucose hypoxia/reoxygenation exposure in primary hippocampal neurons.The results showed that Cav3.2 expression was significantly upregulated in injured hippocampal tissue and primary hippocampal neurons.We further established a Cav3.2 gene-knockout mouse model of cerebral ischemia/reperfusion injury.Cav3.2 knockout markedly reduced infarct volume and brain water content,and alleviated neurological dysfunction after cerebral ischemia/reperfusion injury.Additionally,Cav3.2 knockout attenuated cerebral ischemia/reperfusion injury-induced oxidative stress,inflammatory response,and neuronal apoptosis.In the hippocampus of Cav3.2-knockout mice,calcineurin overexpression offset the beneficial effect of Cav3.2 knockout after cerebral ischemia/reperfusion injury.These findings suggest that the neuroprotective function of Cav3.2 knockout is mediated by calcineurin/nuclear factor of activated T cells 3 signaling.Findings from this study suggest that Cav3.2 could be a promising target for treatment of cerebral ischemia/reperfusion injury.
文摘本研究旨在探讨慢病毒介导的CAV1过表达对HL-60细胞增殖与凋亡的影响。构建慢病毒重组表达载体pcDNA-EF1-CAV1,并与pPACK包装质粒混合物共转染至293TN细胞后,收集病毒液感染HL-60细胞,使CAV1基因在细胞中稳定转染并高表达。采用Western blot方法评价转染后HL-60 CAV1蛋白表达情况。采用CCK-8法、流式细胞术分别检测转染前后HL-60细胞的增殖活性和凋亡情况。结果表明:PCR阳性克隆筛选及核苷酸测序结果证实CAV1基因正确插入表达载体pcDNA-EF1-GFP中。重组慢病毒颗粒Lv-CAV1成功转染HL-60细胞,转染率达90%。Western blot检测结果显示,转染后48 h HL-60细胞中CAV1蛋白表达明显增高。CCK-8检测结果显示转染后48 h HL-60细胞增殖活性降低(P<0.05),流式细胞仪检测结果显示转染后HL-60细胞凋亡率明显增加(P<0.01)。结论:CAV1过表达对HL-60细胞具有抑制细胞的增殖活性、促进细胞凋亡的作用。
