Wu Haitao, Zhou Shuai, Zhao Hongxin, Wang Yuyu, Chen Xiaozhong, Sun Xiaochuan
Department of Neurosurgery, The Affiliated Hospital of Zunyi Medical College, Zunyi, Guizhou 563000, P.R. China.
Department of Neurosurgery, The Affiliated Hospital of Kunming University of Science and Technology, Kunming, Yunnan 654000, P.R. China.
Exp Ther Med. 2018 Feb;15(2):1417-1423. doi: 10.3892/etm.2017.5555. Epub 2017 Nov 23.
The present study aimed to investigate the correlation between apolipoprotein E (APOE) polymorphisms and the intracellular concentration of Ca in astrocytes in the early stages after an injury. The chondroitin sulfate region of three APOE alleles (ε2, ε3 and ε4) was obtained by reverse transcription-polymerase chain reaction (RT-PCR). A recombinant plasmid, pEGFP-N1-APOE, was constructed and identified by sequencing, while astrocytes were isolated from APOE gene-knockout mice and examined using immunocytochemistry. The recombinant plasmid was transfected into the astrocytes using the liposome-mediated method and cell injury models were constructed by a scratch assay. Laser confocal scanning microscopy (LCSM) was used to detect dynamic alterations in intracellular Ca concentration at 12, 24, 48 and 72 h after injury. Compared with the control group, cells transfected with any of the three alleles demonstrated significant increases in the fluorescence intensity of Ca (P<0.05). The fluorescence intensity of Ca was weak at 12 h after injury, with no statistically significant difference detected between any two groups at this time point (P>0.05). However, the fluorescence intensity increased in a time-dependent manner and at 24, 48 and 72 h post injury, the fluorescence intensity of the ε4 allele-containing cells was significantly higher when compared with that of cells harboring the other two alleles (P<0.05). These results indicate that intracellular Ca overloading may contribute to the deterioration of brain cells and poor outcome subsequent to traumatic brain injury in APOE ε4 carriers.
本研究旨在探讨载脂蛋白E（APOE）基因多态性与损伤后早期星形胶质细胞内钙离子浓度之间的相关性。通过逆转录聚合酶链反应（RT-PCR）获得了三种APOE等位基因（ε2、ε3和ε4）的硫酸软骨素区域。构建了重组质粒pEGFP-N1-APOE并进行测序鉴定，同时从APOE基因敲除小鼠中分离出星形胶质细胞并采用免疫细胞化学方法进行检测。采用脂质体介导法将重组质粒转染至星形胶质细胞，并通过划痕试验构建细胞损伤模型。利用激光共聚焦扫描显微镜（LCSM）检测损伤后12、24、48和72小时细胞内钙离子浓度的动态变化。与对照组相比，转染三种等位基因中任何一种的细胞钙离子荧光强度均显著增加（P<0.05）。损伤后12小时钙离子荧光强度较弱，此时各两组间差异无统计学意义（P>0.05）。然而，荧光强度呈时间依赖性增加，在损伤后24、48和72小时，含ε4等位基因的细胞荧光强度显著高于携带其他两种等位基因的细胞（P<0.05）。这些结果表明，细胞内钙离子超载可能导致APOE ε4携带者创伤性脑损伤后脑细胞恶化及预后不良。
