载脂蛋白E基因多态性与星形胶质细胞损伤后兴奋性氨基酸变化的关系

In vitro relationship of apolipoprotein E polymorphisms and excitatory amino acids in astrocyte injury

目的探讨载脂蛋白E(apolipoprotein E,蛋白:ApoE,基因:APOE)基因多态性与星形胶质细胞损伤后兴奋性氨基酸(excitatory amino acids,EAAs)变化的关系。方法提取APOE敲除鼠新生2 d内乳鼠大脑皮层星形胶质细胞,并以GFAP-FITC免疫荧光法对所获细胞进行鉴定。脂质体介导重组体pEGFP-N1-APOEε2、pEGFP-N1-APOEε3、pEGFP-N1-APOEε4分别转染入星形胶质细胞中,RT-PCR鉴定该基因在转染后的细胞中的表达,荧光显微镜观察该基因的亚细胞定位及转染效率。制备3种基因型星形胶质细胞划伤模型,采用氨基酸分析仪分别测其中氨基酸的含量,流式细胞仪检测细胞凋亡。结果星形胶质细胞划伤后谷氨酸(Glu)和天冬氨酸(Asp)的释放较早,在划伤后30 min即达到高峰,在伤后30、60 min APOEε4型显著高于APOEε2、APOEε3型(P<0.05)。细胞早期凋亡率在24 h达到高峰,在伤后12、24 hAPOEε4型显著高于APOEε2、APOEε3型(P<0.05)。结论 APOE以亚型特异性的方式影响星形胶质细胞创伤后EAAs的释放,APOEε4携带体可能通过EAAs毒性作用导致创伤性脑损伤急性期伤情加重及不良预后。

Objective To identify the relationship between the polymorphisms of apolipoprotein E gene（APOE） and the changes of excitatory amino acids（EAAs） in injured astrocytes.Methods Astrocytes were separated from APOE gene-knockout newborn rat cerebral cortex and then identified by GFAP-FITC immunofluorescence staining.The recombinant plasmids pEGFP-N1-APOEε2,pEGFP-N1-APOEε3,and pEGFP-N1-APOEε4 were respectively transfected into the isolated astrocytes with Lipofectamine 2000.Subcellular localization of APOE and transfection efficiency were observed by fluorescent microscopy.RT-PCR was applied to detect the expression of APOE in the astrocytes after transfection.Scratched models of the 3 genotypes astrocytes were produced,and the content of amino acids were measured by amino acid analyzer.Cell apoptosis was detected by flow cytometry（FCM）.Results The level of Glu and Asp in the astrocytes after scratching injury reached a peak at 30 min.APOEε4 transfected astrocytes released higher levels of Glu and Asp compared with those in the other 2 groups at 30 and 60 min after injury（P0.05）.FCM showed that the apoptotic rate reached a peak at 24 h.APOEε4 group had significantly higher apoptotic rate than the other 2 groups at 12 and 24 h after injury（P0.05）.Conclusion APOE affects the contents of EAAs released from the astrocytes after injury in an isoform-specific manner.APOEε4 carriers may induce more severe deterioration and poorer prognosis because of EAAs toxicity.