载脂蛋白E基因多态性对星形胶质细胞损伤早期胞内Ca^2＋浓度的影响  

Effect of apolipoprotein E polymorphisms on intracellular Ca^2＋ concentration in the early stage after astrocyte injury

作　　者：吴海涛[1] 江涌[2] 张晓冬[1] 夏海坚[1] 唐兆华[1] 孙晓川[1] 

机构地区：[1]重庆医科大学附属第一医院神经外科,400016 [2]泸州医学院附属医院神经外科

出　　处：《中华创伤杂志》2010年第8期761-765,共5页Chinese Journal of Trauma

基　　金：国家自然科学基金资助项目（30973087）;重庆市科委自然科学基金资助项目（2007BB5299）;重庆市卫生局医学科技计划重点资助项目（05-1-017）、四川省教育厅青年基金资助项目（08zb049）

摘　　要：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早期胞内Ca^2＋浓度的相关性.方法 （1）通过RT-PCR及定点突变技术扩增人源性APOE三种等位基因的CDS区,并构建pEGFP-N1-APOE重组质粒;（2）APOE基因敲除鼠星形胶质细胞的原代培养、鉴定;脂质体法将重组质粒转染入星形胶质细胞,并筛选稳定表达APOE信息的细胞株;（3）利用划痕致伤构建细胞损伤模型,于伤后12,24,48及72 h时相点,利用激光共聚焦显微镜（LCSM）技术检测各基因型细胞内Ca^2＋的荧光强度.结果 各型细胞相对于自身对照（致伤前）,Ca^2＋荧光强度变化差异有统计学意义（P＜0.05）.伤后12 h,三组细胞内Ca^2＋荧光强度较弱,组间差异无统计学意义（P＞0.05）;伤后24,48及72 h,三组细胞Ca^2＋荧光强度进行性增加,且ε4组明显高于ε2和ε3组（P＜0.05）.结论 损伤后早期,携带APOEε4等位基因的星形胶质细胞内Ca^2＋高于ε2和ε3型,提示ε4携带体可能通过伤后Ca^2＋通道的激活导致急性期伤情加重及不良预后.Objective To investigate the correlation between apolipoprotein E （protein：apoE;gene：APOE） polymorphisms and intracellular Ca2 ＋ concentration in the early stage after astrocyte injury.Methods （ 1 ） The CDS region of three APOE alleles was obtained by using reverse transcription polymerase chain reaction （RT-PCR）. Then, the recombinant plasmid pEGFP-N1-APOE was constructed and identified by sequencing. （2） Astrocytes were separated from APOE gene-knockout mice for immunocytochemical identification. The recombinant plasmid was transfected into the astrocytes with liposome-mediated method to screen the cell lines that could stably express APOE information. （3） Cell injury models were set up by scarification. Laser scanning confocal microscope （LCSM） was used to detect the dynamic changes of intracellular Ca^2＋ at 12, 24, 48 and 72 hours postinjury. Results Compared with the control group （ before injury ）, every allele showed significant changes of fluorescence intensity of Ca2 ＋ （ P 〈0.05）. At 12 hours after injury, the fluorescence intensity of Ca^2＋ was weak, with no statistical difference between three groups （ P 〉 0. 05 ）. At 24,48 and 72 hours postinjury, the fluorescence intensity was increased progressively, with significant higher intensity in ε4 group than the other two groups （P 〈0.05 ）. Conclusions The concentration of intracellular Ca^2＋ in the astrocytes carrying APOEε4 allele is higher than that of those carrying APOEε2 and ε3 alleles, indicating that APOEε4 carriers may activate Ca^2＋ channel and lead to aggravation and poor prognosis of acute injury.

关 键 词：载脂蛋白E 基因多态性 星形胶质细胞 激光共聚焦显微镜 

分 类 号：R686[医药卫生—骨科学]