载脂蛋白E基因多态性对脑损伤后血脑屏障修复的影响

Effect of apolipoprotein E genetic polymorphism in repair of blood brain barrier breakdown following traumatic brain injury

目的探讨载脂蛋白E（apolipoprotein E，APOE）基因不同亚型对创伤性脑损伤（traumatic brain injury，TBI）后血脑屏障（blood brain barrier，BBB）修复的影响及相关机制。方法健康APOE转基因鼠（g3、g4）、APOE敲除鼠及APOE野生鼠各80只，每种基因型鼠按随机数字表法各自分为TBI组（50只）、假手术组（15只）和正常对照组（15只），TBI组又分为TBI 1d组（15只）、TBI 3d组（15只）和TBI 7d组（20只）三个亚组。采用气动TBI撞击器精确打击各基因型鼠头部建立TBI模型。伤后1，3，7d检测伤灶皮层中伊文思蓝（Evans blue，EB）和荧光素钠（fluorescein sodium，NaFI）含量，评价BBB通透性变化。通过干湿重法测定脑组织含水量。伤后7d采用Western blot及qRT-PCR检测不同APOE基因型鼠损伤灶脑微血管内皮细胞紧密连接相关蛋白Occludin及Claudin-5的表达和转录。结果正常对照组中g4鼠和敲除鼠相较于83鼠和野生鼠BBB对EB和NaFI通透性均明显升高。TBI后各基因型鼠BBB通透性均明显增高。各基因型鼠间TBI 1，3d组对EB和NaFI的通透性比较差异无统计学意义。野生鼠及g3鼠TBI 7d组BBB对EB的通透性已降至各自的正常对照组水平（P〉0.05），但敲除鼠及g4鼠BBB对EB的通透性仍高于各自的正常对照组水平（P〈0.01）。敲除鼠及g4鼠BBB对NaFI的通透性明显高于野生鼠及g3鼠（P〈0.01）。各基因型鼠间TBI1，3，7d组脑组织含水量比较差异无统计学意义。Western blot及qRT-PCR结果显示，敲除鼠及g4鼠脑微血管中Occludin及Claudin-5表达水平均明显低于野生鼠及g3鼠（P〈0.05）。结论APOE在维持BBB正常功能及TBI后BBB的修复过程中发挥重要作用。g4可通过作用于紧密连接的修复进而影响BBB损伤后的修复。

Objective To investigate the effect and underlying mechanism of apolipoprotein E （APOE） genetic polymorphism in treating blood brain barrier （BBB） breakdown after traumatic brain injury （TBI）. Methods Human APOE knock-in mice （g3, g4）, APOE knockout mice, and APOE wild-type mice with each numbering 80 were respectively divided into TBI group （ n = 50 ） , sham-operation group （n = 15） and normal control group （n = 15） according to the random number table. TBI group was subdivided at 1 day （n=15）, 3 days （n=15）, and 7 days （n=20）. TBI was induced with a pneumatically operated injury device. BBB permeability to large or small molecules was evaluated by measuring Evans blue （EB） and fluorescein sodium （NaFI） extravasation into the damage area at 1, 3, and 7 days postinjury. Brain water content was determined using the dry-wet method. Western blotting and qRT-PCR for tight junction-associated proteins Occludin and Claudin-5 were performed at 7 days postinjury. Results With respect to normal control group, BBB permeability to EB and NaFI was significantly higher in g4 and APOE knockout mice than in g3 and APOE wild-type mice. There appeared significant increase in BBB permeability to EB and NaFI in TBI group, with insignificant differences among rats of each genotype at 1 and 3 days postinjury （P 〉 0.05）. Whereas at 7 days postinjury, BBB permeability to EB in APOE wild-type and g3 mice returned to the normal level （ P 〉 0.05 ）, but it remained at a high level in APOE knockout and g4 mice （P 〈 0.O1 ）. Meanwhile, BBB permeability to NaFI was significantly higher in g4 and APOE knockout mice than in g3 and APOE wild-type mice （P 〈 O.01 ）. Brain water content was equivalent among rats of each genotype at 1, 3 and 7 days postinjury （ P 〉 0.05 ）. Western blotting and qRT-PCR demonstrated Occludin and Claudin-5 in g4 and APOE knockout mice were significantly lower than those in g3 and APOE wide-type mice （ P 〈 0.05 ）. Conclusion APOE plays an important role in restoration of BBB function after TBI, but g4 may impede the recovery of BBB breakdown after TBI through its effect on tight junction.