Survivin基因敲减对创伤性脑损伤小鼠细胞凋亡和学习记忆功能的影响

Effect of survivin knockdown on apoptosis and spatial learning and memory of mice with traumatic brain injury

目的观察腺病毒载体介导的Survivin基因敲减对创伤性脑损伤（TBI）后成年小鼠海马齿状回凋亡和学习记忆功能的影响。方法选择成年雄性C57BL／6小鼠112只，按随机数字表法分为假手术组、TBI组、阴性对照组和Survivin基因敲减组（敲减组），每组28只。制作小鼠TBI液压打击模型。阴性对照组TBI后立即立体定向注射含有阴性对照RNA的重组腺病毒；敲减组TBI后立即立体定向注射含有shRNA的重组腺病毒。假手术组给予除液压打击外的所有步骤；TBI组单纯接受液压打击。利用实时荧光定量聚合酶链反应（RT—PCR）、Westernblot及免疫荧光检测敲减后Survivin基因在创伤脑组织中的表达，采用TUNEL免疫荧光检测小鼠海马齿状回细胞凋亡的情况。利用Morris水迷宫检测Survivin基因敲减对小鼠学习记忆功能的影响。结果RT—PCR和Westernblot检测显示TBI后3d，敲减组SurvivinmRNA表达量和蛋白水平较阴性对照组明显减低（1．21±0．11VS．4．05±0．30；0．34±0．08vs．1．11±0．17）（P均〈0．01）；免疫荧光检测显示敲减组Survivin阳性细胞数量（267．45±38．41）低于阴性对照组（896．56±102．23）（P〈0．01）；在TBI后3d，敲减组小鼠齿状回TUNEL阳性细胞数量（416．1±29．0）较阴性对照组（250．2±20．8）显著增加（P〈0．01）。Morris水迷宫结果显示敲减组小鼠逃避潜伏期较阴性对照组长（P〈0．05），象限百分比较阴性对照组低（P〈0．01）。结论腺病毒载体介导的Survivin基因敲减可以促进TBI后成年小鼠海马齿状回的凋亡，同时可以导致小鼠学习和记忆功能降低。

Objective To examine the effect of survivin downregulation by adenovirns-mediated RNA interference on apoptosis in hippocampal dentate gyrus and spatial learning and memory of mice with traumatic brain injury （TBI）. Methods A total of 112 adult male mice were allocated into 4 groups （28 animals each） according to the random number table. Fluid percussion model of TBI was used in the study. In negative control group, animals were stereotactically injected with control recombinant adenovirus immediately after injury. In survivin knockdown group, the animals were stereotactically injected with shRNA adenovirus immediately after injury. In sham group, animals were subjected to identical surgery without fluid percussion injury. In TBI group, animals were subjected to isolated fluid percussion injury. Real-time fluorescent quantitative polymerase chain reaction （RT-PCR）, Western blot analysis and immunofluorescence staining were used to detect expression level of survivin in mouse brain tissue. TUNEL assay was used for measuring apoptotic cells in hippocampal denate gyms. Morris water maze test was adopted to evaluate mouse learning and memory after surviving downregulation. Results Expression levels of survivin mRNA and protein in survivin knockdown group reduced at days 3 postinjury compared to negative control group （ 1.21 ± 0. 11 vs. 4.05 ±0.30; 0.34 ± 0.08 vs. 1.11 ± 0. 17, respectively） （P 〈0.01 ）. Immunofluorescence staining showed survivin-positive cells calculated 267.45 ± 38.41 in survivin knockdown group, lower than 896.56 ± 102.23 in negative control group （P 〈 0.01 ）. TUNEL- positive cells in dentate gyrus of the hippocampus in survivin knockdown group increased at days 3 postinjury compared to negative control group （416.1 ±29.0 vs. 250.2 ±20. 8） （P 〈0. 01 ）. Morris water maze test confirmed that survivin downexpression did lead to a statistically significant changes in escape latency （P 〈 0.05 ） and target quadrant （ P 〈 0. 01 ）. Conclusion Decreased expression of survivin may promote apoptotic cell death and result in a negative role in the recovery of learning and memory function of mice after TBI. [ Key words ] Mice, knockout ; Brain injuries ; Apoptosis ; Memory function