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目的 通过观察与认知和学习记忆功能相关的蛋白表达,探讨水通道蛋白4(AQP4)沉默对创伤性脑损伤(TBI)大鼠学习和记忆功能的影响及意义.方法 按随机数字表法将96只健康成年雄性Wistar大鼠分组.① 48只大鼠被分为假手术(Sham)组、TBI组(采用改良Feeney法制模)、AQP4 RNA干扰(RNAi)阴性组〔TBI+无意义的小干扰RNA(siRNA)-AQP4脂质体溶液10 μL〕、AQP4 RNAi组(TBI+siRNA-AQP4脂质体溶液10 μL〕;分别于伤后1、6、12 h各取4只大鼠脑组织,采用蛋白质免疫印迹试验(Western Blot)检测海马组织AQP4、一般性调控阻遏蛋白激酶2(GCN2)、环磷酸腺苷反应元件结合蛋白(CREB)及其磷酸化(p-CREB)蛋白表达.② 另外48只大鼠被分为正常组(Control组)、Sham组、TBI组、AQP4 RNAi组,其中6只于伤后12 h测定脑含水量,另外6只用于Morris水迷宫实验.结果 ① TBI组伤后海马组织AQP4和GCN2蛋白表达较Sham组明显增高,并随时间延长呈逐渐升高趋势,12 h出现统计学差异(AQP4蛋白:5.03±0.09比1,GCN2蛋白:4.01±0.13比1,均1<0.01);CREB和p-CREB表达较Sham组明显降低,并随时间延长呈逐渐下降趋势,12 h出现统计学差异(CREB蛋白:0.38±0.03比1,p-CREB蛋白:0.38±0.03比1,均1<0.01).与TBI组比较,AQP4 RNAi组伤后各时间点AQP4蛋白表达明显下降(1 h:1.02±0.04比2.23±0.05,6 h:1.23±0.03比2.59±0.04,12 h:2.20±0.08比5.03±0.09,均1<0.01),而GCN2、CREB和p-CREB表达则差异无统计学意义.AQP4 RNAi阴性组各蛋白表达变化与TBI组比较差异均无统计学意义.② TBI组脑含水量较Control组和Sham组明显升高〔(83.7±0.4)%比(76.2±0.2)%、(76.2±0.3)%,均1<0.01〕;AQP4 RNAi组脑含水量〔(78.8±0.3)%〕则较TBI组明显下降(1<0.01).TBI组和AQP4 RNAi组伤后第11、13、15天Morris水迷宫实验潜伏期明显延长,探索时间明显缩短;与TBI组比较,AQP4 RNAi组第15天逃避潜伏期明显缩短(s:60.2±11.1比62.0±11.5,1<0.05),探索时间明显延长(s:37.0±8.5比32.7±9.2,1<0.05).结论 TBI后大鼠认知和学习记忆功能受损,与影响认知和学习记忆功能相关的CREB和GCN2蛋白表达变化显著相关;经AQP4 RNAi处理后,虽对大鼠认知和学习记忆功能改善不明显,但可减轻脑水肿.

Objective To observe the protein expression related to cognitive and learning memory function, and to investigate the effect of aquaporin 4 (AQP4) silence on learning and memory function in traumatic brain injury (TBI) rats. Methods Ninety-six healthy adult male Wistar rats were divided into groups according to the random number table. ① Forty-eight rats were divided into sham operation (sham) group, TBI group (by using modified Feeney method), AQP4 RNA interference (RNAi) negative group [TBI+meaningless small interfering RNA (siRNA)-AQP4 liposome solution 10 μL], and AQP4 RNAi group (TBI+siRNA-AQP4 liposome solution 10 μL). In each group, brain tissues of 4 rats were harvested at 1, 6 and 12 hours respectively. The protein expressions of hippocampus AQP4, general control nonderepressible 2 kinase (GCN2), cyclic adenosine monophosphate response element binding protein (CREB) and phosphorylated CREB (p-CREB) were detected by Western Blot. ② In addition, 48 rats were divided into normal control group (control group), sham group, TBI group and AQP4 RNAi group, brain water content were measured in 6 of them after 12 hours of injury, and 6 were used in Morris water maze test. Results ① The protein expressions of hippocampus AQP4 and GCN2 in TBI group were significantly higher than those in sham group, and increased gradually with time with statistical difference at 12 hours (AQP4 protein: 5.03±0.09 vs. 1,GCN2 protein: 4.01±0.13 vs. 1, both 1 < 0.01);the protein expressions of hippocampus CREB and p-CREB were significantly lower than those in sham group, and decreased gradually with time with statistical difference at 12 hours (CREB protein: 0.38±0.03 vs. 1, p-CREB protein:0.38±0.03 vs. 1, both 1 < 0.01). Compared with TBI group, the protein expressions of AQP4 in AQP4 RNAi group was significantly decreased (1 hour: 1.02±0.04 vs. 2.23±0.05, 6 hours: 1.23±0.03 vs. 2.59±0.04, 12 hours: 2.20±0.08 vs. 5.03±0.09, all 1 < 0.01), but there were no significant difference in the expressions of GCN2, CREB or p-CREB. There was no significant difference in the expression of protein between AQP4 RNAi negative group and TBI group.② The brain water content in TBI group was significantly higher than that in control group and sham group [(83.7±0.4)% vs. (76.2±0.2)%, (76.2±0.3)%, both 1 < 0.01]. The brain water content in AQP4 RNAi group [(78.8±0.3)%] was significantly decreased as compared with that in TBI group (1 < 0.01). The latency of Morris water maze test was significantly prolonged in the day 11, 13 and 15 after the injury of the TBI group and AQP4 RNAi group, and the exploration time was significantly shortened. Compared with TBI group, the incubation period of AQP4 RNAi group was significantly shortened at 15 days (s: 60.2±11.1 vs. 62.0±11.5, 1 < 0.05), and the exploration time was significantly prolonged (s: 37.0±8.5 vs. 32.7±9.2, 1 < 0.05). Conclusions The impairment of cognitive and learning memory function in rats after TBI was significantly related to the changes in CREB and GCN2 in cognitive and learning memory function. After RNAi treatment, the cognitive and learning and memory function of rats was not improved obviously, but the brain edema could be alleviated.