siRNA干扰技术联合高压氧对大鼠脑出血后脑水肿及凋亡的影响  

Effects of siRNA interference combined with hyperbaric oxygen treatment on cerebral edema and apoptosis in the brain tissue of rats after hemorrhage

作　　者：潘强[1] 朱琳[2] 张帅[1] 李普贤[1] 刁兴涛[1] 宋纯玉[1] 高勇[1] 司锋[1] 李强[1] 岳晓[3] 

机构地区：[1]泰山医学院附属莱芜医院神经外科,271199 [2]泰山医学院附属莱芜医院ICU,271199 [3]天津市环湖医院,天津市神经外科研究所,300060

出　　处：《中华老年医学杂志》2017年第9期1010-1014,共5页Chinese Journal of Geriatrics

基　　金：国家自然科学基金项目（81201973,81502654）;泰山医学院附属莱芜医院科研项目（YYLX2013016,YYLX2013019,YYLX2014013,YYLX2015028）

摘　　要：目的观察靶向水通道蛋白4（AQP4）的小RNA（siRNA）干扰技术联合高压氧对实验大鼠脑出血后脑水肿及凋亡的影响。方法将大鼠随机分为对照组、高压氧组、AQP-4 siRNA组及联合治疗组，每组24只；凝血酶Ⅶ注射诱发尾状核建立脑出血模型，构建沉默AQP-4 mRNA表达的siRNA质粒；提取第3天脑组织总RNA和总蛋白，用RT—PCR法检测AQP-4的mRNA表达水平；于湿比重法和Evans蓝法测定脑组织含水量及血脑屏障通透性改变；AnnexinV及TUNEL法检测脑细胞凋亡率，Western blot法检测AQP-4、基质金属蛋白2、9（MMP-2、MMP-9），B细胞淋巴瘤／白血病-2（Bcl-2），半胱天冬酶-3（Caspase-3）蛋白表达水平；第3天给予实验动物神经功能缺陷评分（mNSS）。结果AQP-4 siRNA减轻脑出血后脑水肿及沉默AQP-4方面优于高压氧（P〈0．05），二者联合应用在脑出血后降低脑水肿程度方面获最佳效果（P〈0．05）；与对照组比较各组均抑制神经细胞凋亡，以联合组（4．24±0．04）％最明显（F=13．76，P=0．001）；高压氧联合AQP-4 siRNA能降低AQP-4，MMP2、MMP-9，Caspase-3表达（P〈0．05），而增加Bcl-2表达（P〈0．01）。神经功能缺陷评分较对照组均有改善（P〈0．05），以联合组（4．7±1．1）分最理想（F=7．21，P=0．013）。结论靶向siRNA干扰技术联合高压氧可有效减轻脑出血后脑水肿并抑制神经细胞凋亡，促进神经功能恢复，其机制可能与减少AQP-4，MMP-2、MMP-9，caspase-3表达，增加Bcl-2表达有关。Objective To examine the effects and mechanisms of siRNA targeting aquaporin 4 （AQP-4） in combination with hyperbaric oxygen therapy（HBO） on cerebral edema and apoptosis in the brain tissue of rats after hemorrhage. Methods Rats were randomly divided into four groups, the control group, the hyperbaric oxygen group, the AQP-4 siRNA group and the combination therapy group （24 rats）, Thrombin Ⅶ was injected into the caudate nucleus to establish the hemorrhage model. Construction of siRNA targeting aquaporin 4 was conducted. The mRNA expression of AQP-4 was detected by RT-PCR at day 3. Changes in brain moisture and blood-brain barrier perme ability were measured by a wet/dry weight method and Evans blue fluorometry. The nerve cell apoptosis rate was analyzed by Annexin V andTdT-mediated dUTP-biotin nick end labeling （TUNEL）. The expression of proteins including AQP-4, MMP-2, MMP-9, Bel-2 and easpase-3 was detected by Western Blotting, All the animals were given a score for their nerve function at day 3. Results AQP-4 siRNA treatment obtained better effects than HBO in decreasing the brain edema leveland silencing AQP-4 mRNA（P〈0.05）while,the combination therapy group achieved the best results（P〈 0. 05）. Compared with the control group, the percentage of apoptotic cells decreased in all the three treatment groups, with the most marked decrease observed in the combination treatment group（4.24± 0.04） % （F = 13.76, P = 0.001 ）. The expression of AQP-4, MMP-2, MMP-9 and caspase-3 was lower （P〈0. 05） and the expression of Bcl-2 was higher（P〈0. 01）in the combination treatment group than in the other three groups. Compared with the control group, all the other three groups received better scores on nerve function defect evaluation at day 3 after hemorrhage（P〈0.05）, with the combination treatment group again achieving the most favorable score（4.7 ± 1.1） （F = 7.21, P = 0. 013）. Conclusions Targeted siRNA interference combined with hyperbaric oxygen can effectivel

关 键 词：脑出血 RNA干扰 高压氧 水通道蛋白-4 

分 类 号：R743.34[医药卫生—神经病学与精神病学]