阿替普酶对急性脑梗死大鼠血管内皮细胞中Claudin-1和Claudin-5蛋白表达的影响朱云波;李佳佳;马征;赵亮【摘要】目的:探讨阿替普酶对急性脑梗死大鼠溶栓后血管内皮细胞中闭合蛋白1(Claudin-1)和闭合蛋白5(Claudin-5)表达的影响及保护作用机制,为阿替普酶的临床应用提供依据.方法:建立大鼠急性大脑中动脉栓塞模型.54只SD大鼠随机分为假手术组、模型组和阿替普酶溶栓组,每组18只.透射电子显微镜下观察大鼠脑内皮细胞超微结构,荧光免疫组织化学法和Western blotting法检测大鼠血管内皮细胞中Claudin-1和Claudin-5蛋白的表达水平.结果:透射电子显微镜检测,模型组大鼠缺血区域脑体积明显增大,内皮细胞肿胀,部分皮质与周围脑组织界限明显,基膜厚薄均匀,紧密连接结构非常松散、出现断裂和消失;阿替普酶溶栓组大鼠脑梗死区域毛细血管内皮细胞肿胀明显减轻,基膜厚薄不均匀改善,脑毛细血管内皮细胞、内皮细胞之间大部分紧密连接结构断裂情况消失,无紧密连接结构消失.荧光免疫组织化学检测,与模型组比较,阿替普酶溶栓组大鼠血管内皮细胞中Claudin-1和Claudin-5蛋白的表达有明显改善.Western blotting检测,与假手术组比较,模型组大鼠血管内皮细胞中Claudin-1和Claudin-5蛋白表达水平明显减少(P<0.01);与模型组比较,不同时间点阿替普酶溶栓组大鼠血管内皮细胞中Claudin-1和Claudin-5蛋白表达水平均升高(P<0.01).结论:阿替普酶对急性脑梗死大鼠大脑内皮细胞的结构有改善作用,其作用机制可能与阿替普酶能降低大鼠血管内皮细胞中Claudin-1和Claudin-5蛋白表达水平有关.%Objective:To investigate the effect of alteplase on the expressions of Claudin-1 and Claudin-5 proteins in the vascular endothelial cells after thrombolysis in the rats with acute cerebral infarction and its mechanism,and to provide experimentalevidence for its clinical application. Methods: The models of acute thrombosis of middle cerebral artery of the rats were established.Fifty-four SD rats were randomly divided into sham operation group,model group and alteplase group (n= 18).The ultrastructure of brain endothelial cells of the rats was observed under transmission electron microscope.The expression levels of Claudin-1 and Claudin-5 proteins in the vascular endothelial cells of the rats were detected by immunoflurorescence and Western blotting methods.Results:The transmission electron microscope results showed that the brain volume in the ischemic area of the rats in model group was significantly increased and the endothelial cells were swollen,some of the cortical and surrounding brain tissues had obvious boundaries,and the thickness of the basement membrane was uniform and the tightly connected structure was very loose and disappeared;the swelling condition of the capillary endothelial cells in infarcted area of the rats in alteplase group was significantly reduced and the thickness of the basement membrane was improved,and most of the tightly connected structures between the brain capillary endothelial cells and the endothelial cells were loose and the fracture was lost and the structure disappeared.The immunofluorescence results showed that the expressions of Claudin-1 and Claudin-5 proteins in the vascular endothelial cells of the rats in alteplase group were significantly improved compared with model group. The Western blotting results showed the expression levels of Claudin-1 and Claudin-5 proteins in the vascular endothelial cells of the rats in model group were significantly decreased compared with shamoperation group (P < 0.01 ); compared with model group, the expression levels of Claudin-1 and Claudin-5 proteins in the vascular endothelial cells of the rats in alteplase group at different time points were increased (P < 0.01 ). Conclusion: Alteplase can improve the structure of brain endothelial cells in the rats with acute cerebral infarction,and the mechanism may be related to decreasing the expressions of Claudin-1 and Claudin-5 proteins in the vascular endothelial cells of the rats induced by alteplase.【期刊名称】《吉林大学学报（医学版）》【年(卷),期】2017(043)006【总页数】6页(P1137-1141,后插1)【关键词】阿替普酶;急性脑梗死;血管内皮细胞;闭合蛋白1;闭合蛋白5【作者】朱云波;李佳佳;马征;赵亮【作者单位】承德医学院附属医院神经内科,河北承德 067000;承德医学院附属医院神经内科,河北承德 067000;承德医学院附属医院神经内科,河北承德 067000;承德医学院附属医院神经内科,河北承德 067000【正文语种】中文【中图分类】R743.33脑梗死是人类最常见的严重致死疾病，缺血性脑血管病是致残的最常见临床类型，其中急性缺血性脑梗死占1/3～1/4[1]。
因此临床上如何早期积极治疗急性缺血性脑梗死是目前面临的重要问题。
溶栓是目前公认的有效治疗急性缺血性脑梗死的方法[2]。
研究[3]证明：早期及时的溶栓治疗可以减少梗死面积、改善神经功能缺损，也是是目前治疗急性脑梗死的唯一有效手段。
因此利用动物模型来研究脑梗死溶栓机制和溶栓疗效非常重要。
阿替普酶是重组组织型纤溶酶原激活物，为第2代溶栓药物，是基因工程合成产物，其并发症少，溶栓效果好，也是FDA批准的早期溶栓治疗急性缺血性脑梗死的唯一药物[4-5]。
研究[6]证明：阿替普酶对急性脑梗死早期临床改善及晚期预后均有积极作用，而且安全、有效，可降低患者病残率、不增加颅内出血死亡的风险。
但关于阿替普酶对急性脑梗死大鼠血管内皮细胞蛋白作用的报道较少。
本文作者对急性脑梗死大鼠进行溶栓治疗后，研究大鼠血管内皮细胞中相关蛋白闭合蛋白1(Claudin-1)和闭合蛋白5(Claudin-5)的表达水平，为临床治疗脑梗死提供实验数据和理论依据。
2.2 各组大鼠脑缺血区Claudin-1和Claudin-5的表达水平假手术组大鼠脑毛细血管内皮细胞中绿色荧光蛋白Claudin-1和Claudin-5均沿血管内皮正常表达；与模型组比较，阿替普酶溶栓组大鼠脑缺血区Claudin-l和Claudin-5的表达水平明显改善；与假手术组比较，阿替普酶溶栓组大鼠脑缺血区Claudin-1和Claudin-5的表达水平明显下调，且随溶栓时间的延长逐渐下降。
假手术组大鼠脑缺血区Claudin-1和Claudin-5的表达水平分别为0.92和0.91；模型组大鼠脑缺血区Claudin-1和Claudin-5的表达水平分别为0.31、0.33；阿替普酶溶栓组大鼠脑缺血区Claudin-1和Claudin-5的表达水平分别为0.58和0.57。
见图2和3(插页四)。
脑梗死己经成为全世界范围内危害全人类健康的最主要疾病之一，其具有发病率高、致残率极高和致死率高等特点，75%的脑梗死患者遗留有不同程度残疾[9-11]。
在我国人口死因中脑血管病已居第一位，成为神经内科医师防治的重要问题之一。
目前基础研究主要通过动物脑梗死模型，采用溶栓模拟人类治疗过程，从而研究脑梗死病理生理机制。