【作者】 徐妙；

【导师】 黄卫东；

【作者基本信息】 浙江大学 ， 急诊医学， 2008， 博士

【摘要】 研究目的通过建立sD大鼠自由落体脑创伤模型,观察腹腔注射XNJ对大鼠急性颅脑创伤后脑水肿、BBB的损害及AQP4的影响,探讨其脑保护作用及作用机制。研究方法1.实验动物清洁级、健康、成熟sD雄性大鼠108只,体重300～350g。随机分为假手术组、脑外伤组和XNJ治疗组。后两组再分为伤后1、3、5、7d等4个亚组,每亚组12只。2.实验方法采用参照Feeney’S自由落体模型设计并适当改进的打击装置。用20 g重的击锤从30 cm高处自由坠落冲击撞杆,冲击力为600 g·cm,造成脑挫裂伤,制作大鼠脑创伤模型。XNJ治疗组在大鼠脑外伤模型做好后10 min内腹腔内注射XNJ 10mL·kg^-1,每天一次。脑外伤组则腹腔内给予等量0.9%氯化钠溶液。假手术组仅行开颅术,不造成脑损伤。3.标本采集和检测3.1脑组织含水量检测将大鼠伤后第1、3、5、7d分别断头处死后,立即取出伤侧大鼠脑组织,110℃恒温箱烘烤24 h至恒重。用干湿质量法计算脑组织含水量。计算公式为:脑组织含水量（%）=（湿质量-干质量）/湿质量×100%。假手术组术后24 h做同样处理。3.2 BBB定量测定采用EB比色法。各组于处死前1 h通过股静脉注射2%EB(3mL·kg^-1)。取脑组织前,快速开胸暴露心脏,迅速穿刺左心室后,剪右心房一小口,用37℃生理盐水500 mL灌注,至右心房流出清亮液体。取伤灶周边脑组织约0.2 g,加人5 mL甲酰胺,45℃水浴24 h,1500 r·min^-1离心10 min,取上清液用HP8453型分光光度计（λ=635 nm）测其光密度值。用倍比稀释法制作标准曲线,再根据标准曲线求得脑组织EB含量,结果以μg·g^-1（脑组织）表示。3.3 AQP4免疫组化染色各组大鼠在预定时间点在伤灶区周围取脑组织,4%甲醛固定24-48 h,石蜡包埋,然后切片行HE染色和免疫组化检查。以细胞膜呈棕黄色或褐色为AQP4阳性染色。应用NIKON彩色病理图文分析系统进行图像分析。3.4脑组织AQP4 mRNA的实时荧光RT-PCR测定分别取各组不同时间点的损伤周围脑组织进行RT-PCR分析,按Trizol试剂说明提取组织总RNA,逆转录为cDNA,以逆转录所得eDNA为模板进行PCR扩增。AQP4引物:上游5′GGT CCT CAT CTC CCTCTG CTT 3′,下游5′AAC CGT GGT GAC TCC CAA TCC3′,扩增片段长270 bp;β-肌动蛋白引物:上游5′TGT GAT GGT GGG TAT GGG 3′,下游5′TAG AAG CATTTG CGG TGC 3′,扩增片段长度为241 bp。反应体系50 uL,PCR条件:95℃预变性3 min;再95℃变性15 s,58℃退火15 s,72℃延伸30 s,共40个循环。求得各基因的Ct值。采用2^-ΔΔCt方法,即以管家基因β-肌动蛋白的Ct值标准化目的基因Ct值,得到各时点目的基因初始模板相对量,再计算各时点目的基因初始模板相对量与假手术组目的基因初始模板相对量的比值,分析各时点目的基因AQP4mRNA表达相对量的变化。4.统计学处理采用SPSS11.5统计软件包进行处理,资料以均数±标准差（（?）±s）表示,采用单因素方差分析方法（One-Way ANOVA）;P＜0.05为差异有统计学意义。研究结果1.各组大鼠脑含水量比较脑外伤组脑组织含水量伤后1d达高峰,并持续至第3d,第5d开始下降,第7天仍明显高于假手术组（P＜0.05）。XNJ组脑含水量也在第1d达高峰,后逐渐下降,但均明显低于脑外伤组（P＜0.05）;并于第7d降至接近正常,与假手术组比较无显著性差异（P＞0.05）。2.各组大鼠脑组织EB含量比较脑外伤组EB含量伤后1d达高峰,后逐渐下降,各时点均明显高于假手术组（P＜0.05）。XNJ组EB含量伤后1d达高峰,也逐渐下降,各时点均明显低于脑外伤组（P＜0.05）,但均不同程度高于假手术组。3.脑含水量与EB的相关分析脑含水量与EB的变化规律相同,相关分析得,r=0.898,P＜0.01,表明二者具有正性相关。4.AQP4免疫组化染色和AQP4 mRNA光学显微镜下观察,以细胞膜呈棕黄色或褐色为AQP4阳性染色。脑外伤组伤后1d AQP4免疫染色变深,它的mRNA水平上调,并持续至第3d,明显高于假手术组（P＜0.05）;在第5d开始下降,与假手术组比较无显著性差异（P＞0.05）。XNJ治疗后1、3 d的AQP4和AQP4 mRNA较脑外伤组明显上调（P＜0.05）。结论XNJ可以减轻创伤性脑水肿,可能与其减轻BBB破坏,上调AQP4表达有关。更多还原

【Abstract】 ObjectiveIn order to clarify the protective effects and the mechanism of XNJ,TBI rat models were established,brain water content,BBB permeability,AQP4 and AQP4 mRNA were tested. Methods1.Subjects108 male Sprague-Dawley rats approximately 300-350g,were used as subjects and randomly assigned to three groups:sham-operation,TBI and XNJ group.The later two groups were divided into 4 subgroups at 1,3,5 and 7d.2.ModelsTBI in rats was set up by the improved device of Feeney’s weight-dropping model with impact 600 g·cm.Sham-operated rats were anesthetized and had their scalps cut and sutured,but were not submitted to craniectomy or treatment.The intraperitoneal injection of XNJ was performed with 10 mL·kg^-1·d^-1 after the models were set up in the therapy group.TI3I group received only normal saline.3.Measures3.1 Determination of brain water contentEdema was measured at 1,3,5 and 7d after surgery.Briefly,samples from the peri-contusion area were taken and weighed.Then they were placed in an oven and dried at 110℃for 24 h.After drying,all weighing was done on the same balance.The percent water was calculated by the following equation:%Water =[（wet sample weight-dry sample weight）/（wet sample weight）]×100%3.2 Determination of BBB permeability2%Evans blue（EB） dye(3mL·kg^-1) was injected into the femoral vein in one hour before decapitation.The chest was opened 60 minutes later and the heart was perfused with 500 mL saline at 37℃through the left ventricle until the colorless perfusion fluid was obtained from the right atrium.After decapitation,tissue samples about 0.2g were obtained and weighed.Then,the samples were placed in 5ml formamide solution and incubated for 24 hours at 45℃.The optical density（OD） of the EB formamide solution was determined by spectrophotometry at 635 nm,then get the content of EB（μg） according to the standard curve.BBB permeability was expressed as EB per gram of tissue(μg·g^-1). 3.3 Immunohistochemistry for AQP4 protein expressionBrain tissues were fixed in 4%formaldehyde for 24 - 48 h and then embedded in paraffin.Sample sections were cut and stained with hematoxylin and eosin.Adjacent sections were incubated with a primary antibody against AQP4（1:300） for 1 d at 4℃. Sections were sequentially incubated with anti-goat IgG biotynylated secondary antibodies and avidin biotin complex.Sections were finally visualized with 0.01% diaminobenzidine tetrahydrochloride.To test the specificity of the immunohistochemical reaction,control sections were treated with normal goat serum instead of the primary antibody.Nuclei were counterstained by hematoxylin.Samples were photographed and measured by image analysis software.3.4 Real-time quantitative RT-PCR for AQP4 mRNA expressionTotal RNA was isolated from brain with Trizol reagent according to the manufacture’s protocol.The cDNA were amplified with primers based on the sequences of rat AQP4 cDNA.The downstream primer for rat AQP4 was 5′AAC CGT GGT GAC TCC CAA TCC 3′,and the up stream primer was 5′GGT CCT CAT CTC CCT CTG CTT 3′（270 bp）.The downstream primer for humanβ-actin cDNA was5′TAG AAG CAT TTG CGG TGC 3′and the upstream primer was 5′TGT GAT GGT GGG TAT GGG 3′（241bp）.cDNA samples were amplified for 40 cycles（denaturation at 95℃for 15 s,annealing at 58℃for 15 s and extension at 72℃for 30s）.The ABI7500 system software monitored the changes in fluorescence of SYBR Green I dye in every cycle.The threshold cycle（Ct） value for each reaction,reflecting the amount of PCR needed to identify a target gene,and the relative level of AQP4 for each sample were calculated as described.Briefly,β-actin was used for the normalization of the quantity of RNA used.Its Ct value was then subtracted from that of the AQP4 gene to obtain a△Ct value.The difference（△△Ct） between the△Ct values of the samples for the gene target and the△Ct value of the calibrator was determined.The sham-operation group was chosen for calibrators.The relative quantitative value was expressed as 2^-△△Ct, representing the amount of AQP4 expression relative to the calibrators.4.Statistical analysisAll data were expressed as mean±SD.Statistical analysis was carried out with t test by using SPSS 11.5 statistical package.P<0.05 was considered significant.Results1.Effect of XNJ on brain water contentIn TBI group,cerebral edema developed and peaked from 1d to 3d,which were much higher than sham operation group（P<0.05） and decreased significantly by 5 days. At 7d,it was still higher than sham operation group（P<0.05）.XNJ reduced the level of cerebral edema at 1,3,5d as compared to those in TBI group（P<0.05）.At 7d,it was very close to sham operation group（P＞0.05）.2.Effect of XNJ on BBB permeabilityBBB permeability in TBI group increased and peaked from 1d,and then declined gradually at every measure times,which were much higher than sham operation group（P＜0.05）.XNJ injections reduced the leak of BBB as compared to those in TBI group（P＜0.05）.But they were still higher than sham operation group（P＜0.05）.3.BBB permeability was coincident with brain edema development with good correlation （r=0.898,P＜0.01）.4.Effect of XNJ on the expressions of AQP4 mRNA and proteinThe expressions of AQP4 mRNA and protein strongly detected at 1-3d after TBI which were higher than sham operation group（P＜0.05）,then decreased at 5d.XNJ significantly increased AQP4 expressions at 1d-3d post-injury in the brain tissue and much higher than TBI（P＜0.05）.ConclusionXNJ could alleviate cerebral edema following traumatic brain injury via reducing blood-brain barrier damage and the up-regulation of AQP4 expressions.更多还原