【作者】 王威；

【导师】 郑淑慧；

【作者基本信息】 河北医科大学 ， 外科学， 2005， 硕士

【摘要】 目的:脊髓损伤后会发生不同程度的血管源性脊髓水肿,而水肿的程度和持续的时间与继发性脊髓损伤的发生发展密切相关,但目前人们对脊髓水肿的分子生物学机制尚不清楚,随近些年水通道蛋白的发现和对其研究的深入,使人们了解到水通道蛋白与组织水肿关系密切。本实验采用改良Allon 法[1]建立了大鼠脊髓损伤模型,通过大鼠急性脊髓损伤后不同时间点的病理学改变与脊髓含水量的变化,来研究脊髓水肿的发生发展变化规律;用免疫组化法测定正常脊髓和在脊髓损伤后不同时间点水通道蛋白4(AQP-4)的表达变化,来研究水通道蛋白4 在脊髓内的分布特点和脊髓损伤后的表达变化规律;以及通过损伤段脊髓不同时间点含水量的变化与水通道蛋白4表达变化的相关性来研究水通道蛋白4 与脊髓水肿的关系,探讨脊髓损伤后脊髓水肿的分子生物学机制。方法:实验分两部分进行,健康SD 大鼠60 只,体重250～300g,雌雄不限,随机分为两组:含水量测定组30 只,免疫组织化学测定组30 只。两个组内均随机分为假手术组5只,损伤组25 只。损伤组再按脊髓损伤后6h、1d、3d、5d和7d 这5 个时间点随机分为5 组,每组5 只。分别进行损伤段脊髓含水量和水通道蛋白4 表达的免疫组化测定。采用改良Allon 法脊髓致伤的大鼠模型,冲量为5.0×10.0 g cm,更多还原

【Abstract】 Objective: This experiment has set up spinal cord injurymodel of rat with the adoption of Allon method, throughstudying the expression changes of AQP-4 of injured section ofspinal cord in different time after acute spinal cord injury of rathappening and its dependence with the water content of theinjured section of spinal cord in different time, the mechanismof molecular biology of spinal cord oedema after the injury ofspinal cord is to be probed into.Method: 60 healthy SD rat, weight 250 ～300g, nolimitations on male and female, are divided into false operationgroup with 5 rat in it and injury group with 25 rat in it at random,then the injury group is divided into 5 groups with 5 rat in eachgroup according to 6h, 1d, 3d, 5d and 7d after spinal cordsinjury happened to carry on the determination of water contentand immunohistochemistry separately. The spinal cord injurymodel of rat set up with the adoption of improved Allon method;is moderate injury caused by striking T12 with impulse of5.0×10.0 g cm. Bladder is pinched artificially to help to urinateafter striking two times a day. The water content of spinal cordis determined by measuring both wet weight and dry weight:After putting animal to death by cutting head in different time,cut 1. 5 cm spinal cord with injury section in the center, put it onthe analyzing balance with the precision of 1/10000 to weighwet quality, then put it in 110℃electric heat drying cabinet for24h to dry to permanent quality, then weigh dry quality,calculate the content of water of spinal cord according to theformula of Elliot: water content of spinal cord =(wet quality-dry quality) / wet quality ×100%. Use the method ofimmunohistochemistry to determine the expression of AQP-4 indifferent time after the injury of spinal cord: after anaesthetizingby abdominal cavity injection, fixing ventricle--aorta by filling,cutting and fetching the injured section of spinal cords, thenseries desiccating, defating, embeding with paraffin, serialsection with each section 5μm, carring on HE stain andimmunohistochemistry stain separately. Observation marker: (1)water content (%) of spinal cords of rat in normal groups andinjury groups in different time after injuring, (2) Pathologicalchanges of tissues after spinal cord injured in different time (HEstain), (3) changes of AQP-4 expression of normal rat and spinalcord injured rat in different time (use picture analytical systemto determine average degree of spectrum absorption). Resultstatistics use statistical software of SPSS11.0, all measurementdata express in: mean±standard deviation, the comparisonbetween two exponent adopts t-examination, correlationanalysis is to carried out between the determination data ofwater content of spinal cord and the data of AQP-4 expression,P<0.01 means the difference is significant.Result: 6 hours after rat’s spinal cords are injured , thewater content of spinal cord obviously increases than normalgroup (P<0.01), the AQP-4 expression of injured part of spinalcords obviously increases too; the water content of injuredsection of spinal cords reaches peaks 3d after injured, comparedwith the normal group p<0.01, the AQP-4 expression of injuredsection of spinal cords also reaches peaks, compare with thenormal group p<0.01; the water content of spinal cords and theAQP-4 expression of injured section of spinal cords all drops tosome extent 5d after injured compared with 3d after injured, butstill have obvious differences compared with the normal group(p<0.01); the water content of spinal cords and the AQP-4expression of injured section of spinal cords of rat all continuedropping one week after injury compared with 5d after injury,but still higher than normally (P<0.01); The expression ofAQP-4 (average degree of spectrum absorption) and the watercontent of spinal cord (%) in different time after injury appearremarkable positive correlation. (r=0.964 p<0.01=Conclusion: (1)The injured section appears oedema afterthe spinal cords of rat injured, it becomes worse gradually,reaches the peak 3d after injury, lightens gradually afterwards,the injured section of spinal cords stills has oedema 7d afterinjuring. (2)The AQP-4 expression in the oedema section ofinjured spinal cords is obviously increased after rat’s spinalcords are injured, reaches the peak 3d after injury, reducesgradually afterwards, The AQP-4 expression in the oedema更多还原