【作者】 刘旭冬；

【导师】 于灵芝；

【作者基本信息】 山东大学 ， 麻醉学， 2007， 硕士

【摘要】 目的：结合前期有关大鼠脊髓缺血再灌注损伤后血清中内皮抑素(endostatin，ES)和血管内皮生长因子(vascular endothelial growth factor，VEGF)的相关性研究结果，探讨大鼠脑缺血再灌注损伤后脑组织内皮抑素与VEGF的表达和相关性。方法：将48只雄性成年wistar大鼠随机分为2组：假手术组即A组(n=8)、脑缺血模型组即B组(n=40)。其中B组又根据再灌注后观察时间不同分为5个亚组：B1～B5组，分别对应再灌注后观察6小时、1天、2天、4天、7天，每组n=8。采用改良的Zea Longa线栓法，建立大鼠左侧大脑中动脉栓塞(MCAO)缺血再灌注模型。A组大鼠手术后1天、B组大鼠再灌注达到相应的观察时间点时，将大鼠处死后断头取脑，取梗死灶脑组织进行实验研究。方法如下：1、参照Zea Longa 5分制评分标准，分别在再灌注后各观察时间段对大鼠进行神经功能评分。2、断头取脑后利用triphenyl tetrazolium chloride(TTC)染色观察各组大鼠脑梗死情况。3、RT-PCR(real time polymerase chain reaction)技术检测大鼠脑组织中VEGF以及内皮抑素的mRNA表达，计算二者的比值即E／V值。所有数据均采用(?)±s表示，采用SPSS13.0分析软件，计量资料采用t检验及方差分析，三组以上均数有显著性差异时，采用两两比较q检验，神经功能评分与E／V值相关性作直线相关分析，以P＜0.05为有统计学意义。结果：1、大鼠神经功能评分A组大鼠再灌注一天内未出现任何神经功能症状，评分为0分；B组中，再灌注6h、1d时，7只大鼠爬行时向右侧转圈(2分)，一只向右侧倾倒(3分)，评分2.125±0.354；再灌注2d，大鼠神经功能明显异常，其中5只大鼠无自主活动(4分)，3只向右侧倾倒(3分)，评分3.625±0.518；而再灌注4d、7d时评分多为2分，评分分别2.375±0.518、2.250±0.463。相对于A组，B组中各亚组神经功能评分均增高(P＜0.01)。2、TTC染色结果TTC染色后，A组大鼠双侧大脑呈现均匀的红色，未见白色梗死灶；B组大鼠右侧大脑和A组一致，左侧显示不同程度的缺血情况，出现苍白色的梗死灶。再灌注6h，大鼠左脑略肿胀，出现小面积梗死灶；再灌注1d时梗死灶较明显，且多见于皮质；再灌注2d时缺血情况最为严重，左脑呈现大片苍白色梗死灶；再灌注4d与7d时脑水肿有所减轻，但梗死灶仍然清晰可见。3、RT-PCR技术检测脑组织中VEGF mRNA以及内皮抑素mRNA的表达。以A组为参照，VEGF mRNA表达量为1，B组大鼠脑组织中VEGF mRNA表达变化明显，再灌注6h时即有所升高(2.499±0.399，P＜0.05)，随着时间的延长，在1d时达到高峰(4.243±0.352)，与A组相比，P＜0.01，差异有统计学意义；再灌注2d时有所回落(1.321±0.215)，而4d(1.647±0.264)、7d(1.920±0.253)时又有再次升高的趋势(P＜0.05)。而B组内比较，再灌注1d时VEGF的mRNA表达相对其余亚组均较高(P＜0.05)，其余各亚组间差别不大。而内皮抑素mRNA的表达较VEGF迟，相对于A组(表达量为1)，再灌注6h时无明显变化(0.989±0.308，P＞0.05)，但1d时明显升高(3.229±0.247，P＜0.01)，2天时达高峰(7.301±0.242，P＜0.01)，4d(4.614±0.372)、7d(4.414±0.291)时有所回落，但仍维持在较高水平，与A组比较表达明显增高(P＜0.01)。各亚组间比较，再灌注2d时内皮抑素mRNA的表达相对最高(P＜0.01)。B组内皮抑素与VEGF的比值即E／V值，A组E／V值为1，再灌注6h(0.389±0.066)、1d(0.762±0.036)时略有降低，此后逐渐升高，2d时E／V值上升明显(5.588±0.786，P＜0.01)，4d(2.850±0.377)、7d(2.323±0.257)时有所下降，相对A组仍然较高(P＜0.05)。各亚组间比较，再灌注2d时E／V值高于其余组(P＜0.01)，而4d、7d时高于6h、1d组(P＜0.05)。经直线相关分析，脑缺血再灌注损伤后大鼠神经功能评分和E／V值正相关程度密切(r=0.811)。结论：脑缺血90分再灌注后，大鼠神经功能明显异常，尤其是再灌注2d时，神经功能评分最高。根据再灌注时间的不同，大鼠左侧脑组织呈现不同程度的缺血状况，2d时可见大片苍白色梗死灶。脑组织中VEGF mRNA的表达，再灌注6h即有升高，并且在1d时达到高峰，但2d后明显回落；而内皮抑素mRNA的表达较VEGF迟，2d时最高，此后略有回落。二者的比值即E／V在再灌注早期有所降低，但在2d时上升明显，随后有所下降。E／V值与大鼠神经功能评分是密切相关的。总之，大鼠脑缺血再灌注过程中，内皮抑素与VEGF的平衡性，即E／V值高低的变化，与大鼠脑损伤而导致的神经功能异常紧密相关，循环中血管生成促进因子与抑制因子之间的平衡可能是影响脑缺血损伤程度和愈后的关键因素。而再灌注后早期尤其是24h内，VEGF升高而内皮抑素并无显著提高，可能是微循环重建的一个关键时期。更多还原

【Abstract】 Objective: Based on the previous studies of correlation between endostatin(ES) and vascular endothelial growth factor(VEGF) in blood serum of rats induced by spinal cord ischemia-reperfusion injury, we detect the expression and associativity between ES and VEGF in rats subjected to cerebral ischemia-reperfusion injury. Methods: The models of focal brain reperfusion injury by middle cerebral artery occlusion (MCAO) were established in wistar rats according to the advanced Zea Longa’s method. Rats were randomly divided into two groups: sham- operated group(group A) and cerebral ischemia-reperfusion injury group ( group B). Group A (n=8) underwent the same surgical procedure but without the introduction of the filament into the MCA. Group B(n=40) was divided into 5 subgroup(n=8 for each) and the subgroups were anesthetized and euthanized by decapitation at 6h, 1d, 2d, 4d, and 7d after reperfusion respectively.1. Neurologic examination was Clinically performed on each rat after 6h, 1d, 2d, 4d, 7d reperfusion according to the Zea Longa’s scoring method.2. At the given end point,rats were sacrificed and brain tissue biopsy was immediately taken. Cerebral slices were stained with triphenyl tetrazolium choride (TTC) and observed under light microscope;3. Expression levels of VEGF mRNA and ES mRNA in brain tissue were quantified with real time Polymerase chain reaction (RT-PCR) and ratios of ES/VEGF were calculated.Measurement data was presented as mean ± SD. Data analysis was performed by one way ANOVA followings t test and q test,and analysis of linear correlation was used to evaluate the correlation between neurological function score and the ratio ofE/V with SPSS 13.0. P < 0.05 was considered statistically significant.Results :1 .Neurological deficits of the rats:Compared with group A, the neurological deficits were significant in group B at each time point. In group B1, seven rats dextrad circled during crawl and one tumbled rightward, and score was 2.125 ± 0. 354. Group B2 exhibited slightly difference to group B1.In group B3, five rats lost autonomic activities and three tumbled rightward, and score was 3.625±0. 518. Most rats in group B4 and B5 scored 2 points, and scores was 2.375 ± 0. 518 and 2.250±0.463.2. TTC staining of cerebral slices:Cerebral slices of rats in group A showed no infarct; so did the right hemispheres of rats in group B .Six hours after reperfusion, left cerebrums of group B began to swell and showed small infarction. On 1 day post-reperfusion, infarction area appeared more obvious and most infarction were in the cortical substance. And with the deterioration of ischemia, larger infarction area appeared at day 2.Then the encephaledema relieved, while the infarction were still obvious after 4 days.3. Expression of VEGF mRNA and ES mRNA in brain tissues with RT-PCR.The VEGF mRNA expression quantity of rats in group A was set as 1. Compared with those in group A, VEGF mRNA expression in brain tissues of group B elevated significantly: the quantities upregulated as early as 6 hours after reperfusion (2.499±0.399, P<0.05 ) and peaked at 1d point (4.243±0.352, P<0.01) followed by a noticeable decrease at 2d point (1.321±0.215) .Interestingly, the level of VEGF mRNA elevated at 4d after reperfusion (1.647±0.264) and kept a higher level at 7d point (1.920±0.253, P<0.05). The quantity level of those in subset B2 was distinguished contrast to those of other subsets (P<0.05) .There were no statistical difference observed among subgroup B1, B3, B4, and B5.Compared to group A who’s ES mRNA was set as 1, the expression of ES mRNA remained a normal level (0.989±0.308,P>0.05) at 6h point. The level went up dramaticlly after 1 d post reperfusion (3.229±0.247, P<0.01), and the peak value was distinguished at 2d point (7.301±0.242,P<0.01) .From 4d to 7d after reperfusion, the ES mRNA expression still kept a higher level compared with those of group A (P<0.01). Quantity of subset B3 exhibited a statistically significant compared with those of other subsets (P<0.01).In group B, values of ES/VEGF decreased at both 6h and 1d points, but increased gradually until 2d after reperfusion (5.588±0.786, P<0.01).But it is still at high levels at 4d point (2.850±0.377,P<0.05) and 7d point (2.323±0.257,P<0.05) .Valueof subgroup B3 was distinguished compared with those of other subgroups (P<0.01) and values of subgroup B4 and B5 was significantly higher contrast to those of group B1 and B2(P<0.05).The correlation analysis showed a strong relationship between neurological function scores of the rats subjected to ischemic reperfusion injury and their E/V values (r=0.811) . ConclusionSubjected to reperfusion injury following 90 minutes of cerebral ischemia, rats central nervous systems begin to work abnormally, especially 2 days after reperfusion when neurological function scores rise to the peak; left cerebrums showed various signs of ischemia and large area of infarction could be observed. Expressions of VEGF mRNA expression in brain tissues increase as early as 6 hours after reperfusion and peak at 1d point followed by a noticeable down regulation at 2d point however still higher than the sham group. Expressions of ES mRNA don’t upregulate until 1d after reperfusion and reach the peak at 2d point. Ratio of E/V shows a falling trend during the early time of reperfusion, but it goes up significantly at day 2 and remains a high level later. Neurological function scores shows closely related to the ratio of E/V.In conclusion, during the procedure of brain ischemia reperfusion of rats, the balance of ES and VEGF, which may be reflected from the value of E/V, is tightly correlated with the neurological dysfunctions. Balance of enhancing and inhibiting factors of angiogenesis may be the key factor affecting severity and prognosis of injury after cerebral ischemic reperfusion. Since there are noticeable upregulation of VEGF mRNA expression and no significant elevation of ES during the early period and especially before 2 days after reperfusion, this period may be of crucial importance to the rebuilding of microcirculation.更多还原