[目的]本课题利用基因敲除小鼠研究β-Arrestin在血管和大脑中的作用。课题分为三个部分：第一部分是β-Arrestin的表达以及基因敲除小鼠的鉴定；第二部分是β-Arrestin对小鼠血管收缩功能的影响；第三部分是β-Arrestin对小鼠缺血性脑卒中的影响。[方法]第一部分：β-Arrestin的表达以及基因敲除小鼠的鉴定。按要求分别将引入的β-Arrestin1和β-Arrestin2杂合子种鼠分别交配后大量繁殖。待幼鼠4周大时,β-Arrestin1小鼠提取尾巴DNA、测定DNA浓度后做real time-PCR鉴定基因型；β-Arrestin2小鼠的尾巴DNA直接做PCR鉴定基因型。取8-12周大的C57BL6小鼠以及不同基因型小鼠麻醉处死后,迅速取胸主动脉、心脏和大脑,组织经过匀浆、蛋白浓度测定,然后做Western blot,分析β-Arrestin1和β-Arrestin2蛋白在上述三种组织的表达情况。第二部分：β-Arrestin对小鼠血管收缩功能的影响。该部分主要研究β-Arrestin1或β-Arrestin2敲除小鼠的胸主动脉收缩舒张功能的改变。取8-12周大的不同基因型(野生型、杂合子和纯合子)敲除小鼠,脱臼处死后取胸主动脉,迅速去掉血管外膜和内皮,每条胸主动脉可切成2条5mm的血管环,在离体血管灌流系统内稳定后,分别给予新福林(Phe)、血管紧张素Ⅱ(AngⅡ)的刺激,考察其收缩反应。第三部分：β-Arrestin对小鼠缺血性脑卒中的影响。该部分主要研究分别敲除两种β-Arrestin对小鼠大脑永久缺血后的影响。取8-12周大的三种基因型(野生型、杂合子和纯合子)敲除小鼠和C57BL6小鼠做大脑中动脉栓塞(MCAO)手术；基因敲除小鼠24小时后进行神经学评分；评分后麻醉小鼠,将大脑切片后用2%TTC染色,拍照后计算大脑梗死体积(缺血区面积与全脑面积百分比)；C57BL6小鼠24h后取缺血区半暗带和对侧脑组织,Western blot检测β-Arrestin1和β-Arrestin2表达量的改变。原代培养敲除小鼠(纯合子和野生型)神经元,糖氧剥夺实验(OGD)处理后检测细胞存活率、细胞活性及细胞毒性。明确β-Arrestin1和β-Arrestin2是否对缺血性脑卒中产生影响。[结果]第一部分：1. β-Arrestin1在小鼠大脑、心脏和胸主动脉表达较为丰富,β-Arrestin2在大脑和心脏有表达,而在胸主动脉表达较低。第二部分：1.小鼠胸主动脉环最适静息张力为1.5g;2. β-Arrestin1和β-Arrestin2敲除小鼠胸主动脉环对AngⅡ反应性较低,且与野生型的反应性无差别；3. β-Arrestin1杂合子小鼠胸主动脉环对Phe张力略高于野生型,无统计学差异；β-Arrestin2纯合子小鼠胸主动脉环对Phe张力与野生型没有差异。第三部分：1.成年C57BL6小鼠永久性MCAO后24h时,β-Arrestin1和β-Arrestin2在缺血区半暗带蛋白表达上调,β-Arrestin1上调程度更明显；2.敲除β-Arrestin1, MCAO后24小时杂合子小鼠和野生型小鼠的大脑梗死体积相近、神经功能评分一致,而纯合子小鼠大脑梗死体积变大、神经学评分增加,术后24小时死亡率增加：3.敲除β-Arrestin2, MCAO后24小时杂合子小鼠和野生型小鼠大脑梗死体积相近,但比野生型小鼠大脑梗死体积减少,纯合子神经功能评分稍低但与野生型组没有统计学差异,术后死亡率不变。4.敲除β-Arrestin1, OGD处理2h引起的神经元死亡细胞数增多,细胞活力降低,释放LDH增加。[结论]1.敲除β-Arrestin不影响小鼠胸主动脉对Phe诱导的收缩反应。2.小鼠胸主动脉对血管紧张素Ⅱ诱导的收缩反应很低,该现象与β-Arrestin无关。3. β-Arrestin1对缺血性脑卒中具有保护作用,是一种内源性保护因子。4.敲除β-Arrestin2降低小鼠MCAO后脑梗死体积,对神经功能没有明显改善。
[Objective]Our project was to apply knockout mice to investigate the role of β-Arrestin playing in vessels and brain. And project was divided into three parts:the first one is genotype identifying of β-Arrestin knockout mice and expression of β-Arrestin; the second one is the effect of β-Arrestin in vascular function; the last one is the effect of β-Arrestin on ischemic stroke after MCAO.[Method]Part Ⅰ:Genotype identifying of β-Arrestin knockout mice and expression of β-Arrestin Mating the β-Arrestin land β-Arrestin2heterozygous breeders, and expanding the posterity.4weeks after born, extracting tail DNA of knockout mice, measuring DNA concentration and run realtime-PCR for β-Arrestin las well as PCR for β-Arrestin2to idenfy the genotype respectively.8-12weeks after born, C57BL6and three genotypes of mice were sacrificed with anesthesia, and then thoracic aorta, heart and brain were extracted to analysis the expression of β-Arrestin1and β-Arrestin2by western blot. Part Ⅱ:Study on effects of β-Arrestin in vascular function;Systolic and diastolic function of thoracic aorta extracted fromp-Arrestinl and β-Arrestin2mice was detected in this study.8-12weeks old, three types of genotype (homozygote, heterozygote and wildtype) knockout mice were killed by dislocated and then thoracic aorta was extracted, vascular adventitia and endothelial were excluded quickly and the aorta were cut into several rings. Bathing in isolated organ perfused system, aorta rings were excited by phenylephrine or angiotensin Ⅱ changes in tension were recorded.Part Ⅲ:Study on effects of β-Arrestin on brain after MCAOThis part investigated the effect of β-Arrestin on infarct area and neurological score after permanent MCAO.8-12weeks old, three types of genotype (homozygote, heterozygote and wildtype) knockout mice were performed by permanent MCAO, after24hours, neurological score and infarct area of brain slices were measured. Changes of β-Arrestin1 and β-Arrestin2expression after permanent MCAO in ischemic penumbra of C57BL6mice were detected by western blot. Primary cultured neurons extracted wildtype and homozygote mice were subjected to oxygen-glucose deprivation; cell number, cell viability and cell injury were detected by different methods.[Result]Part Ⅰ:β-Arrestinl is high expression in thoracic aorta, heart and brain extracted from C57BL6, β-Arrestinl heterozygote and wildtype β-Arrestinl mice, but without homozygote mice. β-Arrestin2expresses in brain of C57BL6, heterozygote and wildtype β-Arrestin2mice, less in thoracic aorta, but without in tissues extracted from homozygote β-Arrestin2mice.Part Ⅱ:1, The optimal resting tension of thoracic aorta ring was1.5g.2, Low reactivity to Angiotensin Ⅱ was detected in aorta rings extracted from β-Arrestin1and β-Arrestin2knockout mice, and had no difference between wildtype mice.3, Tension to phenylephrine of heterozygote mice aorta ring was higher than wildtype ring in P-Arrestinl mice, but without statistical difference; there was no statistical difference between homozygote and wildtype aorta ring tension.Part Ⅲ:1, β-Arrestin1and β-Arrestin2were up-regulated in the ischemic penumbra of C57BL6at24hours after MCAO;2, Heterozygote and wildtype β-Arrestinl mice had the similar infarct area and neurological score, but homozygote mice had bigger infarct area and increased neurological score;3, As to β-Arrestin2, heterozygote and wildtype mice had the similar infarct area and neurological score, but infarct area in homozygote mice was smaller with statistical difference, as well as decreased neurological score without statistical difference;4, knockout β-Arrestinl gained the number of dead neuron, decreased the cell viability and increased the cell injury after OGD performance.[Conclusion]1. β-Arrestin didn’t affect the systolic reactivity induced by phenylephrine.2. Mice thoracic aorta ring had low systolic reactivity to Angiotensin Ⅱ, and had no relation to β-Arrestin. 3. β-Arrestin1had protective effect on ischemic stroke, and was an endogenous protective factor.4. Knockout β-Arrestin2could reduce the infarct area but had no effect on the neurological function.