丁基苯酞调控大鼠急性脑梗死AQP4表达的多b值扩散成像评价

发布时间：2018-06-21 02:21

本文选题：大鼠 + 脑缺血　；参考：《郑州大学》2016年硕士论文

【摘要】：背景和目的急性脑缺血是临床常见疾病,脑缺血继发的脑水肿与颅内压增高、脑疝等病理过程密切相关,是引起脑缺血患者急性期死亡的主要原因之一,也对脑缺血疾病过程及预后具有重要的价值。脑缺血引起水肿的分子级联机制包括细胞膜离子泵的丢失、细胞肿胀,继发自由基及蛋白酶的形成堆积,进一步导致细胞膜破坏,造成不可挽回的损害。自从Agre等在1993年发现水通道蛋白(Water channel protein,WCP)以来,越来越多的基础和临床研究确定了其在水分子跨细胞膜转运过程中的重要作用,尤其是水通道蛋白-4(Aquaporin 4,AQP4)越来越多的被学者们认为与脑水肿的病理过程密切相关。传统磁共振扩散加权成像(Diffusion weighted imaging,DWI)成像的理论基础是水分子在组织细胞间的自由扩散运动,传统理念认为主要是因为组织中细胞膜、细胞间质等对水分子自由运动的限制,以及水分子在扩散过程中部分与细胞或细胞间隙的组织上大分子交换。细胞膜上具有丰富的、不同亚型的水通道蛋白分布。并且,水分子在细胞内及细胞间隙自由扩散运动的速率与其通过细胞膜水通道蛋白(AQPs)的速率存在显著差异。水通道蛋白理论的建立对传统DWI成像机理提出了一定挑战,并能让人们在分子医学、分子影像学以及分子生物学等方面重新了解DWI技术,也为DWI进一步的临床研究及应用提供了一定的理论基础。丁基苯酞是我国在脑血管疾病领域的第一个拥有自主知识产权的国家一类新药,该药物对缺血性脑卒中的保护作用已经得到许多基础及临床试验的证明。丁基苯酞可通过改善微循环灌注、抑制脑细胞凋亡等来保护缺血性脑损伤,也有研究认为其可调控AQP-4的表达保护缺血性脑水肿,但机制尚不明确。本实验通过大鼠急性脑缺血脑水肿多b值DWI成像分析所测参数的动态演变规律;探讨丁基苯酞对大鼠脑缺血后AQP-4的调控机制。材料与方法1、制作大鼠大脑中动脉梗阻模型(middle cerebral artery occlusion,MCAO),分为模型组、丁基苯酞组、假手术组。模型组及丁基苯酞组按照插入线栓的时间分再为1小时、3小时、6小时、12小时、24小时组,每组10只。2、采用美国GE 750 3.0T超导磁共振扫描仪器及大鼠专用线圈。扫描序列包括T1加权成像(T1-weighted imaging,T1WI)、T2加权成像(T2-weighted imaging,T2WI)、T2WIFLAIR、b值=1000s/mm2扩散加权成像(DWI)、多b值DWI:梯度扩散因子(b)值为取0、50、100、150、200、300、500、800、1000、1300、1500、1700、2000、2500、3000、3500、4000和4500 s/mm2 18个b值点。3、在造模后3小时三组分别选取5只大鼠,进行神经功能学评分,然后断头取脑,进行干湿重分析。各个时间点扫完图像后每小组10只大鼠进行免疫组织化学AQP4及PKC的半定量分析。4、标准ADC值ADCst处理与标准DWI图像,多b值DWI处理采用三组件数学模型,b值范围0-1500 s/mm2(2000 s/mm2)采用双指数模型(IVIM)得出D(纯扩散系数,反映水分子的自由运动),D~*(伪扩散系数,反映快扩撒或以灌注基础的分子扩);b值2000-4500 s/mm2(≥2000 s/mm2)时测得ADCuh。所有数据均测三次求平均值。5、所有统计分析均采用SPSS 17.0软件包完成。统计结果中定量资料以均数±标准差(SDX±)表示,并进行正态性、方差齐性检验。神经功能评分比较采用秩和检验,三组两两间比较应用LSD法,以P0.05表示差异有统计学意义。结果1.在造模3h后丁基苯酞组与MCAO组比较,其神经功能学评分值有所降低(Z=-2.01,P=0.044);各个组在造模后3小时测得脑组织含水量相互比较,MCAO组与丁基苯酞组、MCAO组于假手术组、丁基苯酞组与假手术组差异具有统计学意义(P1=0.00,P2=0.01,P3=0.01)。2.丁基苯酞组、MCAO组患侧ADCst值12小时下降至最低而后轻度上升,两组不同时间点异具有统计学意义(P10.05);D~*、D、ADCuh值在MCAO组中于术后6h下降至最低,而后轻度上升,而在丁基苯酞组中于术后12h下降至最低,而后轻度上升;1-6h中,D~*、D、ADCuh值在丁基苯酞组与MCAO组差异具有统计学意义(P10.05);D~*、D、ADCuh值在各个时间点上MCAO组与假手术组、丁基苯酞组与假手术组差异具有统计学意义P20.05、P30.05);1-6h中MCAO组,ADCuh变化最为明显(30%)。3.MCAO组及丁基苯酞组1小时后AQP4、PKC表达开始增加,并于24小时达到最高值。MCAO组及丁基苯酞组与假手术组差异具有统计学意义(P2、P30.05),1-6h MCAO组与丁基苯酞组相比各个时间点的表达有差异且有统计学意义(P10.05)。AQP4、PKC的表达在1-6h时间段内最为明显。结论1.D~*值能够反映部分微循环灌注信息,为活体急性缺血后脑组织中微循环变化提供有价值的信息。2.ADCuh值监测脑梗死超急性期脑水肿的变化优于ADCst、D~*、D。ADCuh可能反映部分水通道蛋白信息。3.丁基苯酞可下调PKC及AQP-4表达,综合改善脑缺血性脑水肿;丁基苯酞可能通过PKC通路调控AQP-4表达需要进一步去研究。
[Abstract]:Background and objective acute cerebral ischemia is a common clinical disease. Cerebral edema secondary to cerebral ischemia is closely related to the pathological process such as intracranial hypertension and brain hernia. It is one of the main causes of acute cerebral ischemia death, and it also has important value for the process and prognosis of cerebral ischemia. The loss of the cell membrane ion pump, the swelling of the cells, the formation and accumulation of secondary free radicals and protease, further causing cell membrane destruction and irreparable damage. Since the discovery of Water channel protein (WCP) in 1993, more and more basic and clinical studies have determined its transcellular membrane transport in water molecules. The important role of the process, especially the water channel protein -4 (Aquaporin 4, AQP4), is believed to be closely related to the pathological process of brain edema. The theoretical basis of the traditional magnetic resonance diffusion-weighted imaging (Diffusion weighted imaging, DWI) imaging is the free diffusion movement between the water molecules in the tissue cells, and the traditional concept is considered to be the basis of the traditional theory. It is mainly due to the restriction of the free movement of water molecules by the cell membrane in the tissue, the intercellular mass and the free movement of water molecules, and the exchange of large molecules on the tissue in the cell or cell gap in the diffusion process. The cell membrane has a rich, different subtype of aquaporin distribution. And the water molecules spread freely in the cell and in the space. The rate of movement is significantly different from the rate of the cell membrane water channel protein (AQPs). The establishment of the theory of aquaporin is a challenge to the traditional DWI imaging mechanism, and can make people understand the DWI technology in molecular medicine, molecular imaging and molecular biology, and further clinical research and application for DWI. It provides a theoretical basis. Butylphthalide is the first country with independent intellectual property rights in the field of cerebrovascular disease. The protective effect of this drug on ischemic stroke has been proved by many basic and clinical trials. Butylphthalide can be used to improve microcirculation perfusion and inhibit apoptosis of brain cells. To protect ischemic brain injury, there is also a study that it can regulate the expression of AQP-4 to protect ischemic brain edema, but the mechanism is still not clear. In this experiment, the dynamic evolution of parameters measured by the multiple b value DWI imaging analysis of acute cerebral ischemia brain edema in rats and the regulation mechanism of AQP-4 after cerebral ischemia in rats by butylphthalide were discussed. Materials and methods 1, The rat model of middle cerebral artery occlusion (MCAO) was divided into model group, butylphthalide group and sham operation group. The model group and butylphthalide group were divided into 1 hours, 3 hours, 6 hours, 12 hours, 24 hour group and 10.2 in each group, using American GE 750 3.0T MRI scanning instrument and large The scanning sequence includes T1 weighted imaging (T1-weighted imaging, T1WI), T2 weighted imaging (T2-weighted imaging, T2WI), T2WIFLAIR, b values, =1000s/mm2 diffusion weighted imaging (DWI), and the value of the multiple values gradient diffusion factor is 4500. Value point.3, 5 rats were selected in the three groups 3 hours after the model, and then the neurologic score was scored. Then the head was taken to take the brain to carry out the dry and wet weight analysis. After the images were scanned at each time point, 10 rats in each group were used for the semi quantitative analysis of the immunohistochemical AQP4 and PKC, the standard ADC value ADCst treatment and the standard DWI image, and the multi b value DWI processing three. The component mathematical model, the b value range 0-1500 s/mm2 (2000 s/mm2) uses a double exponential model (IVIM) to obtain D (pure diffusion coefficient reflecting the free movement of water molecules), D~* (pseudo diffusion coefficient, reflecting fast spreading or molecular expansion on the basis of perfusion); b value 2000-4500 s/mm2 (> 2000 s/mm2) and all ADCuh. all data measured by ADCuh. three times average value.5, all The statistical analysis was performed with SPSS 17 software package. The quantitative data in the statistical results were expressed with mean standard deviation (SDX +), normal and homogeneity test. The scores of the neurological function were compared with the rank sum test, the three groups were compared with the LSD method, and the difference was statistically significant by P0.05. Results 1. in the 3H group after the model 3H Compared with the MCAO group, the score of the neurologic score was decreased (Z=-2.01, P=0.044), and the water content of the brain tissue was compared in each group 3 hours after the model, group MCAO and butylphthalide group, the group MCAO in the sham group, the difference between the butylphthalide group and the sham group was statistically significant (P1=0.00, P2=0.01, P3=0.01) in the.2. butyl phthalide group, and the MCAO group was developed. The side ADCst value decreased to a minimum and then slightly increased in 12 hours. The two groups had statistical significance at different time points (P10.05); D~*, D, ADCuh value decreased to the lowest in MCAO group and then slightly increased, while in the butylphthalide group, 12h decreased to the lowest and then slightly increased; 1-6h, D~*, D, and ADCuh values in the butylphthalide group The difference of the group was statistically significant (P10.05); the values of D~*, D, and ADCuh were in the MCAO group and the sham operation group at all time points, the difference between the Ding Ji phthalide group and the sham group was statistically significant P20.05, P30.05); the MCAO group in 1-6h was the most obvious (30%).3.MCAO group and Ding Ji phenthalide group after 1 hours, the expression began to increase, and reached 24 hours. The difference between the highest.MCAO group and the butyl phthalide group and the sham group was statistically significant (P2, P30.05). The expression of each time point in the 1-6h MCAO group was different from that of the butylphthalide group and had statistical significance (P10.05).AQP4, and the expression of PKC was most obvious in the 1-6h time period. The nodal 1.D~* value could reflect the partial microcirculation perfusion information. The changes of microcirculation in cerebral tissue after acute ischemia provide valuable information.2.ADCuh value monitoring the changes of cerebral edema in hyperacute phase of cerebral infarction better than ADCst, D~*, D.ADCuh may reflect partial water channel protein information.3. Ding Jiben phthalein can downregulate PKC and AQP-4 expression and improve cerebral hemorrhagic brain edema; butylphthalide may be modulated through PKC pathway Control of AQP-4 expression needs to be further studied.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R743.3

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