本文選題：大鼠 + 腦缺血　； 參考：《鄭州大學(xué)》2016年碩士論文

【摘要】：背景和目的急性腦缺血是臨床常見疾病,腦缺血繼發(fā)的腦水腫與顱內(nèi)壓增高、腦疝等病理過程密切相關(guān),是引起腦缺血患者急性期死亡的主要原因之一,也對腦缺血疾病過程及預(yù)后具有重要的價值。腦缺血引起水腫的分子級聯(lián)機制包括細(xì)胞膜離子泵的丟失、細(xì)胞腫脹,繼發(fā)自由基及蛋白酶的形成堆積,進(jìn)一步導(dǎo)致細(xì)胞膜破壞,造成不可挽回的損害。自從Agre等在1993年發(fā)現(xiàn)水通道蛋白(Water channel protein,WCP)以來,越來越多的基礎(chǔ)和臨床研究確定了其在水分子跨細(xì)胞膜轉(zhuǎn)運過程中的重要作用,尤其是水通道蛋白-4(Aquaporin 4,AQP4)越來越多的被學(xué)者們認(rèn)為與腦水腫的病理過程密切相關(guān)。傳統(tǒng)磁共振擴散加權(quán)成像(Diffusion weighted imaging,DWI)成像的理論基礎(chǔ)是水分子在組織細(xì)胞間的自由擴散運動,傳統(tǒng)理念認(rèn)為主要是因為組織中細(xì)胞膜、細(xì)胞間質(zhì)等對水分子自由運動的限制,以及水分子在擴散過程中部分與細(xì)胞或細(xì)胞間隙的組織上大分子交換。細(xì)胞膜上具有豐富的、不同亞型的水通道蛋白分布。并且,水分子在細(xì)胞內(nèi)及細(xì)胞間隙自由擴散運動的速率與其通過細(xì)胞膜水通道蛋白(AQPs)的速率存在顯著差異。水通道蛋白理論的建立對傳統(tǒng)DWI成像機理提出了一定挑戰(zhàn),并能讓人們在分子醫(yī)學(xué)、分子影像學(xué)以及分子生物學(xué)等方面重新了解DWI技術(shù),也為DWI進(jìn)一步的臨床研究及應(yīng)用提供了一定的理論基礎(chǔ)。丁基苯酞是我國在腦血管疾病領(lǐng)域的第一個擁有自主知識產(chǎn)權(quán)的國家一類新藥,該藥物對缺血性腦卒中的保護作用已經(jīng)得到許多基礎(chǔ)及臨床試驗的證明。丁基苯酞可通過改善微循環(huán)灌注、抑制腦細(xì)胞凋亡等來保護缺血性腦損傷,也有研究認(rèn)為其可調(diào)控AQP-4的表達(dá)保護缺血性腦水腫,但機制尚不明確。本實驗通過大鼠急性腦缺血腦水腫多b值DWI成像分析所測參數(shù)的動態(tài)演變規(guī)律;探討丁基苯酞對大鼠腦缺血后AQP-4的調(diào)控機制。材料與方法1、制作大鼠大腦中動脈梗阻模型(middle cerebral artery occlusion,MCAO),分為模型組、丁基苯酞組、假手術(shù)組。模型組及丁基苯酞組按照插入線栓的時間分再為1小時、3小時、6小時、12小時、24小時組,每組10只。2、采用美國GE 750 3.0T超導(dǎo)磁共振掃描儀器及大鼠專用線圈。掃描序列包括T1加權(quán)成像(T1-weighted imaging,T1WI)、T2加權(quán)成像(T2-weighted imaging,T2WI)、T2WIFLAIR、b值=1000s/mm2擴散加權(quán)成像(DWI)、多b值DWI:梯度擴散因子(b)值為取0、50、100、150、200、300、500、800、1000、1300、1500、1700、2000、2500、3000、3500、4000和4500 s/mm2 18個b值點。3、在造模后3小時三組分別選取5只大鼠,進(jìn)行神經(jīng)功能學(xué)評分,然后斷頭取腦,進(jìn)行干濕重分析。各個時間點掃完圖像后每小組10只大鼠進(jìn)行免疫組織化學(xué)AQP4及PKC的半定量分析。4、標(biāo)準(zhǔn)ADC值A(chǔ)DCst處理與標(biāo)準(zhǔn)DWI圖像,多b值DWI處理采用三組件數(shù)學(xué)模型,b值范圍0-1500 s/mm2(2000 s/mm2)采用雙指數(shù)模型(IVIM)得出D(純擴散系數(shù),反映水分子的自由運動),D~*(偽擴散系數(shù),反映快擴撒或以灌注基礎(chǔ)的分子擴);b值2000-4500 s/mm2(≥2000 s/mm2)時測得ADCuh。所有數(shù)據(jù)均測三次求平均值。5、所有統(tǒng)計分析均采用SPSS 17.0軟件包完成。統(tǒng)計結(jié)果中定量資料以均數(shù)±標(biāo)準(zhǔn)差(SDX±)表示,并進(jìn)行正態(tài)性、方差齊性檢驗。神經(jīng)功能評分比較采用秩和檢驗,三組兩兩間比較應(yīng)用LSD法,以P0.05表示差異有統(tǒng)計學(xué)意義。結(jié)果1.在造模3h后丁基苯酞組與MCAO組比較,其神經(jīng)功能學(xué)評分值有所降低(Z=-2.01,P=0.044);各個組在造模后3小時測得腦組織含水量相互比較,MCAO組與丁基苯酞組、MCAO組于假手術(shù)組、丁基苯酞組與假手術(shù)組差異具有統(tǒng)計學(xué)意義(P1=0.00,P2=0.01,P3=0.01)。2.丁基苯酞組、MCAO組患側(cè)ADCst值12小時下降至最低而后輕度上升,兩組不同時間點異具有統(tǒng)計學(xué)意義(P10.05);D~*、D、ADCuh值在MCAO組中于術(shù)后6h下降至最低,而后輕度上升,而在丁基苯酞組中于術(shù)后12h下降至最低,而后輕度上升;1-6h中,D~*、D、ADCuh值在丁基苯酞組與MCAO組差異具有統(tǒng)計學(xué)意義(P10.05);D~*、D、ADCuh值在各個時間點上MCAO組與假手術(shù)組、丁基苯酞組與假手術(shù)組差異具有統(tǒng)計學(xué)意義P20.05、P30.05);1-6h中MCAO組,ADCuh變化最為明顯(30%)。3.MCAO組及丁基苯酞組1小時后AQP4、PKC表達(dá)開始增加,并于24小時達(dá)到最高值。MCAO組及丁基苯酞組與假手術(shù)組差異具有統(tǒng)計學(xué)意義(P2、P30.05),1-6h MCAO組與丁基苯酞組相比各個時間點的表達(dá)有差異且有統(tǒng)計學(xué)意義(P10.05)。AQP4、PKC的表達(dá)在1-6h時間段內(nèi)最為明顯。結(jié)論1.D~*值能夠反映部分微循環(huán)灌注信息,為活體急性缺血后腦組織中微循環(huán)變化提供有價值的信息。2.ADCuh值監(jiān)測腦梗死超急性期腦水腫的變化優(yōu)于ADCst、D~*、D。ADCuh可能反映部分水通道蛋白信息。3.丁基苯酞可下調(diào)PKC及AQP-4表達(dá),綜合改善腦缺血性腦水腫;丁基苯酞可能通過PKC通路調(diào)控AQP-4表達(dá)需要進(jìn)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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R743.3

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