【作者】 刘东梅；

【导师】 娄凤兰； 张岫美；

【作者基本信息】 山东大学 ， 护理学， 2014， 博士

【摘要】 研究背景及研究目的中风为全球性常见病和多发病,具有发病率高,致残率高,死亡率高,严重危害人们的健康,给国家,社会,家庭及个人带来了沉重的精神和生活负担。到目前为止,中风的发病原因及发生发展的分子生物学机制尚未明确,缺乏对多数患者安全有效的治疗措施和药物。tPA,是唯一被美国FDA批准的用于治疗中风的药物。但其治疗时间窗仅局限于发病后的4.5h内。超过此时间段应用,则会导致严重的出血及脑水肿等不良反应。现实中,由于诸多因素的影响,绝大多数的病人在发病后的4.5h不能到达医疗机构,因此,也就失去了使用tpA进行溶栓治疗的机会,并且,tpA可能具有神经毒性和或导致神经元的凋亡反应等不良反应。因此,中风的治疗成为全球医务工作者研究的重点之一。在目前已有的中风发病机制中,中风病人脑区内的神经元发生的凋亡反应引起了越来越多学者的重视,并被认为是治疗中风的重要的靶向目标之一。葛根素是从中药葛根中提取的异黄酮类化合物,临床上用于治疗心、脑血管等疾病的已有20余年。但是,葛根素不良反应较多,且脂溶性低,不能有效的通过血脑屏障,限制了其在临床的广泛使用。乙酰葛根素是在葛根素的基础上经结构改造引入侧链得到的新型异黄酮类化合物,与葛根素相比,乙酰葛根素的脂溶性显著提高,能够通过血脑屏障。已有体外动物实验表面,乙酰葛根素能显著提高缺氧复氧神经元的细胞活力,降低细胞凋亡率,提高胞内bcl-2和Bax的表达,提示对缺氧缺糖神经元具有保护作用,是一个具有开发前景的药物。本实验在课题组前期工作的基础上进一步研究乙酰葛根素对脑缺血再灌注损伤的作用。我们选用大鼠胎鼠的海马神经元作为研究对象,采用体外培养的神经元缺氧缺糖再灌注法模型,旨在观察受缺氧缺糖损伤的海马神经元中神经元活力,神经元凋亡发生率和凋亡相关因子的变化以及乙酰葛根素对上述所述项目的影响。本实验通过动态检测缺氧缺糖再灌注神经元活力,凋亡发生率以及乙酰葛根素的作用,筛选出观测最佳时间点,并以此时间点作为后续实验指标标本收集点,测定凋亡相关印迹的变化以及乙酰葛根素的作用,观察该药物对大鼠胎鼠海马神经元的抗凋亡作用并对涉及到的机制做深入研究,为研究开发乙酰葛根素用于治疗缺血性脑血管疾病提供实验依据。本实验分为二个部分：第一部分：乙酰葛根素对氧糖剥夺诱导海马神经元损伤的细胞活力及形态学影响第二部分：乙酰葛根素对氧糖剥夺诱导海马神经元凋亡相关因子的影响第一部分：乙酰葛根素对氧糖剥夺诱导海马神经元损伤的细胞活力及形态学影响目的采用体外分离培养海马神经元,缺氧缺糖后复氧复糖建立缺血再灌注模型,利用形态学上和细胞活力的变化,观察乙酰葛根素对糖氧剥离诱导海马神经元损伤的影响,并筛选出后续实验的最佳时间点。方法取孕17.5-18.5天的wistar大鼠的胎鼠,剥离海马神经元进行原代培养。每天于倒置显微镜下观察神经元生长状况,每三天半量换液一次,至生长8d后取活力好的神经元进行后续实验。实验分组及OGD/R根据药物浓度的不同实验共分5组：1.正常对照组Neurobasal medium(NB)+B27培养至8天后,换成无B27的NB的培养液一天,后吸弃原培养液,换成温暖的含糖D-Hank’s液,置入C02培养箱,180min后换成NB完全培养液(不加B27)继续孵育至预定时间。2.低糖低氧再灌注损伤组无糖D-Hank’s液预先放入厌氧培养箱(95%N2/5%C02)中30min,使氧的浓度<1%。神经元以无糖D-Hank’s液清洗4次,每次为原培养液体积的2/3,使葡萄糖终浓度小于1mM。换无糖D-Hank’s后将神经元放入厌氧罐内,自开始换液计时至180mmin,吸弃无糖D-Hank’s,换以NB完全培养液(不加B27),置入C02培养箱继续孵育至预定时间,以此模拟体内缺血再灌注的过程。3.低浓度乙酰葛根素组换无糖D-Hank’s液及OGD结束改为NB完全培养液(不加B27)均时添加入乙酰葛根素,使终浓度为0.1μM,其余处置同2组。4.中浓度乙酰葛根素组换无糖D-Hank’s液及OGD结束改为NB完全培养液(不加B27)均时添加入乙酰葛根素,使终浓度为0.4gM,其余处置同2组。5.高浓度乙酰葛根素组换无糖D-Hank’s液及OGD结束改为NB完全培养液(不加B27)均时添加入乙酰葛根素,使终浓度为1.6gM,其余处置同2组。在OGR-12h、24h和36h分别采用倒置显微镜,MTT法和DAPI法动态的观测了不同时间段中神经元形态、活力及神经元发生凋亡的变化,在对上述检测方法重复三次测量并统计分析结果后选择OGD/R-24h作为后续实验的主要时间点；OGD/R-24h,采用末端脱氧核苷酸酶转移酶介导的duTP缺口末端标记术(TUMEL)法检测神经元发生凋亡的状况并观察不同浓度的乙酰葛根素对神经元凋亡的影响；结果1.神经元经OGD处理再灌注12,24h及36h,光学显微镜下观察,神经元胞体的折光性下降,轴突缩短,甚至消失,部分胞体溶解,仅可见散在的细胞核。加入0.1μM,0.4μM和1.6μM的乙酰葛根素则能明显改善OGD/R对神经元的形态学损伤,减少受损神经元数目,表明乙酰葛根素对缺糖缺氧引起的神经元损伤具有保护作用。2.在体外OGD/R-12h,24h及36h可直接引起神经元损伤,MTT法检测神经元的活力降低,0.1μM,0.4μM和1.6μM的乙酰葛根素对体外OGD/R引起胎鼠海马神经元的损伤有保护作用,可提高神经细胞的活力,高浓度组的乙酰葛根素效力明显优于中浓度和低浓度组。3. DAPI染色法显示,OGD/R-12h,24h及36h后,神经元中染色质固缩,向外周集聚并呈成颗粒状的凋亡细胞数明显增多。高,中和低浓度的乙酰葛根素对OGD/R-12h均无保护作用,OGD/R-24h和36h二个时间点中,0.41μM和1.6μM的乙酰葛根素能降低凋亡的阳性细胞数,但0.1μM的乙酰葛根素未能显示有保护作用,并且,统计结果显示,在OGD/R-24h, DAPI阳性细胞数达到最高峰,超过24h, DAPI染色的阳性细胞数逐渐减小,故本实验选择OGD/R-24h作为后续指标的主要观测时间点。4. OGD/R-24h可引起海马神经元发生凋亡,凋亡指数升高。TUNEL法显示0.1μM,0.4μM和1.6μM的乙酰葛根素能显著降低神经元中凋亡阳性细胞数,使得细胞凋亡指数下降,显示了对缺氧缺糖神经元的保护作用。结论1.不同时间段的OGD/R可使体外培养的海马神经元受损,活力下降,并可致神经元发生凋亡,凋亡发生的高峰时间为OGD/R-24h。2.乙酰葛根素可以减轻神经元的损伤,提高神经元的活力,维持神经元结构的完整性3.乙酰葛根素在0.1μM,0.4μM和1.6μM的浓度可减少凋亡的阳性细胞数,显示其对缺氧缺糖再灌注损伤的保护作用。第二部分：乙酰葛根素对糖氧剥夺诱导海马神经元凋亡相关因子的影响目的利用第一部分实验筛选出的药物效应时间点,从分子水平检测乙酰葛根素对凋亡相关因子变化的影响,为一线乙酰葛根素临床治疗脑缺血再灌注损伤提供实验依据。方法神经元的体外培养及模型建立同第一部分。实验时间点为OGD/R-6h,24h。实验分组为低糖低氧再灌注损伤组,低浓度乙酰葛根素组,中浓度乙酰葛根素组,高浓度乙酰葛根素组。OGD/R-6h,采用荧光分光光度法观察caspase-8活力的变化及加入乙酰葛根素后蛋白水解酶活力的改变；OGD/R-24h采用荧光分光光度法观察caspase-3活力的变化及加入乙酰葛根素后此种蛋白水解酶活力的改变；OGD/R-24h,处理后的神经元经离心收集蛋白,运用免疫印迹法检查神经元缺氧缺糖损伤后凋亡相关蛋白Fas-L, FADD, TNF-α及Hsp70的变化及添加乙酰葛根素后对上述蛋白水平的影响；OGD/R-24h,利用半定量RT-PCR法检测HIF-1αmRNA, NF-κBmRNA和P53mRNA的表达和乙酰葛根素的影响。结果1.0.1μM,0.4μM和1.6μM的乙酰葛根素能改善OGD/R引起的胎鼠海马神经元中caspase活力的变化。荧光分光光度法中,OGD/R-6h使得caspase-8蛋白水解酶的活力显著升高,而不同浓度的乙酰葛根素组可使升高的caspase-8活力下降。OGD/R-24h, Caspase-3的活力升高,与对照组相比,低、中和高浓度的乙酰葛根素使得升高的Caspase-3活力不同程度的降低。2. OGD/R-24h,海马神经元中Fas-L, FADD和TNF-α表达量增高,免疫印迹法显示Fas-L, FADD和TNF-α条带面积及灰度均升高。加入不同浓度的乙酰葛根素后,结果显示上述蛋白的合成均减少,显示对OGD/R-24h后神经元的保护作用。3.乙酰葛根素能增强海马神经元抗损伤的能力。OGD/R-24h, Hsp70合成增加,不同浓度的乙酰葛根素使得的Hsp70升高更加明显。4. OGD/R-24h可诱导海马神经细胞表达HIF-1αmRNA, NF-κBmRNA和P53mRNA的表达增加。加入0.1μM,0.4μM和1.6μMM的乙酰葛根素后24小时行半定量RT-PCR,电泳结果显示HIF-1αmRNA, NF-κBmRNA和P53mRNA条带面积及灰度均降低,结果具有统计学意义。表明乙酰葛根素可以使OGD/R-24h诱导海马神经细胞增加HIF-1αmRNA, NF-κB mRNA和P53mRNA的表达减少。结论不同时间段的OGD/R可使体外培养的海马神经元受损, Caspase-3及Caspase-8的活力升高,Fas-L, FADD和TNF-α的蛋白合成增加,Hsp70的蛋白合成降低,HIF-1αmRNA, NF-κBmRNA和P53mRNA的表达升高,导致细胞凋亡的发生。乙酰葛根素可以减轻神经元的损伤,0.1μM,0.4μM和1.6μM的乙酰葛根素可使升高的Caspase-3及Caspase-8的活力降低,Fas-L, FADD和TNF-α的蛋白合成下降,Hsp70的蛋白合成升高,并减少HIF-1αmRNA, NF-κBmRNA和P53mRNA的表达,表明乙酰葛根素对缺糖缺氧再灌注所致的海马神经元损伤具有明显的保护作用,可抑制损伤所致的神经元凋亡的发生,减少凋亡的阳性细胞数。乙酰葛根素可通过影响凋亡相关因对海马神经元缺血再灌注损伤发挥脑保护作用。意义乙酰葛根素属新型异黄酮类化合物,我们的结果显示葛根素乙酰对缺糖缺氧模型鼠神经细胞的抗凋亡保护作用,提示乙酰葛根素在防治脑缺血再灌注损伤方面具有广阔的开发前景。更多还原

【Abstract】 Background and purposeStrok is a common disease in the World and is the first leading cause of death in China. It is characteristered by high rate of incidence, high disability and high mortality. Every year, country and family pay many for treatment this disease. However, the biochemical events that occurs after stroke do not well understand up-to-date, as a result, there lack of effective drugs and measures targeted the therapy for stroke. Tissue-type plasminogen activator (t-PA) is the only therapy approved by the Food and Drug Administration (FDA) for the treatment of acute ischemic stroke, but the therapeutic time window is limited within4.5hours of the onset of ischemic stroke, Beyond4.5h after stroke onset, t-PA administration show increased risk of hemorrhagic conversion and cerebral edema in the infarcted area of brain. For various reasons, the overwhelming majority of patients did not arrive medical institutions after stroke onset, so, they do not benefit from this therapeutic strategy. And, it is well known that tPA has neurotoxicity and could lead to neuronal apoptosis. The therapy of stroke has become one of the most important aims for the global medical workers. At present, the apoptosis that occurs in the ischemic had arised more and more attentions and been thought one of the targeted theragy for the stroke.Puerarin is an isoflavones that extracted from the traditional Chinese medicine, Pueraria lobata (Willd) Ohwi, It has been pasted more than20years since study Puerarin in laboratory to clinical cardiovascular and cerebrovascular diseases treatment. But puerarin has a poor lipid-soluble and can not permeate through blood-brain barrier (BBB), this shortcoming limits its extensive use in clinical. Acetylpuerarin, a new isoflavone compound, whose structure was modified based on puerarin, has been investigated. Acetylpuerarin has higher solubility in lipid than puerarin and can permeate BBB easily. Several studies have revealed that acetylpuerarin has protective effects against brain ischemia-reperfusion injury by increasing the expression of bcl-2and Bax, inhibiting cell apoptosis. Acetylpuerarin has a broad prospects in the treatmet of ischemic stroke.In this study, we furture explore the effect of acetylpuerarin on the ischemia/reperfusion. OGD/R-induced hippocampal neurons injured model were used to study the vibility, apoptosis and apoptic relating factors and the protective effects of acetylpuerarin. We choose a better time point by statistical analysis for the lasted study. We hope, our founding may do some use in the research and development of acetylpuerarin.Our study includes the following two parts:1. Effect of acetylpuerarin on the cell viability and morphology of hippocampal neurons following oxygen-glucose deprivation/reperfusion in rats.2. Acetylpuerarin reduces apoptosis of hippocampal neurons following oxygen-glucose deprivation/reperfusion in rats. Part I Effect of acetylpuerarin on the cell viability and morphology of hippocampal neurons following oxygen-glucose deprivation/reperfusion in ratsObjectivHippocampal neurons were cultured and OGD/R model was established, the effect of acetylpuearin on the cell viability and morphology following oxygen-glucose deprivation/reperfusion were detected by MTT and inverted microscope. In this part, we choose a best main point for the future research.MethodsHippocampi were removed from the brains of embryonic day18and Cells were cultured in a medium. Half volume of the medium was changed per3days for each. The following experiments were performed after8days.The neurons were divided into five groups according to the concentration of acetylpuearin:1.Control group:The primary embryonic hippocampal neurons were incubated in NB with B27for8days, then culture medium was removed and changed into warmLy D-Hank’s balanced salt solution with glucose and placed in5%CO2incubator for180min. After that, the neurons were incubated in NB without B27at37℃for scheduled time.2. Oxygen-glucose deprivation and reperfusion (O/R) group:The D-Hank’s balanced salt solution without glucose was placed in OGD chamber(95%N2/5%CO2)for30min and the oxygen was less than1%. The neuron were then rinsed with OGD buffer4times and transferred to an OGD chamber(95%N2/5%CO2)for3hours. After OGD treatment, the neurons were incubated in NB without B27for scheduled time.3. Low concentration of acetylpuearin for OGD/R:as soon as the OGD were taken and replaced in NB without B27, acetylpuerarin was added to medium to0.1μM and incubated for scheduled time. Other treating processes were the same as that of the OGD/R groups. 4. Middle concentration of acetylpuearin for OGD/R:as soon as the OGD were taken and replaced in NB without B27, acetylpuerarin was added to medium to0.4μM and incubated for scheduled time. Other treating processes were the same as that of the OGD/R groups.5. High concentration of acetylpuearin for OGD/R:as soon as the OGD were taken and replaced in NB without B27, acetylpuerarin was added to medium to1.6μM and incubated for scheduled time. Other treating processes were the same as that of the OGD/R groups.OGR/R-12h,24h and36h, we use inverted microscope, MTT and DAPI to observed the morphology, viability and apoptosis of neurons. Through statistical analysis, we choose OGD/R-24h as a main point for the future research;The effect of acetylpuerarin on hippocampal neuron apoptosis induced by OGD/R-24h were determined with TUNEL (terminal-deoxynucleotidyl transferase mediated nick end labeling) staining.Results1. With inverted microscope, we observed, that the neurons refraction were decreased, projections were shorten, or even disappear, part of the cell had dissolved, scatterd nucleus.0.1μM,0.4μM and1.6μM acetylpuerarin can significantly improve the morphological injury after the OGD/R, acetylpuerarin show a neuron protection against OGD/R injury.2. OGD/R-12h,24h and36h decreased the vibility of neurons. Acetylpuerarin could improve neuronal viability, the value of high concentration of acetylpuerarin treatment increasing neuron survival rates was more obvious than those of middle and low concentrations of acetylpuerarin treatments.3. OGD/R treatment resulted in a time-dependent increase in numbers of apoptotic cells with a peak time at24h. Acetylpuerarin could decrease neuron apoptosis in high and middle concentration treatment except for OGD/R-12h, The low concentration of acetylpuerarin has no significant changes for all three scheduled time. 4. Acetylpuerarin with the concentration of0.1μM,0.4μM and1.6μM μM could inhibit the apoptosis of the injured neurons induced by OGD/R-24h significantly. The influence of acetylpuerarin on hippocampal neuronal cell death with OGD/R was determined with TUNEL staining. Results showed that acetylpuerarin decreased the apoptotic index.Conclusion1. OGD/R could damage the cultured hippocampal neurons at different points in vitro, decrease the cell viability and cause neurons apoptosis, the peak time of apoptosis is OGD/R-24h.2. Acetylpuerarin can alleviate neuronal damage, enhance the vitality of neurons and maintain the structural integrity of neurons.3. Acetylpuerarin in the concentration of0.1μM,0.4μM and1.6μM could reduce the number of apoptosis positive cells, show a protective effect on the ischemic-reperfusion injury. Part Ⅱ Acetylpuerarin reduces hippocampal neuron apoptosis following oxygen-glucose deprivation/reperfusion in ratsObjectiveUsing the point from the first part, the effect of acetylpuerarin on the apoptosis related factors were detection from the change of molecular level, provide effective theory basis for the clinical treatment of cerebral ischemia/reperfusion injury.MethodHippocampal neurons cultured and model established as same as the part Ⅰ. The point of experiment were OGD/R-6h,24h. The groups were oxygen-glucose deprivation and reperfusion (O/R) group, Low concentration of acetylpuearin for OGD/R, middle concentration of acetylpuearin for OGD/R, high concentration of acetylpuearin for OGD/R, respectively.The activities of caspase-8and caspase-3of hippocampal neurons were detected by fluorescence spectrophotometry; The effect of acetylpuerarin on OGD/R-24-h-induced Fas-L, FADD, TNF-αand Hsp70were determined by western blot on cultured hippocampal neuron in vitro.It were used the methods of RT-PCR to investigate the level of HIF-lamRNA, NF-κB mRNA and P53mRNA and the influence of acetylpuerarin on hippocampal neuronal cell death with OGD/R-24h.Results1.0.1μM,0.4μM and1.6μM acetylpuerarin could inhibit the activities of caspase-8and caspase-3with OGD/R. The examination with fluorescence spectrophotometry method showed that acetylpuerarin treatment led to an decrease in the expression of enzymatic activity of caspase-8and caspase-3.2. The expression of Fas-L, FADD and TNF-awere examined through western blotting and the results showed0.1μM,0.4μM and1.6μMacetylpuerarin could decrease the Fas-L, FADD and TNF-α after OGD/R was added to the cultured.3. After OGD/R-24h, the level of Hsp70increased, whereas Hsp70significantly increased when neurons cultured with0.1μM,0.4μM and1.6μM acetylpuerarin. 4.0.1μM,0.4μM and1.6μM acetylpuerarin could inhibited the expression of HIF-1α mRNA and P53mRNA24h after OGD/R was added to the cultured cell. Through the reverse transcriptase polymerase chain reaction, we find the area and the mean gray value of HIF-1α mRNA, NF-κBmRNA and P53mRNA all decrease.ConclusionOGD/R could injuried hippocampal neurons at different point, increasing the viability of caspase-3and8, the level of Fas-L, FADD and TNF-aprotein were increasing, decreasing the level of Hsp70, increasing the expressing of HIF-la mRNA, NF-κB mRNA and P53mRNA. Acetylpuerarin had protective effects against injury induced by OGD/R in hippocampal neurons via attenuating the level of Fas-L, FADD and TNF-α, and down-regulating the activities of caspase-8and caspase-3, as well as the level of HIF-1α mRNA, NF-κBmRNA and P53mRNA, and enhanced the expression of the Hsp70. These results show that the protection of acetylpuerarin is related its anti-apoptosis and effect some apoptosis related factors.更多还原