【作者】 许贤平；

【导师】 杨期东； 杨晓苏；

【作者基本信息】 中南大学 ， 神经病学， 2010， 博士

【摘要】 背景及目的：脑卒中具有高发病率、高死亡率、高致残率的临床特点,缺血性卒中为其主要类型,严重危害着人类的健康。脑缺血神经元死亡的分子机制特别复杂,目前已发现三条主要的细胞通路与其有关,分别为谷氨酸受体的过度激活、氧化应激及细胞凋亡。LIM蛋白是一类包含有2个或以上LIM结构域的蛋白,LMO4为最近发现的核转录协作因子,属于LIM蛋白家族的一员,通过对转录因子的激活或抑制来调控其他蛋白的功能,在基因表达、细胞分化和发育、细胞骨架的形成中发挥重要作用。pCREB、pStat3也属于核转录因子,部分研究表明二者对脑缺血神经元损伤有保护作用。在脑缺血的细胞模型中,LMO4通过调节IL-6/Stat3细胞信号通路促进体外缺氧神经细胞的存活,而pCREB又可作为上游成分调节LMO4的转录活性。本研究拟在脑缺血再灌注模型中观察LMO4、pCREB、pStat3在缺血半暗带的表达变化及定位,并探讨LMO4与后两者之间的关系及其对脑缺血细胞存活的作用,为缺血性卒中脑保护药物的开发提供新的分子靶点。方法：健康成年雄性Sprague-Dawley(SD)大鼠84只,随机分为假手术组及缺血再灌注1h、3h、6h、12h、24h、48h组,每组12只,6只用于分子生物学检测,余6只用于组织学实验。线栓法制备局灶性大脑中动脉闭塞(MCAO)模型,缺血2小时后恢复再灌住,以Longa5级标准评分法评价神经功能缺损,分别在各再灌注时间点处死动物。常规HE染色观察组织病理学改变；尼氏染色观察神经元的存活；Map2抗体染色观察神经元树突；TUNEL法检测缺血脑组织的凋亡细胞；免疫荧光检测、LMO4、pCREB、pStat3的在缺血半暗带皮层细胞的表达数量及部位；免疫荧光双标观察LMO4与Map2、pCREB、pStat3、TUNEL阳性细胞之间以及pCREB.pStat3与NEUN的共表达情况。RT-PCR法测定假手术组及再灌后不同时间点LMO4mRNA在缺血半暗带的表达水平；western-blot法测定假手术组及再灌后不同时间点LMO4和pStat3、pCREB三分子在缺血半暗带中的蛋白表达水平。结果：①模型成功率为76.60%,死亡率为13.83%,主要死亡原因为蛛网膜下腔出血、脑水肿；脑组织TTC染色显示成功模型大鼠出现皮层和纹状体梗死,而假手术组大鼠未见梗死。②模型组HE染色可见典型的坏死灶、神经元丢失和组织水肿,梗死区细胞总数目呈减少趋势,假手术组细胞形态正常；③模型组尼氏染色可见细胞肿胀,尼氏体消失,缺血半暗带皮层区存活神经元数目随再灌时间逐渐减少(P<0.05),48小时最低(P<0.01),假手术组神经元形态正常；④模型组树突长度变短,直径变小,缺血半暗带皮层区MAP2阳性细胞OD值随再灌时间逐渐减少,48小时最低(P<0.01),假手术组神经元树突形态正常；⑤TUNEL阳性细胞主要分布在梗死周围区,凋亡细胞数再灌后6小时开始增多(P<0.05),24小时达高峰,一直持续到48小时(P<0.01)；⑥与假手术组比较,缺血侧半暗带皮层组织LMO4mRNA水平再灌后3小时开始升高,24小时达高峰,48小时明显下降(P<0.05或P<0.01)；⑦与假手术组比较,缺血侧半暗带皮层组织LMO4蛋白水平再灌后3小时开始升高,24小时达高峰,48小时明显下降,pCREB、pStat3蛋白水平6小时开始升高,24小时达高峰,48小时逐渐下降(P<0.05或P<0.01)；⑧与假手术组比较,LMO4、pCREB、pStat3阳性细胞数再灌后6小时开始升高,24小时达高峰,48小时显著下降(P<0.05或P<0.01)；⑨免疫荧光双标显示LMO4和pCREB仅表达于神经元,pStat3在神经元和胶质细胞均可表达；LMO4主要表达于细胞核,少量位于细胞浆,pCREB和pStat3仅表达于细胞核；在缺血半暗带皮层区,LMO4与TUNEL阳性细胞呈分离表达,LMO4与pCREB完全共表达,而与pStat3部分共表达。结论：①脑缺血再灌后诱导LMO4、pCREB及pStat3表达升高,并呈动态变化趋势,可能为神经细胞对缺血再灌注损伤的一种内部适应性保护机制。②LMO4可能通过与pCREB之间的相互作用及调节pStat3性来促进缺血神经元的存活。背景及目的：依达拉奉是一种合成的新型自由基清除剂和抗氧化剂,可改善脑缺血后引起的神经功能缺损,并且对迟发性神经细胞死亡也有抑制作用。依达拉奉对脑缺血的保护作用较为明确,但其具体的分子机制尤其是抗凋亡的机制并不十分清楚。凋亡为一种基因调控的细胞程序性死亡,而LMO4作为核转录协助因子可调节基因的表达。LMO4基因敲除小鼠出现各种发育障碍,提示其对上皮细胞及神经细胞的抗凋亡作用。本研究拟进一步探讨依达拉奉对脑缺血的保护机制及初步探讨其对LMO4表达的影响。方法：健康成年雄性SD大鼠36只,随机分为假手术对照组(n=12)、脑缺血模型组(n=12)及依达拉奉治疗组(n=12)。以线栓阻断一侧大脑中动脉构建局灶性脑缺血模型,缺血2小时后恢复再灌住,再灌注24小时后处死动物,药物组在脑缺血再灌注后即刻腹腔注射依达拉奉3mg／kg,模型组注射等量的生理盐水。常规HE染色、尼氏染色观察病理学改变并评价神经元的存活;TUNEL法检测缺血侧脑组织的凋亡细胞；RT-PCR、western-blot、免疫荧光检测三组缺血半暗带皮层LMO4蛋白和mRNA的表达水平,并进行组间比较。结果：①假手术组细胞形态基本正常,模型组和药物组均有细胞坏死,但后者程度减轻,形态正常的细胞数较前者增多(P<0.01)；尼氏染色示缺血半暗带皮层存活神经元数目药物组较模型组显著增多(P<0.05)：②假手术组几乎无TUNEL阳性细胞,模型组和药物组梗死周围皮层均可见TUNEL阳性细胞,但后者明显减少(P<0.01)；③假手术组有少量LMO4mRNA和蛋白的表达,模型组皮层半暗带区较多LMO4mRNA和蛋白的表达,与模型组比较,药物组表达水平显著提高(P<0.05或P<0.01)；④假手术组可见少量LMO4阳性细胞,模型组缺血半暗带皮层可见大量LMO4阳性细胞,与模型组比较,药物组LMO4阳性细胞数更多(P<0.01)。结论：依达拉奉促进LMO4的表达升高,可能是其脑保护作用机制之一。更多还原

【Abstract】 Background and objective:Stroke is characterized by high incidence, high mortality and high disability, whereas ischemic stroke has been the most common type and severely harmful to human health. The molecular mechanism of neuronal death is extremely complex under cerebral ischemia, three main cellular pathways of which has been found including over activation of glutamate receptor, oxidative stress and apoptosis. The LIM proteins are characterized by 2 or more LIM domains. LMO4 is a recently discovered transcription co-factor that belongs to LIM family. LMO4 could regulate the function of other proteins through activating or suppressing the corresponding transcription factors, playing crucial roles in gene expression, cell differentiation and development, and the formation of cytoskeleton. Both pCREB and pStat3 belongs to nuclear transcription factors also, partial studies have demonstrated that both contribute to protection of injured neurons in cerebral ischemia. In the cell models of cerebral ischemia, LMO4 can facilitate survival of hypoxic neurons in vitro by regulating the IL-6/Stat3 signal pathway, and pCREB mediates the transcriptional activity of LMO4 as an upstream molecular element. In this study we will speculate the temporal expression and localization of LMO4, pCREB and pStat3 in animal models of cerebral ischemia-reperfusion, and explore the relation of LMO4 with pCREB and pStat3 and the role of LMO4 in ischemic neuronal survival, hence providing new molecular target for introducing drugs of ischemic stroke.Methods:84 Sprague-Dawley rats of healthy adult male were randomly divided into seven of sham operation group, and ischemic-reperfusion 1h,3h,6h,12h,24h,48h group. Each group had 12 rats, of which 6 were used for molecular biology study and the rest for histology study. Suture methods were used to make the focal middle cerebral artery occlusion model and the ischemic brain was reperfused 2 hours after occlusion. The neurological deficit score was rated by Longa scale. Animals were killed at various reperfusion intervals. Routine HE staining was used to speculate the histopathological change. Nissle staining was used to evaluate the neuronal survival. MAP2 staining was used to detect the dendrite. TUNEL methods was used to detect the apoptotic cell. Immunoflurence was used to detect the quantity and location of、LMO4、pCREB、pStat3 on the penumbra cortex. Double immunoflurence was used to speculate the colocalization between LMO4 and MAP2, pCREB, pStat3, TUNEL positive cells respectively and between NEUN and pCREB, pStat3 respectively. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the LMO4 mRNA level of penumbra tissue in sham group and ischemic subgroups. Western-blot was used to detect the LMO4, pCREB and pStat3 protein level of penumbra tissue in sham group and ischemic subgroups.Results:①the qualified rate was 76.60% for model inclusion and the 48h mortality rate was 13.83%. Death was mainly caused by hemorrhage and brain edema. TTC staining of the brain tissue revealed cortex and caudate putamen infarction in the qualified rat models and no infarction in the sham group rats.②HE staining revealed typical necrosis, neuronal loss, tissue edema in ischemic models, number of total cells decreased in infarction area, whereas cells in the sham group kept intact.③Nissle staining revealed that cells swelled and nissle body of the neuronal cells disappeared, the number of surviving neuron decreased with perfusion time on the penumbra cortex (P<0.05), reached the lowest level at 48h (P<0.01), whereas cells in the sham group kept intact.④The length and diameter of the dendrites decreased, OD signal of the MAP2 positive cells decreased with perfusion time on the penumbra cortex in ischemic group, reached the lowest level at 48h (P< 0.01), whereas the dendrites in the sham group kept intact.⑤TUNEL positive cells were mainly located in the peri-infarction area, the number of apoptotic cells increased at 6h after reperfusion (P< 0.05), peaked at 24h, and persisted at 48h (P<0.01).⑥Compared to the sham group, LMO4 mRNA level of the penumbra cortex in ischemic group increased at 3h after reperfusion, peaked at 24h, and obviously declined at 48h (P<0.05 or P<0.01);⑦Compared to the sham group, LMO4 protein level of the penumbra cortex in ischemic group increased at 3h after reperfusion, peaked at 24h, and obviously declined at 48h, pCREB and pStat3 protein level increased at 3h after reperfusion, peaked at 24h, and gradually declined at 48h (P<0.05 or P<0.01);⑧Compared to the sham group, the number of LMO4, pCREB, pStat3 positive cells in ischemic group increased at 6h after reperfusion, peaked at 24h, and gradually declined at 48h (P<0.05 or P<0.01);⑨Double immunoflurence indicated that both LMO4 and pCREB were only expressed in neurons, whereas pStat3 was expressed in neurons and glial cells; LMO4 was mainly localized in nucleus, a little part in cytoplasm, both pCREB and pStat3 were only localized in nucleus; the expression of LMO4 was segregated from the TUNEL positive cells in the penumbra cortex in ischemic group; LMO4 was completely coexpressed with pCREB, whereas was partially coexpressed with pStat3.Conclusions:①Cerebral ischemia reperfusion induces the overexpression of LMO4, pCREB and pStat3, with a dynamic profile, which may be an endogenous adaptive protective mechanism of neuronal cells triggered by ischemia reperfusion injury.②LMO4 may contribute to ischemic neuronal survival through interacting with pCREB and regulating the activity of pStat3. Background and objective:Edaravone is a kind of newly synthesized radical scavenger and anti-oxidant, and can improve the neurological deficit after cerebral ischemia. The neuroprotective role of edaravone in cerebral ischemia has been proved by many studies, however, the exact molecular mechanism for which remains unclear till now, especially the anti-apoptotic mechanism. Apoptosis is a kind of programmed cell death highly regulated by genes. LMO4 can mediate the expression of many other genes as a transcription co-factor. The LMO4 deficient mouse presents with all kinds of development deficits, suggesting its anti-apoptotic effect on epithelial cell and neuronal cell. This study was to further explore the molecular mechanism of edaravone protecting the ischemic neurons and the effect of edaravone on LMO4 expression.Methods:36 SD rats of healthy adult male were randomly divided into three of sham control group (n=12), ischemic model group (n=12) and edaravone treated group (n=12). Focal cerebral ischemia model was made by suture occluding one side of middle cerebral artery. Blood flow was restored after 2 hours of occlusion and the animals were killed at 24h after perfusion. Rats received intraperitoneal injections of edaravone for the drug group and same volume of physiological saline for the model group upon reperfusion. Routine HE and Nissle staining was used to speculate the histopathological change and evaluate the neuronal survival. TUNEL methods was used to detect the apoptotic cell. RT-PCR, western-blot and immunoflurence were used to detect the levels of LMO4 protein and mRNA on the penumbra cortex in the three groups, the final data were compared among different groups.Results:①Cellular morphology was normal in the sham group. Necrotic cell was seen both in the model group and the drug group, but less severe in the latter, with morphologically normal cells more common in the latter (P<0.01); Nissle staining revealed that the number of surviving neurons on the penumbra cortex was significantly higher in the drug group than in the model group (P<0.05);②No TUNEL positive cell was seen in the sham group, but was seen both in model group and the drug group, with the number much less in the latter (P<0.01);③The levels of LMO4 protein and mRNA were low in the sham group, however, that of the penumbra cortex were high in the model group and significantly higher in the drug group (compared to the model group, P<0.05 or P<0.01);④A few LMO4 positive cells were seen in the sham group, large number of LMO4 positive cells were seen on the peri-infarction cortex in the model group, and much larger were seen in the drug group (compared to the model group, P<0.01).Conclusions:Edaravone can enhance the overexpression of LMO4, which may be one of the mechanisms underlying the neuroprotection of this drug.更多还原