【作者】 李庆云；

【导师】 李冀；

【作者基本信息】 黑龙江中医药大学 ， 方剂学， 2007， 博士

【摘要】 脑梗死（又称缺血性脑卒中）是严重危害人类健康的多发病和常见病，其病因繁多，病机复杂，地黄饮子是临床治疗缺血性中风的有效方剂。本课题研究地黄饮子对脑缺血再灌注损伤的脑保护作用及其作用机理，并对该方进行拆方研究，探讨地黄饮子及其方中补肾法和化痰法在治疗脑梗死作用机理等方面的配伍意义。目的：研究地黄饮子对脑缺血再灌注损伤脑保护作用的机理及配伍意义。方法：改良线栓法制备MCAO再灌注大鼠模型，将地黄饮子拆方（分为补肾组和化痰组），采用评分法和TTC染色法观察神经功能缺损恢复情况和脑梗死体积的变化；通过HE染色、TUNEL法和免疫组化法观察病理形态学变化、细胞凋亡及凋亡调控基因Bcl-2和NF-κBp65的表达；采用免疫组化法和RT-PCR法观察Nestin、SCF、PSA-NCAM和TGF-β₁的表达，探讨地黄饮子及其各拆方组对MCAO再灌注模型大鼠的脑保护作用及其作用机理。结果：（1）地黄饮子对神经功能缺损的恢复有明显地促进作用，拆方Ⅰ组优于拆方Ⅱ组（P＜0.05），二拆方组之间有协同作用。（2）地黄饮子能够显著减少脑梗死体积，拆方Ⅰ组优于拆方Ⅱ组（P＜0.05），二拆方组之间有协同作用。（3）HE染色光镜下观察MCAO再灌注模型大鼠脑组织病理形态学改变，可见地黄饮子能够减轻缺血性脑损伤神经细胞的损害，对脑缺血神经细胞起到保护作用。（4）地黄饮子有较好地抑制细胞凋亡的作用，拆方Ⅰ组优于拆方Ⅱ组（P＜0.05），二拆方组之间有协同作用。（5）地黄饮子可促进Bcl-2蛋白的表达，抑制NF-κBp65蛋白的表达，拆方Ⅰ组促进Bcl-2表达作用优于拆方Ⅱ组（P＜0.05），拆方Ⅰ组和拆方Ⅱ组抑制NF-κBp65表达作用相当（P＞0.05），二拆方组之间有协同作用。（6）地黄饮子能够促进Nestin的表达，拆方Ⅰ组优于拆方Ⅱ组（P＜0.05），二拆方组之间有协同作用。（7）地黄饮子可促进SCFmRNA、PSA-NCAMmRNA和TGF-β₁的表达，拆方Ⅰ组优于拆方Ⅱ组（P＜0.05），二拆方组之间有协同作用。结论：（1）地黄饮子对MCAO再灌注模型大鼠神经功能缺损的恢复有明显地促进作用，能够显著减少脑梗死体积，表明该方具有确切地脑保护作用。（2）地黄饮子有较好地抑制细胞凋亡的作用，可促进Bcl-2的表达，升高TGF-β₁的表达，抑制NF-κBp65的表达，表明其抑制神经细胞凋亡的作用与调控Bcl-2、TGF-β₁和NF-κBp65的表达有关。（3）地黄饮子能够促进Nestin的表达，表明该方有明显地促进神经干细胞增殖与分化的作用，并可促进SCFmRNA、PSA-NCAMmRNA和TGF-β₁的表达，表明其促进神经干细胞增殖与分化的作用与调控SCF、PSA-NCAM和TGF-β₁的表达有关。（4）通过拆方研究表明，补肾法和化痰法均有一定地脑保护作用，其中补肾法作用较突出。说明不同治法之间，其作用并非简单相加，而是一种协同作用，从而提示地黄饮子补肾法和化痰法配伍的合理性。更多还原

【Abstract】 Cerebral Infarction with various etiologies and complicated pathogenesis is the common and frequently encountered disease which seriously harms the health of human being,while Rehmanniae Potion is the effective prescription for treating the blood-lacking stroke.This topic studies the cerebral protection of Rehmanniae to ischemical reperfusion injury and Potion as well as its mechanism action.Besides,the topic also approaches the compatibility significance between the treatment of invigorating kidney and dissipating phlegm.Objective:To study the mechanism action and its compatibility significance on protection of Rehmanniae Potion to cerebral ischemical reperfusion injury.Method:Focal cerebral ischemia was induced by intraluminal middle cerebral artery （MCA） occlusion method using a nylon monofilament thread in rats. Rehmanniae Potion is separated into groups of invigorating kidney and dissipating phlegm.The neurologic impairment recuperation and cerebral infarction volume are observed with the score method and TTC staining method.HE staining and electron microscope were used to observe the changes of histomorphology.The expression of Bcl-2、NF-κBp65、TGF-β₁ and Nestin protein were measured by immunohistochemistry technique respectively.The expression of NestinmRNA、SCFmRNA、PSA-NCAMmRNA and TGF-β₁mRNA were measured by reverse transcription-polymerase chain reaction.Result:（1）Rehmanniae Potion has the obvious auxoaction on recuperation of neurologic impairment,and it can reduce cerebral infarction volume,lessen neurologic impairmen,repress apoptosis, promote Nestin, SCFmRNA、PSA-NCAMmRNA and TGF-β1 expression, which is of obvious difference with control group （ P<0.05）.GroupⅠsurpasses groupⅡ（P<0.05）and the two groups have the synergistic effect.（2）Rehmanniae Potion can promote Bcl-2 expression and inhibit NF-KBp65 expression which is of obvious difference with the control group（P<0.05）.GroupⅠsurpasses groupⅡon the promotion of Bcl-2 expression（P<0.05）,which has the correspondent action on NF-κBp65 repression with groupⅡ,the two groups have the synergistic effect.Conclusion:（1）Rehmanniae Potion has the obvious auxoaction on neurologic impairment recuperation and reduces cerebral infarction volume which indicates its certain action of brain protection.（2）Rehmanniae Potion can lessen neurologic impairment, repress apoptosis,promote Bcl-2 expression,heighten TGF-β₁ expression and inhibit NF-κBp65 expression,which indicates that the action on repressing apoptosis is related to the regulation on Bcl-2、TGF-β₁and NF-κBp65 expression.（3）Rehmanniae Potion can promote Nestin expression,which indicates that it can promote the nerve stem cell proliferation and differentiation,while it can promote SCFmRNA、PSA-NCAMmRNA and TGF-β₁ expression, which indicates that the action of promoting the nerve stem cell proliferation and differentiation is related to the regulation on SCF、PSA-NCAM and TGF-β₁ expression.（4）Both invigorating kidney and dissipating phlegm treatment have the brain protection action and invigorating kidney treatment is superior, which indicates that the different treatments are not a simple addition but a synergistic action which hints the rationality of compatibility.更多还原