加减薯蓣丸对血管性痴呆大鼠海马神经重构干预的研究

发布时间：2018-04-03 21:40

  本文选题：加减薯蓣丸　切入点：血管性痴呆　出处：《湖北中医药大学》2017年博士论文

【摘要】：[目的]神经重构是血管性痴呆(Vascular dememtia,VD)发生及维持的重要机制,包括神经元丢失、神经胶质增生、轴突及树突重构、突触联系紊乱及变性等。本课题通过研究加减薯蓣丸对VD大鼠海马神经细胞凋亡与炎症的干预,探索加减薯蓣丸减少海马椎体元丢失及预防神经重构的作用;研究加减薯蓣丸对VD大鼠海马神经胶质细胞中少突胶质细胞及星型胶质细胞增生干预及促进神经纤维再生的作用,探索其对VD神经胶质及神经纤维重构的干预;研究加减薯蓣丸对VD大鼠海马神经突触的保护并促进突触重建作用,探索其增强神经突触联系,预防突触重构的作用。从干预神经重构的三个方面的作用进一步探讨加减薯蓣丸对VD的治疗及改善智能的机制。[方法]选取SPF级SD雄性大鼠40只,分为假手术组、模型组与药物组三组。模型组与药物组采用改良双血管阻断法制备VD模型,假手术组除不阻断双侧颈总动脉外余操作同手术组。手术一周后药物组以加减薯蓣丸浓缩制剂(由湖北省中医院制剂室制备,剂量按1ml/100g体重)灌胃治疗45天,另两组以生理盐水灌胃,剂量、疗程同药物组。记录三组大鼠在术前、术后与取材前体重变化,观察大鼠精神、饮食、进水等情况。以水迷宫行为学实验(包括定位航行实验与空间探索实验)评定三组大鼠学习记忆能力。水迷宫实验结束后处死大鼠,对大鼠海马取材,取新鲜海马组织以进行电镜观察及RT-PCR检测;取灌注海马组织以进行HE染色与免疫组化染色。HE染色重点观察大鼠海马CA1区组织改变,包括椎体细胞层、辐射层、腔隙层、及多形细胞层改变;免疫组化测定海马CA1区Bax、Bcl-2与NF-κB以分析VD大鼠海马神经细胞凋亡与炎症调控,反应神经保护与预防神经重构;检测Olig-2、GFAP以分析少突胶质细胞与星型胶质细胞增生情况以反映海马神经胶质重构;检测SYP、PSD-95、MAP-2分析海马突触蛋白改变以研究突触生成、突触破坏与重建;采用RT-PCR分析海马组织GAP-43m RNA表达以进一步研究神经再生与突触重建的基因调控;采用透射电镜重点观察突触超微结构的改变。[结果]一般情况观察:在改良2-VO手术结束后,手术组大鼠进食、进水明显减少,精神差、活跃程度明显低下、嗜睡等现象。一周后体重比假手术组明显下降(F值:19.535,P:0.000),术后经过2周左右的康复及喂养,手术组快速恢复,在进食、饮水等方面明显增多,经过45天的饲养,在取材当天称重,三组体重已无差异(F值:0.010,P:0.990)。水迷宫实验中,各组逃避潜伏期及逃避距离在实验最初两天相互间没有统计学差异,第三天开始,假手术组逃避距离及逃避潜伏期与模型组相比下降较多,达到显著性差异(P0.05)。第四天药物组逃避距离及逃避潜伏期下降较快,与假手术组接近,二者与模型组相比达到显著性差异(P0.05,P0.05)。在空间搜索实验中,与模型组相比,假手术组及药物组跨越平台次数均较模型组多,达到显著性差异(P0.01及P0.05)、平台所在象限游行时间及游行距离多,达到显著性差异(P0.05及P0.01)。三组总游行距离及速度没有统计学差异。HE染色观察:假手术组海马CA1区锥体细胞排列3-5层,层次分明,相邻细胞排列整齐致密,细胞质均质红染,胞核大而圆,核仁清晰。辐射层呈较规则的辐射状排列,胶质细胞较少,可见呈圆形核的少突胶质细胞。分子层排列致密,细胞间腔隙较少。模型组海马CA1区细胞数目减少,细胞排列紊乱稀疏,椎体细胞分1-3层,细胞形态不完整,胞浆内可见空泡,细胞核深染、固缩,呈三角形或不规则形,核仁不明显,部分细胞核消失。辐射层明显疏松,极性紊乱,延续到分子层和腔隙层。腔隙层纤维错乱,缝隙较多较大。可见胶质细胞增生。药物组海马CA1区神经元排列较为整齐,椎体细胞明显增多,排列5-7层,结构相对正常。辐射层致密,排列较规整。分子层及腔隙层可见大量椎体细胞及胶质细胞增生。RT-PCR测定GAP-43m RNA表达来看,假手术组表达最低,模型组较之明显升高,达到极显著性差异(P0.01)。而药物组进一步升高,与假手术组及模型组相比均达到统计学差异(P0.01及P0.05)。免疫组化结果:1.凋亡及炎症指标:Bax表达:模型组最高,与药物组及假手术组相比均达到极显著性差异(P�d0.01,P0.01);假手术组最低,与药物组相比达到极显著性差异(P�d0.01)。Bcl-2:药物组Bcl-2表达最高,与假手术组及模型组相比均达到极显著性差异(P�d0.01,P0.01);假手术组Bcl-2表达比模型组高,达到极显著性差异(P�d0.01)。NF-κB:药物组和假手术组均明显低于模型组,达到极显著性差异(P�d0.01),药物组高于假手术组,达到显著性差异(P�d0.05)。2.神经胶质细胞增生指标:Olig-2表达:假手术组表达最多,药物组次之,二者与模型组相比达到极显著性差异(P�d0.01,P0.01);药物组与假手术组相比达到显著性差异(P�d0.05)。模型组表达最低。GFAP表达:假手术组最低,与另外两组相比均有极显著性差异(P�d0.01,P0.01);药物组次之,与模型组相比达到极显著性差异(P�d0.01);模型组表达最高。3.突触相关蛋白表达:MAP-2表达:药物组及假手术组高于模型组,达到显著性差异(P�d0.01,P�d0.05)。药物组高于假手术组,达到极显著性差异(P�d0.01);SYP表达:药物组表达明显高于模型组及假手术组,达到极显著性差异(P�d0.01,P0.01);假手术组也明显高于模型组(P�d0.01);PSD-95表达:药物组及假手术组明显高于模型组,达到极显著性差异(P�d0.01,P0.01);药物组最高,与假手术组相比,也达到极显著性差异(P�d0.01)。突触的电镜观察:假手术组:突触间隙清晰,突触前后膜轮廓完整。突触小泡较多,突触曲面规则,突触前后膜吻合良好,后膜可见明显增厚。线粒体完整,内质网上核糖体较多。模型组:突触间隙模糊,突触前后膜肿胀,空化。难以区分突触前后膜。突触小泡减少,线粒体固缩,较少。内质网脱颗粒。药物组:突触前后膜结构完整。突触间隙清晰可见,突触小泡较多,线粒体结构完整,含量丰富。内质网颗粒较多。[结论]加减薯蓣丸具有明显治疗VD作用,通过本课题研究表明其作用机制在于:1.通过在缺血环境中对抗海马神经元凋亡、抑制神经炎症、促进神经元再生、保护并恢复神经-血管单元固有构筑,预防神经重构;2.抑制星型胶质细胞增生,促进少突胶质细胞增生及轴突髓鞘化,干预胶质重构以保证神经信息的高效传导;3.保护缺血情况下的突触损伤与变性、促进突触再生与重建、保证突触可塑性及神经信息准确传递。
[Abstract]:[Objective] nerve reconstruction of vascular dementia (Vascular dememtia VD) is an important mechanism for generating and maintaining, including neuronal loss, gliosis, axonal and dendritic remodeling, synapse disorder and degeneration. Through the research and the Shuyuwan on God in the hippocampus of VD rats by cell apoptosis and inflammation of the intervention, explore modified Shuyuwan reduce hippocampal pyramidal element loss and the preventive effect of neural remodeling; to study the effect of Shuyuwan less proliferation of glial cells and oligodendrocytes and star of astrocytes in the hippocampus of VD rats in the intervention and promote the regeneration of nerve fibers, to explore the intervention of VD glial and nerve fiber remodeling; protection of Dioscorea modified pill on synapses of hippocampal nerve of VD rats and explore its role in promoting synaptic reconstruction, enhanced synaptic connections, prevent synaptic remodeling. From three aspects of neural remodeling intervention With the further study mechanism. Methods of treatment and improve intelligence of Dioscorea modified Pill on VD's SPF] selected 40 male SD rats were divided into sham operation group, model group and drug group three groups. Model group and drug group using a modified double vessel occlusion model of VD by the legal system, the sham operation group was not blocked by bilateral carotid artery operation with surgery group. More than a week after the surgery to add medicine group Shuyuwan concentrated preparation (preparation, preparation by Hubei Provincial Traditional Chinese Medical Hospital preparation room according to the dose of 1ml/100g body weight) by gavage for 45 days, the other two groups with normal saline, dose, treatment with the drug group. The rats in the three groups in operation records before and after surgery and were taken before the change of weight, rats spirit, diet, water and so on. The water maze (including place navigation test and spatial probe test) ability of learning and memory of rats. The end of the three groups were evaluated after water maze test rats were sacrificed on rat hippocampus Based in rathippocampus for electron microscopy and RT-PCR assay; hippocampus tissue perfusion with HE staining and immunohistochemical staining.HE key changes were observed in CA1 region of hippocampus tissue of rats, including the pyramidal cell layer, radiation layer, lacunar layer, and polymorphic layer change; immunohistochemical determination of Bax in hippocampal CA1 region Bcl-2, NF- and B to analyze the kappa nerve cell apoptosis and inflammatory reaction in hippocampus of rats with VD control, neural protection and prevention of neural remodeling; detection of Olig-2, GFAP in the analysis of oligodendrocyte and astrocyte hyperplasia to reflect hippocampal glial remodeling; detection of SYP, PSD-95, MAP-2 analysis of synaptic protein in the hippocampus to generate change study on synapses, synaptic damage and reconstruction; RT-PCR was used to analyze the expression of RNA gene in hippocampus of GAP-43m to further study on the regulation of nerve regeneration and synaptic reconstruction were observed by transmission electron microscopy; synaptic junction Change the structure of the general situation. Results: at the end of the observation after modified 2-VO operation, operation group rats eating water significantly reduced, poor spirit, active degree is obviously low, drowsiness phenomenon. One week after the weight than in the sham operation group decreased significantly (F-measure: 19.535, P:0.000), postoperative rehabilitation and after feeding about 2 weeks, operation group, fast recovery, eating, drinking and feeding increased significantly after 45 days, in the day were weighed, has no difference between the three groups (F-measure weight: 0.010, P:0.990). The water maze test, the escape latency and escape distance in the first two days of each other without experiment statistical differences between the start of the third day, the sham operation group escape distance and escape latency compared with the model group decreased more significant difference (P0.05). The fourth day drug group escape distance and the escape latency decreased rapidly, and close to the sham operation group, the two phase compared with the model group Significant differences (P0.05, P0.05) in the search space. In the experiment, compared with the model group, sham operation group and drug group over platform times compared with model group, significant difference (P0.01 and P0.05), the platform quadrant time and distance parade parade, significant difference (P0.05 and P0.01). The three groups had no statistically significant differences between the parade distance and speed.HE staining observation: sham operation group of pyramidal cells in hippocampal CA1 region are arranged in 3-5 layers, distinct, adjacent cells arranged in dense cytoplasm, homogeneous red staining, large round nucleus, clear nucleolus. Radiation layer radiating a regular arrangement of glial cells less visible, circular nucleus oligodendrocytes. Molecular layer of dense, intercellular space is less. The model group in hippocampus CA1 area of reducing the number of cells, cells arranged irregularly sparse, pyramidal cells are divided into 1-3 layers, the cell morphology is not complete, cytoplasmic vacuoles, fine Hyperchromatic nucleus, pyknosis, triangular or irregular, nucleolus, some nuclei disappeared. Radiation layer was loose, polarity disorder, extended to the molecular layer and lacunar layer. Lacunar layer fiber disorder, gap larger. Visible glial cell proliferation. Groups of neurons in hippocampal CA1 region of drugs were well arranged, pyramidal cells increased significantly, arrangement of 5-7 layer structure is relatively normal. The radiation layer is dense, regular arrangement. The molecular layer and lacunar layer shows a large number of pyramidal cells and glial cell proliferation was determined by.RT-PCR GAP-43m RNA expression, the sham operation group was the lowest, compared with the model group increased significantly and reached the extremely significant difference (P0.01). The drug group increased, compared with the sham operation group and model group were statistically different (P0.01 and P0.05). The results of immunohistochemistry: 1. apoptosis and inflammatory index: the expression of Bax: compared with the model group, drug group and sham operation group 鍧囪揪鍒版瀬鏄捐憲鎬у樊寮，

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