本文選題：加減薯蕷丸　切入點(diǎn)：血管性癡呆　出處：《湖北中醫(yī)藥大學(xué)》2017年博士論文

【摘要】：[目的]神經(jīng)重構(gòu)是血管性癡呆(Vascular dememtia,VD)發(fā)生及維持的重要機(jī)制,包括神經(jīng)元丟失、神經(jīng)膠質(zhì)增生、軸突及樹突重構(gòu)、突觸聯(lián)系紊亂及變性等。本課題通過研究加減薯蕷丸對(duì)VD大鼠海馬神經(jīng)細(xì)胞凋亡與炎癥的干預(yù),探索加減薯蕷丸減少海馬椎體元丟失及預(yù)防神經(jīng)重構(gòu)的作用;研究加減薯蕷丸對(duì)VD大鼠海馬神經(jīng)膠質(zhì)細(xì)胞中少突膠質(zhì)細(xì)胞及星型膠質(zhì)細(xì)胞增生干預(yù)及促進(jìn)神經(jīng)纖維再生的作用,探索其對(duì)VD神經(jīng)膠質(zhì)及神經(jīng)纖維重構(gòu)的干預(yù);研究加減薯蕷丸對(duì)VD大鼠海馬神經(jīng)突觸的保護(hù)并促進(jìn)突觸重建作用,探索其增強(qiáng)神經(jīng)突觸聯(lián)系,預(yù)防突觸重構(gòu)的作用。從干預(yù)神經(jīng)重構(gòu)的三個(gè)方面的作用進(jìn)一步探討加減薯蕷丸對(duì)VD的治療及改善智能的機(jī)制。[方法]選取SPF級(jí)SD雄性大鼠40只,分為假手術(shù)組、模型組與藥物組三組。模型組與藥物組采用改良雙血管阻斷法制備VD模型,假手術(shù)組除不阻斷雙側(cè)頸總動(dòng)脈外余操作同手術(shù)組。手術(shù)一周后藥物組以加減薯蕷丸濃縮制劑(由湖北省中醫(yī)院制劑室制備,劑量按1ml/100g體重)灌胃治療45天,另兩組以生理鹽水灌胃,劑量、療程同藥物組。記錄三組大鼠在術(shù)前、術(shù)后與取材前體重變化,觀察大鼠精神、飲食、進(jìn)水等情況。以水迷宮行為學(xué)實(shí)驗(yàn)(包括定位航行實(shí)驗(yàn)與空間探索實(shí)驗(yàn))評(píng)定三組大鼠學(xué)習(xí)記憶能力。水迷宮實(shí)驗(yàn)結(jié)束后處死大鼠,對(duì)大鼠海馬取材,取新鮮海馬組織以進(jìn)行電鏡觀察及RT-PCR檢測(cè);取灌注海馬組織以進(jìn)行HE染色與免疫組化染色。HE染色重點(diǎn)觀察大鼠海馬CA1區(qū)組織改變,包括椎體細(xì)胞層、輻射層、腔隙層、及多形細(xì)胞層改變;免疫組化測(cè)定海馬CA1區(qū)Bax、Bcl-2與NF-κB以分析VD大鼠海馬神經(jīng)細(xì)胞凋亡與炎癥調(diào)控,反應(yīng)神經(jīng)保護(hù)與預(yù)防神經(jīng)重構(gòu);檢測(cè)Olig-2、GFAP以分析少突膠質(zhì)細(xì)胞與星型膠質(zhì)細(xì)胞增生情況以反映海馬神經(jīng)膠質(zhì)重構(gòu);檢測(cè)SYP、PSD-95、MAP-2分析海馬突觸蛋白改變以研究突觸生成、突觸破壞與重建;采用RT-PCR分析海馬組織GAP-43m RNA表達(dá)以進(jìn)一步研究神經(jīng)再生與突觸重建的基因調(diào)控;采用透射電鏡重點(diǎn)觀察突觸超微結(jié)構(gòu)的改變。[結(jié)果]一般情況觀察:在改良2-VO手術(shù)結(jié)束后,手術(shù)組大鼠進(jìn)食、進(jìn)水明顯減少,精神差、活躍程度明顯低下、嗜睡等現(xiàn)象。一周后體重比假手術(shù)組明顯下降(F值:19.535,P:0.000),術(shù)后經(jīng)過2周左右的康復(fù)及喂養(yǎng),手術(shù)組快速恢復(fù),在進(jìn)食、飲水等方面明顯增多,經(jīng)過45天的飼養(yǎng),在取材當(dāng)天稱重,三組體重已無差異(F值:0.010,P:0.990)。水迷宮實(shí)驗(yàn)中,各組逃避潛伏期及逃避距離在實(shí)驗(yàn)最初兩天相互間沒有統(tǒng)計(jì)學(xué)差異,第三天開始,假手術(shù)組逃避距離及逃避潛伏期與模型組相比下降較多,達(dá)到顯著性差異(P0.05)。第四天藥物組逃避距離及逃避潛伏期下降較快,與假手術(shù)組接近,二者與模型組相比達(dá)到顯著性差異(P0.05,P0.05)。在空間搜索實(shí)驗(yàn)中,與模型組相比,假手術(shù)組及藥物組跨越平臺(tái)次數(shù)均較模型組多,達(dá)到顯著性差異(P0.01及P0.05)、平臺(tái)所在象限游行時(shí)間及游行距離多,達(dá)到顯著性差異(P0.05及P0.01)。三組總游行距離及速度沒有統(tǒng)計(jì)學(xué)差異。HE染色觀察:假手術(shù)組海馬CA1區(qū)錐體細(xì)胞排列3-5層,層次分明,相鄰細(xì)胞排列整齊致密,細(xì)胞質(zhì)均質(zhì)紅染,胞核大而圓,核仁清晰。輻射層呈較規(guī)則的輻射狀排列,膠質(zhì)細(xì)胞較少,可見呈圓形核的少突膠質(zhì)細(xì)胞。分子層排列致密,細(xì)胞間腔隙較少。模型組海馬CA1區(qū)細(xì)胞數(shù)目減少,細(xì)胞排列紊亂稀疏,椎體細(xì)胞分1-3層,細(xì)胞形態(tài)不完整,胞漿內(nèi)可見空泡,細(xì)胞核深染、固縮,呈三角形或不規(guī)則形,核仁不明顯,部分細(xì)胞核消失。輻射層明顯疏松,極性紊亂,延續(xù)到分子層和腔隙層。腔隙層纖維錯(cuò)亂,縫隙較多較大�？梢娔z質(zhì)細(xì)胞增生。藥物組海馬CA1區(qū)神經(jīng)元排列較為整齊,椎體細(xì)胞明顯增多,排列5-7層,結(jié)構(gòu)相對(duì)正常。輻射層致密,排列較規(guī)整。分子層及腔隙層可見大量椎體細(xì)胞及膠質(zhì)細(xì)胞增生。RT-PCR測(cè)定GAP-43m RNA表達(dá)來看,假手術(shù)組表達(dá)最低,模型組較之明顯升高,達(dá)到極顯著性差異(P0.01)。而藥物組進(jìn)一步升高,與假手術(shù)組及模型組相比均達(dá)到統(tǒng)計(jì)學(xué)差異(P0.01及P0.05)。免疫組化結(jié)果:1.凋亡及炎癥指標(biāo):Bax表達(dá):模型組最高,與藥物組及假手術(shù)組相比均達(dá)到極顯著性差異(P㩳0.01,P0.01);假手術(shù)組最低,與藥物組相比達(dá)到極顯著性差異(P㩳0.01)。Bcl-2:藥物組Bcl-2表達(dá)最高,與假手術(shù)組及模型組相比均達(dá)到極顯著性差異(P㩳0.01,P0.01);假手術(shù)組Bcl-2表達(dá)比模型組高,達(dá)到極顯著性差異(P㩳0.01)。NF-κB:藥物組和假手術(shù)組均明顯低于模型組,達(dá)到極顯著性差異(P㩳0.01),藥物組高于假手術(shù)組,達(dá)到顯著性差異(P㩳0.05)。2.神經(jīng)膠質(zhì)細(xì)胞增生指標(biāo):Olig-2表達(dá):假手術(shù)組表達(dá)最多,藥物組次之,二者與模型組相比達(dá)到極顯著性差異(P㩳0.01,P0.01);藥物組與假手術(shù)組相比達(dá)到顯著性差異(P㩳0.05)。模型組表達(dá)最低。GFAP表達(dá):假手術(shù)組最低,與另外兩組相比均有極顯著性差異(P㩳0.01,P0.01);藥物組次之,與模型組相比達(dá)到極顯著性差異(P㩳0.01);模型組表達(dá)最高。3.突觸相關(guān)蛋白表達(dá):MAP-2表達(dá):藥物組及假手術(shù)組高于模型組,達(dá)到顯著性差異(P㩳0.01,P㩳0.05)。藥物組高于假手術(shù)組,達(dá)到極顯著性差異(P㩳0.01);SYP表達(dá):藥物組表達(dá)明顯高于模型組及假手術(shù)組,達(dá)到極顯著性差異(P㩳0.01,P0.01);假手術(shù)組也明顯高于模型組(P㩳0.01);PSD-95表達(dá):藥物組及假手術(shù)組明顯高于模型組,達(dá)到極顯著性差異(P㩳0.01,P0.01);藥物組最高,與假手術(shù)組相比,也達(dá)到極顯著性差異(P㩳0.01)。突觸的電鏡觀察:假手術(shù)組:突觸間隙清晰,突觸前后膜輪廓完整。突觸小泡較多,突觸曲面規(guī)則,突觸前后膜吻合良好,后膜可見明顯增厚。線粒體完整,內(nèi)質(zhì)網(wǎng)上核糖體較多。模型組:突觸間隙模糊,突觸前后膜腫脹,空化。難以區(qū)分突觸前后膜。突觸小泡減少,線粒體固縮,較少。內(nèi)質(zhì)網(wǎng)脫顆粒。藥物組:突觸前后膜結(jié)構(gòu)完整。突觸間隙清晰可見,突觸小泡較多,線粒體結(jié)構(gòu)完整,含量豐富。內(nèi)質(zhì)網(wǎng)顆粒較多。[結(jié)論]加減薯蕷丸具有明顯治療VD作用,通過本課題研究表明其作用機(jī)制在于:1.通過在缺血環(huán)境中對(duì)抗海馬神經(jīng)元凋亡、抑制神經(jīng)炎癥、促進(jìn)神經(jīng)元再生、保護(hù)并恢復(fù)神經(jīng)-血管單元固有構(gòu)筑,預(yù)防神經(jīng)重構(gòu);2.抑制星型膠質(zhì)細(xì)胞增生,促進(jìn)少突膠質(zhì)細(xì)胞增生及軸突髓鞘化,干預(yù)膠質(zhì)重構(gòu)以保證神經(jīng)信息的高效傳導(dǎo);3.保護(hù)缺血情況下的突觸損傷與變性、促進(jìn)突觸再生與重建、保證突觸可塑性及神經(jīng)信息準(zhǔn)確傳遞。
[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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