本文選題：睡眠剝奪　切入點：證候模型　出處：《湖北中醫(yī)學(xué)院》2008年博士論文

【摘要】： 目的:本課題結(jié)合臨床建立老年血虧陰虛型失眠證候動物模型,并將該復(fù)合型模型與傳統(tǒng)單因素睡眠剝奪模型進行對照,遵循中醫(yī)因-脈-證-治的思路進行評價:首先對“因”進行分析,即根據(jù)中醫(yī)病因病機來評價造模因素的科學(xué)性;然后對“脈”進行觀測,即觀測模型較直接、直觀、重要的中醫(yī)體表特征,并確認這種模型的證候?qū)傩?接著對“證”進行探討,即通過分子生物學(xué)等現(xiàn)代科技手段對體表特征所反映的老年血虧陰虛型失眠病理機制進行概括;最后用驗方酸棗仁湯“治”來進行反證,探討對應(yīng)治法驗方酸棗仁湯的作用及其可能機制,并建立基于代謝網(wǎng)絡(luò)變化的中藥復(fù)方系統(tǒng)生物學(xué)藥效評價平臺。 方法:在對酸棗仁湯的促眠作用進行實驗研究之后,將健康Wistar大鼠100只(雌雄各半、體重200g±20g)隨機分成5組,每組20只:環(huán)境對照組;睡眠剝奪組(單因素模型組);證候模型組(復(fù)合因素模型組);酸棗仁湯大劑量組;酸棗仁湯小劑量組。其中環(huán)境對照組給予其它組睡眠剝奪時相似的水環(huán)境;單因素模型組采用自制多平臺水環(huán)境法剝奪睡眠48h制備;復(fù)合因素模型組及酸棗仁湯大、小劑量組均采用頸背部皮下注射D-半乳糖6w、然后腹腔同時注射環(huán)磷酰胺和氫化可的松5d、接著用自制多平臺水環(huán)境法剝奪睡眠48h制備復(fù)合模型,造模歷時共7w;酸棗仁湯大、小劑量組在造模4w后開始同時灌胃給藥3w,其它組均給予等容積生理鹽水。指標(biāo)檢測具體方法如下: 1、通過同步檢測體重、攝食量、腋溫、及顯微放大拍照爪、尾、口唇等部位,對各組實驗大鼠的中醫(yī)體表特征進行比較; 2、用組織化學(xué)法檢測各組實驗大鼠大腦皮質(zhì)、海馬部位自由基和一氧化氮合酶(NOS)水平的變化; 3、采用血液細胞分析儀檢測各組實驗大鼠血細胞計數(shù)的變化; 4、采用Morris水迷宮實驗檢測各組實驗大鼠學(xué)習(xí)記憶能力的改變; 5、透射電鏡下觀察各組實驗大鼠大腦皮質(zhì)部位超微結(jié)構(gòu)的病理改變; 6、采用高相液相法檢測各組實驗大鼠大腦皮質(zhì)及下丘腦部位谷氨酸(Glu)及γ-氨基丁酸(GABA)含量的變化; 7、采用免疫組化染色和mRNA半定量RT-PCR法檢測分析各組實驗大鼠大腦皮質(zhì)及海馬部位γ-氨基丁酸A受體(GABA_AR)a_1和γ_2亞單位的表達改變; 8、采用免疫組化染色法檢測各組實驗大鼠大腦皮質(zhì)部位星形膠質(zhì)細胞的表達改變。 結(jié)果: 1、與空白對照組比較,舒樂安定組、酸棗仁湯大、小劑量組均能顯著延長閾上劑量戊巴比妥鈉致小鼠睡眠時間(P＜0.01),顯著增加閾下劑量戊巴比妥鈉致小鼠入睡率(P＜0.01)。且酸棗仁湯大劑量組作用比小劑量組更明顯,但無統(tǒng)計學(xué)意義(P＞0.05); 2、中醫(yī)體表特征顯示,與環(huán)境對照組比較,睡眠剝奪組出現(xiàn)失眠癥狀,表現(xiàn)為重度疲勞狀態(tài);與睡眠剝奪組比較,證候模型組在出現(xiàn)失眠癥狀的同時,出現(xiàn)面色無華,閉目,血呈暗紅,嗜睡,食欲明顯下降,四肢無力,毛蓬豎少光澤等血虧陰虛體表特征,且體重、攝食量顯著下降(P＜0.01),而腋溫的下降無統(tǒng)計學(xué)意義(P＞0.05);與證候模型組比較,酸棗仁湯大、小劑量組對血虧陰虛體表特征有改善作用,但無統(tǒng)計學(xué)意義(P＞0.05); 3、與證候模型組比較,其它各組大鼠大腦皮質(zhì)、海馬丙二醛(MDA)含量、超氧化物歧化酶(SOD)和NOS活性均明顯降低(P＜0.01,P＜0.05)。且酸棗仁湯大、小劑量組呈現(xiàn)一定量效關(guān)系(P＜0.05); 4、與證候模型組比較,其它各組大鼠全血中紅細胞計數(shù)、白細胞計數(shù)、血小板計數(shù)及血紅蛋白含量顯著升高(P＜0.01)。且酸棗仁湯大、小劑量組呈現(xiàn)一定量效關(guān)系(P＜0.05); 5、在定位航行實驗中,與證候模型組比較,環(huán)境對照組和酸棗仁湯大、小劑量組大鼠實驗4d及2d的潛伏期均顯著縮短(P＜0.01)。在空間搜索實驗中,各組平臺象限的游泳距離占總距離百分比、池壁20%和40%區(qū)域游泳距離百分比的實驗結(jié)果表現(xiàn)出與定位航行實驗各組一致的趨勢;與證候模型組比較,環(huán)境對照組和酸棗仁湯大、小劑量組跨過原平臺位置次數(shù)明顯升高(P＜0.01)。兩種模型組間相比無明顯差異(P＞0.05); 6、睡眠剝奪48h后,透射電鏡下顯示:睡眠剝奪組大鼠大腦皮質(zhì)神經(jīng)元細胞異染質(zhì)輕微聚集,趨邊排列,核周間隙輕微增寬,大部分線粒體中高度水腫,線粒體基質(zhì)及線粒體脊消失;證候模型組大鼠大腦皮質(zhì)神經(jīng)元細胞間胞質(zhì)水腫,連接增寬,可見單個神經(jīng)元細胞異染質(zhì)凝聚,趨邊,呈現(xiàn)凋亡形態(tài),細胞器消失;酸棗仁湯大、小劑量組能改善大鼠神經(jīng)元細胞超微結(jié)構(gòu)的病理改變; 7、與環(huán)境對照組比較,睡眠剝奪組大鼠大腦皮質(zhì)及下丘腦部位氨基酸類神經(jīng)遞質(zhì)Glu、GABA含量均明顯增高(P＜0.01),但GABA增加更為明顯,Glu/GABA比例減小;與環(huán)境對照組比較,證候模型組大鼠Glu、GABA含量均明顯增高(P＜0.01),但Glu增加更為明顯,Glu/GABA比例增大;與證候模型組比較,酸棗仁湯大、小劑量組大鼠大腦皮質(zhì)及下丘腦部位氨基酸類神經(jīng)遞質(zhì)Glu、GABA含量顯著減少,但Glu減少更為明顯,Glu/GABA比例減小,并呈現(xiàn)一定量效關(guān)系(P＜0.05); 8、與證候模型組比較,環(huán)境對照組和酸棗仁湯大、小劑量組大鼠大腦皮質(zhì)及海馬部位的GABA_ARa_1和γ_2亞單位免疫化學(xué)累積光密度明顯較低(P＜0.01),皮質(zhì)部位GABA_ARa_1和γ_2 mRNA的表達均顯著下調(diào)(P＜0.01)。而與睡眠剝奪組比較,證候模型組表達相對較弱(P＜0.05); 9、與證候模型組比較,環(huán)境對照組和酸棗仁湯大、小劑量組大鼠睡眠剝奪48h后星形膠質(zhì)細胞標(biāo)志物—膠原纖維酸性蛋白(GFAP)在皮質(zhì)部位表達明顯較弱;與睡眠剝奪組比較,證候模型組表達亦相對較強。 結(jié)論: 1、酸棗仁湯具有明顯的鎮(zhèn)靜催眠作用,且大劑量作用強于小劑量; 2、模型大鼠的體表特征顯示,D-半乳糖+環(huán)磷酰胺、氫化可的松+睡眠剝奪這一復(fù)合模型較接近臨床老年血虧陰虛型失眠患者的表現(xiàn),是一種可行、可靠的復(fù)合型動物模型; 3、老年大鼠的學(xué)習(xí)記憶能力和大腦皮質(zhì)病理損傷與睡眠剝奪、血虧陰虛證候具有相關(guān)性。自由基水平增加、全血象減少是老年血虧陰虛型失眠的病理機制之一。反證驗方酸棗仁湯可有效改善證候模型大鼠的學(xué)習(xí)記憶能力及修復(fù)腦損傷,其作用機理可能與抗自由基、補血作用有關(guān); 4、大腦皮質(zhì)及下丘腦部位Glu、GABA含量及Glu/GABA比值均升高可能是老年血虧陰虛型失眠的神經(jīng)生物學(xué)病理機制。酸棗仁湯治療老年血虧陰虛型失眠的作用機制可能與通過下調(diào)大腦皮質(zhì)及下丘腦部位Glu、GABA的含量及Glu/GABA的比值來保護腦組織有關(guān)。 5、睡眠剝奪可明顯上調(diào)大鼠腦內(nèi)皮質(zhì)及海馬部位GABA_ARa_1和γ_2亞單位表達。酸棗仁湯治療老年血虧陰虛型失眠的作用機制可能與下調(diào)大鼠大腦皮質(zhì)及海馬部位GABA_ARa_1和γ_2亞單位的表達有關(guān)。 6、睡眠剝奪可上調(diào)大鼠大腦皮質(zhì)部位星形膠質(zhì)細胞標(biāo)志物—膠質(zhì)纖維酸性蛋白(GFAP)的表達,提示星形膠質(zhì)細胞為可興奮細胞,且參與睡眠調(diào)節(jié)。大腦皮質(zhì)部位GFAP的表達明顯增強可能是老年血虧陰虛型失眠的又一病理機制。酸棗仁湯治療老年血虧陰虛型失眠的作用機制可能還與下調(diào)大腦皮質(zhì)部位GFAP的表達有關(guān)。
[Abstract]:Objective: This study combined with the clinical establishment of elderly blood deficiency and Yin insomnia syndrome animal model, and the composite model and the traditional single factor model of sleep deprivation group, traditional Chinese medicine for vein syndrome - treatment of evaluation: first of all, "because" analysis, namely according to the pathogenesis of scientific evaluation modeling of factors; then observe the "pulse", which is more intuitive, direct observation model, the Chinese surface important characteristics, and confirm the syndrome attribute of the model; then discusses the "syndrome", namely the elderly insomnia mechanisms with blood deficiency and Yin by molecular biology and other modern technologies of surface features the reflection was summarized; finally, the prescription szrt "cure" for evidence to the contrary, on the corresponding treatment prescription szrt effect and the possible mechanism, and the establishment of Chinese herbal compound system on the basis of metabolic network changes Biological efficacy evaluation platform.
Methods: after the experiments in the sleep promoting effect of Suanzaoren Decoction, the 100 healthy Wistar rats (Ci Xiong female, weight 200g + 20g) were randomly divided into 5 groups, 20 rats in each group: control group; sleep deprivation group (univariate model group); syndrome model group (model group compound factors); Suanzaoren Decoction high dose group; Suanzaoren Decoction low dose group. The control group was given the other group environment when sleep deprived similar water environment; single factor model group using the self-made multi platform water environment law 48h sleep deprivation preparation; multiple factors model group and szrt of large and small dose group were treated with neck subcutaneous injection of D- galactose 6W, then intraperitoneal injection of cyclophosphamide and hydrocortisone and 5D, then using the self-made multi platform water environment law of sleep deprivation model prepared by 48h model, for a total of 7W; szrt of large and small dose groups also began intragastric administration of 3W in 4W after modeling, the The group was given equal volume of saline. The specific methods were as follows:
1, by measuring the body weight, food intake, axillary temperature, and micromagnifying photo claw, tail, lip and other parts, the body surface characteristics of the experimental rats in each group were compared.
2, the changes in the level of free radicals and nitric oxide synthase (NOS) in the cerebral cortex, hippocampus and hippocampus were detected by histochemical method.
3, blood cell analyzer was used to detect the changes of blood cell count in each group.
4, the Morris water maze test was used to detect the changes in the learning and memory ability of the rats in each group.
5, under the transmission electron microscope, the pathological changes of the ultrastructure of the cortex of the cerebral cortex of the rats were observed.
6, the changes in the content of glutamic acid (Glu) and gamma aminobutyric acid (GABA) in the cerebral cortex and hypothalamus of the rats were detected by high phase liquid phase.
7, immunohistochemical staining and mRNA semi quantitative RT-PCR were used to detect the expression of GABA A receptor (GABA_AR) a_1 and gamma _2 subunits in each group of rats.
8, the expression of astrocytes in the cerebral cortex of each group was detected by immunohistochemical staining.
Result:
1, compared with the blank control group, diazepam group and szrt of large and small dose group could significantly extend the threshold dose of pentobarbital induced sleep time in mice (P < 0.01), significantly increased the subthreshold dose of sodium pentobarbital induced sleep rate (P < 0.01). Szrt large agent the amount of group was more obvious than low-dose group, but no statistical significance (P > 0.05);
2, the Chinese surface characteristics showed that, compared to environmental control group, sleep deprivation group appear insomnia symptoms, severe fatigue performance of the state; and the sleep deprived group, syndrome model group in insomnia symptoms appear at the same time, lusterless complexion, eyes closed, blood is dark, lethargy, weakness, appetite decreased significantly. Peng hair less shiny blood deficiency such as vertical surface characteristics, and the body weight, food intake decreased significantly (P < 0.01), but no significant decline in axillary temperature (P > 0.05); compared with syndrome model group, Suanzaoren Decoction, small dose group has good effect on blood deficiency of surface characteristics, but no statistical significance (P > 0.05);
3, compared with the syndrome model group, cerebral cortex of other rats hippocampus malondialdehyde (MDA) content, superoxide dismutase (SOD) and NOS activity were significantly decreased (P < 0.01, P < 0.05). Szrt of large and small dose group showed a dose effect relationship (P < 0.05);
4, compared with the syndrome model group, red blood cell count other rats'whole blood, white blood cell count, platelet count and hemoglobin content increased significantly (P < 0.01). Szrt of large and small dose group showed a dose effect relationship (P < 0.05);
5, in the navigation experiment, compared with syndrome model group, control group and szrt, experimental 4D and 2D low dose group rat latency shortened significantly (P < 0.01). In the search space in the experiment, each platform quadrant swimming distance percentage of the total distance, the wall of the pool 20% the 40% area and swimming distance percentage of the experimental results show the positioning navigation experiments consistent trend; compared with syndrome model group, control group and szrt of large and small dose groups across the original platform location of significantly increased (P < 0.01). The two groups showed no significant difference (P > 0.05);
6, sleep deprivation after 48h transmission electron microscope showed that sleep deprivation rat cerebral cortex neurons of heterochromatin margination array, slight aggregation, nuclear week gap widened slightly, the majority of mitochondrial highly edema, mitochondrial matrix and mitochondrial crista disappeared; syndrome model group rat cortical neurons cell cytoplasm edema, connecting widened, visible single neurons with different chromatin condensation, margination, showed apoptotic morphology, cell disappeared; szrt of large and small dose group could improve the ultrastructure of neurons pathological changes in rat;
7, with comparison to the control group, sleep deprivation group rat cerebral cortex and hypothalamus of amino acid neurotransmitters Glu, GABA content increased significantly (P < 0.01), but GABA was significantly increased, Glu/GABA ratio decreased; and in comparison to the control group, the rats in group Glu syndrome model, GABA significantly increased (P < 0.01), but Glu was significantly increased, the proportion of Glu/GABA increased; compared with syndrome model group, Suanzaoren Decoction and small dose group rat cerebral cortex and hypothalamus of amino acid neurotransmitters Glu, GABA were significantly reduced, but Glu reduced more significantly, the ratio of Glu/GABA decreased, and a certain dose effect relationship (P < 0.05);
8, compared with the syndrome model group, control group and szrt, GABA_ARa_1 and gamma subunit _2 immunohistochemistry in small dose group rat cerebral cortex and hippocampus of the accumulated optical density was significantly lower (P < 0.01), the expression of cortex GABA_ARa_1 and gamma mRNA _2 were significantly lower (P < 0.01). Compared to the sleep deprived group, syndrome model group showed weaker expression (P < 0.05);
9, compared with the syndrome model group, control group and szrt of large and small dose groups of rats after sleep deprivation 48h astrocyte marker glial fibrillary acidic protein (GFAP) expression was significantly weaker in the cortex; and the sleep deprived group, the expression of syndrome model group is relatively strong.
Conclusion:
1, acid zizzizen decoction has obvious sedative and hypnotic effect, and the effect of large dose is stronger than that of small dose.
2, show the surface characteristics of the rat model, D- galactose + cyclophosphamide and hydrocortisone + sleep deprivation, this composite model is closer to the elderly patients with blood deficiency and Yin insomnia, is a feasible and reliable animal model of compound;
3, the ability of learning and memory and cerebral cortex injury in aged rats with sleep deprivation, associated with Yin deficiency syndrome in blood. The level of free radicals increased, whole blood reduction is one of the pathological mechanism of insomnia in elderly patients with blood deficiency and Yin. But the ability of learning and memory and improve the repair of brain injury syndrome model rats disproving prescription Suanzaoren Decoction and its mechanism may be related to anti free radical effect on blood;
4, cerebral cortex and hypothalamus of Glu were increased, GABA content and Glu/GABA ratio might be a neurobiological pathological mechanism of insomnia in elderly blood deficiency and Yin. The mechanism of Suanzaoren Decoction in the treatment of senile insomnia, blood deficiency and Yin may be related to the cerebral cortex and hypothalamus through down-regulation of Glu contents and Glu/GABA ratio of GABA to protect the brain.
5, sleep deprivation could significantly increase the cortex and hippocampus in rat brain GABA_ARa_1 and gamma subunit _2 expression. The mechanism of Suanzaoren Decoction in the treatment of senile insomnia, blood deficiency and Yin may be associated with the down-regulation of expression in rat cerebral cortex and hippocampus GABA_ARa_1 and gamma subunit _2.
6, sleep deprivation can be parts of the cerebral cortex increased rat astrocyte marker glial fibrillary acidic protein (GFAP) expression, suggesting that astrocytes to excitable cells and is involved in the regulation of sleep. The expression of GFAP in cerebral cortex increased and a part of the pathogenesis may be blood loss in elderly insomnia Yin. The mechanism of Suanzaoren Decoction in the treatment of senile insomnia, blood deficiency and Yin may be associated with the down-regulation of GFAP expression in brain cortex.

【學(xué)位授予單位】：湖北中醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R-332;R285.5

【引證文獻】

相關(guān)期刊論文 前1條

1 黃攀攀;王平;李貴海;游秋云;李浩;程靜;彭圓;;老年陰虛失眠動物模型的建立與評價[J];中華中醫(yī)藥學(xué)刊;2010年08期

相關(guān)博士學(xué)位論文 前1條

1 黃攀攀;從“心藏神”探討《內(nèi)經(jīng)》睡眠理論及天王補心丹干預(yù)老年失眠大鼠作用機制研究[D];湖北中醫(yī)藥大學(xué);2010年

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本文編號：1686969