本文選題：腦缺血再灌注 + 痰熱腑實(shí)��； 參考：《寧夏醫(yī)科大學(xué)》2017年碩士論文

【摘要】：腦梗死造成的神經(jīng)功能損害對人類的健康有著嚴(yán)重的影響,使患者出現(xiàn)殘疾的幾率已位居第一,病死率位居第三,僅低于癌癥和心肌梗死。據(jù)世界衛(wèi)生組織預(yù)計(jì)在每年大約有1500萬患有腦血管相關(guān)疾病,缺血性腦血管病是其發(fā)病率的4/5。腦梗死的患病已經(jīng)逐漸年輕化,其復(fù)發(fā)、致殘和死亡的機(jī)率均較高,不但使患者的生活質(zhì)量下降,也給家庭和社會造成了沉重的負(fù)擔(dān),給我國醫(yī)療衛(wèi)生產(chǎn)生了巨大壓力。腦部血流減少后,腦組織會產(chǎn)生一系列炎性級聯(lián)效應(yīng),包括炎癥因子的表達(dá)、氨基酸毒性的興奮、神經(jīng)元細(xì)胞死亡、周圍去極化四個(gè)不同的機(jī)制,且各機(jī)制在發(fā)生時(shí)間上可相互影響、互成因果。其中炎癥反應(yīng)是損傷級聯(lián)反應(yīng)發(fā)生過程中氧自由基、細(xì)胞因子及促炎酶共同促發(fā)的結(jié)果。細(xì)胞因子既可通過作用于內(nèi)皮細(xì)胞,將受體激活,從而使白細(xì)胞進(jìn)入腦內(nèi),又可以直接刺激小膠質(zhì)或星形膠質(zhì)細(xì)胞釋放有害成分,最終形成腦損傷。病與證結(jié)合既體現(xiàn)了中醫(yī)的基本理論,又結(jié)合了西醫(yī)學(xué)理論與動物實(shí)驗(yàn)學(xué)的知識,運(yùn)用西醫(yī)學(xué)的病因理論制備了動物的疾病,同時(shí)將中醫(yī)的證候建立在疾病的模型中,使動物模型中的證候與疾病相融合,使其具有較好的穩(wěn)定性和可靠性,并且可以動態(tài)的分階段的、系統(tǒng)的進(jìn)行觀察與研究,因此病證結(jié)合動物模型的研究已成為現(xiàn)代中醫(yī)藥研究模式的一個(gè)重要發(fā)展趨勢,對腦梗死的疾病發(fā)病機(jī)制及用藥探索有著重大的實(shí)際應(yīng)用意義。目的:1.通過檢測痰熱腑實(shí)腦缺血再灌注模型大鼠血脂血糖等指標(biāo),明確腦缺血痰熱腑實(shí)模型應(yīng)激性代謝功能的變化,觀察扎里奴思方對腦梗死代謝功能紊亂的調(diào)節(jié)作用。2.通過觀察腦缺血再灌注后痰熱腑實(shí)型大鼠神經(jīng)功能和神經(jīng)元細(xì)胞的病理變化,明確扎里奴思方對神經(jīng)細(xì)胞的保護(hù)作用。3.通過觀察腦缺血再灌注痰熱腑實(shí)型模型大鼠經(jīng)再灌注后炎性相關(guān)因子表達(dá)的情況,從扎里奴思方對其變化的影響,明確其可能的作用機(jī)制;方法:Sprague Dawley大鼠,雄性,250只,SPF級,將其隨機(jī)分為正常組、假手術(shù)組、單純腦梗死組、痰熱腑實(shí)組、尼莫地平組、扎里奴思方組共六個(gè)組,其中正常組10只,其余各組分為1天、3天、7天3個(gè)取材時(shí)間點(diǎn)每個(gè)時(shí)間點(diǎn)15只大鼠;正常組、假手術(shù)組、單純腦梗死組給予生理鹽水灌胃,其余各組大鼠通過藥物灌胃制備痰熱腑實(shí)證候模型,痰熱腑實(shí)模型完成后行大腦中動脈阻塞(MCAO)手術(shù),術(shù)后2小時(shí)進(jìn)行缺血再灌注;對模型大鼠的宏觀表現(xiàn)進(jìn)行觀察,對大鼠的神經(jīng)功能采用Longa 5級4分法進(jìn)行評分,大鼠腦組織神經(jīng)元細(xì)胞采用蘇木精-伊紅(Hematoxylin and Eeosin staining-HE)染色法觀察;檢測模型大鼠總膽固醇(total cholesterol,TC)、甘油三酯(triglyceride,TG)、血糖(glucose,GLU)、低密度脂蛋白(low-density lipoprotein,LDL)等能量代謝指標(biāo)的變化;運(yùn)用免疫組化測定各取材點(diǎn)缺血側(cè)腦組織中TNF-α、白介素6、VCAM-1、ICAM-1陽性細(xì)胞數(shù),實(shí)時(shí)熒光定量(Real-time PCR,RT-PCR)法測定各因子m RNA的表達(dá),探討扎里奴思方對痰熱腑實(shí)腦缺血模型大鼠炎性級聯(lián)反應(yīng)及能量代謝的影響及其神經(jīng)保護(hù)機(jī)制。結(jié)果:1.痰熱腑實(shí)模型大鼠在腦缺血再灌注后血脂及血糖等指標(biāo)的變化及扎里奴思方對其的影響相比正常組,單純腦梗死組大鼠甘油三酯、血糖均有顯著的增高(P0.05);痰熱腑實(shí)模型組大鼠血糖較假手術(shù)組和單純腦梗死組升高(P0.05);總膽固醇、甘油三酯、低密度脂蛋白相比假手術(shù)組明顯上升(P0.05);與痰熱腑實(shí)1天組比較,尼平組TC、TG、LDL-C顯著降低(P0.05);痰熱腑實(shí)3天組比較尼莫地平3天組TC、TG、LDL-C差異顯著(P0.05);扎方組1天、3天、7天組與痰熱腑實(shí)組相比TC、TG、LDL-C均有顯著降低(P0.05);HDL-C有明顯的升高(P0.05);GLU在尼平組和扎方組3天、7天時(shí)顯著減少(P0.05)。2.痰熱腑實(shí)腦缺血模型大鼠再灌注后神經(jīng)細(xì)胞損傷及扎里奴思方的保護(hù)作用假手術(shù)組大鼠未出現(xiàn)評分升高的表現(xiàn),經(jīng)過MCAO手術(shù)后的大鼠較假手術(shù)組和正常組相比,神經(jīng)功能評分明顯升高;單純腦梗死組及痰熱腑實(shí)組大鼠與假手術(shù)組相比,1天、3天、7天的神經(jīng)功能評分均明顯升高(P0.01),尤以1天組大鼠上升最為明顯,且痰熱腑實(shí)模型組神經(jīng)功能缺損較單純腦梗死組明顯;給藥后,扎里奴思方組和尼莫地平組大鼠在各時(shí)間點(diǎn)神經(jīng)功能評分均開始降低,并隨給藥時(shí)間延長,評分下降明顯(P0.05);尼莫地平組神經(jīng)功能評分較扎里奴思方組略高。3.痰熱腑實(shí)模型大鼠腦缺血再灌注后腦組織神經(jīng)元細(xì)胞形態(tài)的改變及扎里奴思方對其的影響扎里奴思方對痰熱腑實(shí)腦缺血再灌注大鼠缺血側(cè)腦細(xì)胞的影響:HE染色結(jié)果顯示,與單純腦梗死組相比,假手術(shù)組海馬組織結(jié)構(gòu)排列整齊,胞體、核仁清晰,染色均勻,神經(jīng)元細(xì)胞排列緊密;模型組海馬組織細(xì)胞結(jié)構(gòu)松散,排列散亂,神經(jīng)元細(xì)胞消失較多,部分神經(jīng)細(xì)胞胞核變小,細(xì)胞間質(zhì)內(nèi)可見大小不等的空泡,細(xì)胞核深染且核仁消失,皺縮,界限不清,大部分有退形性改變,神經(jīng)細(xì)胞有嗜酸性改變;扎方組和尼平組神經(jīng)細(xì)胞排列稍亂,仍可見細(xì)胞固縮,其周邊可見神經(jīng)細(xì)胞輕度腫脹,胞漿、胞膜、胞核尚能分清,程度較模型組明顯減輕,扎方組較尼莫地平組改善明顯。4.痰熱腑實(shí)模型大鼠腦缺血再灌注后炎癥因子的表達(dá)及扎里奴思方對其的影響4.1扎里奴思方對TNF-α表達(dá)的影響TNF-α在假手術(shù)組中表達(dá)較少,單純腦梗死組與假手術(shù)組相比陽性細(xì)胞數(shù)增多明顯(P0.01);隨著時(shí)間的延長,TNF-α陽性細(xì)胞數(shù)呈下降趨勢,與假手術(shù)組相比仍較高;尼平組與扎方組相對痰熱腑實(shí)組來說,TNF-α陽性細(xì)胞數(shù)都有所下降(P0.01),以扎里奴思方1d,3d時(shí)下降更為明顯(P0.05);模型組各時(shí)間點(diǎn)比假手術(shù)組TNF-αm RNA表達(dá)升高顯著(P0.01);尼平組和扎方組相比模型組TNF-αm RNA表達(dá)降低明顯(P0.01);扎里奴思方組較尼莫地平各組TNF-αm RNA表達(dá)有所降低,其中扎里奴思方1、7天降低明顯(P0.05)。4.2扎里奴思方對IL-6β表達(dá)的影響單純腦梗死組IL-6陽性細(xì)胞數(shù)比假手術(shù)組增多明顯(P0.05);扎里奴思方組各時(shí)間點(diǎn)與單純腦梗死組及痰熱腑實(shí)組比較差異亦均有顯著性(P0.05);與尼平組比較扎方組在1天、3天時(shí)均具有顯著差異(P0.05);在假手術(shù)組中IL-6 m RNA表達(dá)較低,模型組各時(shí)間點(diǎn)相比假手術(shù)組,IL-6 m RNA表達(dá)顯著升高(P0.01),在3天時(shí)達(dá)到高峰;尼莫地平組和扎里奴思方組相比模型各組,IL-6 m RNA表達(dá)明顯降低(P0.01);扎方各組相比尼平1天、3天、7天組,IL-6 m RNA表達(dá)降低明顯(P0.05)。4.3扎里奴思方對ICAM-1表達(dá)的影響假手術(shù)組中ICAM-1陽性細(xì)胞較少表達(dá),單純腦梗死組相比,假手術(shù)組大鼠缺血側(cè)腦組織中陽性細(xì)胞表達(dá)升高明顯(P0.01);在術(shù)后1天表達(dá)最多,扎方組與尼平組大鼠缺血側(cè)腦組織中陽性細(xì)胞表達(dá)減少且與模型組各組相比差異明顯(P0.01);扎方組相比尼平組,在1天、3天時(shí)減少明顯;腦缺血后1天、3天、7天,模型組ICAM-1 m RNA的表達(dá)明顯升高,3天后開始減少(P0.01);痰熱腑實(shí)組在7天時(shí)較單純腦梗死組表達(dá)稍有增多;扎方各組和尼平各組與非給藥組比較,ICAM-1 m RNA的表達(dá)都明顯降低,(P0.01,P0.05);扎方各組與尼平1天、3天、7天組比較ICAM-1 m RNA表達(dá)降低明顯(P0.05)。4.4扎里奴思方對VCAM-1表達(dá)的影響假手術(shù)組中VCAM-1陽性細(xì)胞表達(dá)較少,單純腦梗死組與假手術(shù)組相比,各時(shí)間點(diǎn)VCAM-1細(xì)胞陽性表達(dá)明顯增多(P0.05);扎方組和尼莫地平組各時(shí)間點(diǎn)VCAM-1細(xì)胞陽性表達(dá)明顯減少(P0.05);扎里奴思方與尼莫地平組相比,1天組VCAM-1細(xì)胞陽性表達(dá)降低尤其顯著(P0.05);VCAM-1 m RNA在假手術(shù)組中已有少量表達(dá),模型組各時(shí)間點(diǎn)假手術(shù)組相比,VCAM-1 m RNA顯著升高(P0.01);相比非用藥組,扎方組和尼平組VCAM-1m RNA表達(dá)明顯降低(P0.01,P0.05);扎方各組相比尼平1天、3天、7天組VCAM-1 m RNA表達(dá)明顯降低(P0.05)。結(jié)論:1.痰熱腑實(shí)模型大鼠在腦缺血再灌注反應(yīng)后應(yīng)激性出現(xiàn)血糖、TG、膽固醇、LDL的升高,HDL的降低,應(yīng)用扎里奴思方后大鼠血糖及血脂水平均有不同程度的改善,說明其對梗死后脂質(zhì)的代謝有促進(jìn)作用。2.痰熱腑實(shí)模型大鼠在腦缺血損傷后神經(jīng)細(xì)胞出現(xiàn)不同程度的損傷并表現(xiàn)出神經(jīng)功能缺失癥狀,扎里奴思方可促進(jìn)受損細(xì)胞的恢復(fù)并改善其神經(jīng)功能缺損程度。3.大鼠經(jīng)腦缺血再灌注后炎性因子的表達(dá)明顯增多,運(yùn)用扎里奴思方后可明顯降低其表達(dá),說明其可能是通過抑制炎性級聯(lián)反應(yīng)發(fā)揮腦保護(hù)作用。
[Abstract]:The neurological impairment caused by cerebral infarction has a serious impact on human health. The risk of disability is ranked first. The mortality rate is third, only lower than cancer and myocardial infarction. According to the WHO, about 15 million have cerebrovascular related diseases each year. Ischemic cerebrovascular disease is the 4/5. of its incidence. The incidence of cerebral infarction has gradually become younger, its recurrence, disability and mortality are high, which not only reduces the quality of life of the patients, but also causes a heavy burden on the family and society, causing great pressure to the health and health of our country. After the decrease of blood flow in the brain, the brain tissue produces a series of inflammatory cascade effects, including inflammatory factors. Expression, excitatory activity of amino acid toxicity, neuronal cell death, and peripheral depolarization of four different mechanisms, and each mechanism can interact with each other in time. The inflammatory reaction is the result of oxygen free radicals, cytokines and proinflammatory enzymes in the process of injury cascade. The skin cell, which activates the receptor, enables the leukocytes into the brain, and can directly stimulate the microglia or astrocytes to release harmful components and eventually form brain damage. The combination of disease and syndrome combines the basic theory of traditional Chinese medicine and the knowledge of Western medicine theory and animal experiment, and makes use of the etiological theory of Western medicine to prepare the animal. At the same time, the syndrome of Chinese medicine is established in the model of the disease, and the syndrome in the animal model is fused with the disease, so it has good stability and reliability, and it can be observed and studied systematically. Therefore, the research of disease syndrome combined with animal model has become a model of modern Chinese medicine research. An important development trend is of great practical significance for the pathogenesis of cerebral infarction and the exploration of drug use. 1. through the detection of blood lipid and blood glucose in the model rats of phlegm heat and cerebral ischemia reperfusion model, the change of stress metabolic function of cerebral ischemia phlegm heat Fu Fu model was clearly defined, and the metabolic function of cerebral infarction was observed by Sali Nu Fang. .2. by observing the pathological changes of nerve function and neuron cell in the rat model of phlegm heat syndrome after cerebral ischemia and reperfusion, the protective effect of Zi Li Si Fang on the nerve cells was determined by observing the expression of inflammatory related factors in the rat model of cerebral ischemia reperfusion phlegm heat syndrome model rats after reperfusion, from the point of view of.2.. The influence of Fang on its change, clear its possible mechanism of action; methods: Sprague Dawley rats, male, 250, SPF grade, randomly divided into normal group, sham operation group, simple cerebral infarction group, phlegm hot Fu Fu Group, nimodipine group, and the group of nimodipine, 10 of the normal group, the other groups are divided into 1 days, 3 days, 7 days 3 timber time. There were 15 rats at each time point, the normal group, the sham operation group and the simple cerebral infarction group were given the normal saline for gastric perfusion, the other rats were given the phlegm heat syndrome model through the medicine gavage. After the phlegm heat syndrome was completed, the middle cerebral artery occlusion (MCAO) operation was performed, and the ischemia reperfusion was carried out at 2 hours after the operation, and the macroscopic performance of the model rats was carried out. The neural function of rats was evaluated by Longa 5 grade 4 method. The neuron cells in the rat brain were observed by hematoxylin eosin (Hematoxylin and Eeosin staining-HE) staining, and the total cholesterol (total cholesterol, TC), triglyceride (triglyceride, TG), blood glucose (glucose, GLU), low density lipoprotein (LDL), and low density lipoprotein (LDL) were detected in the rat model. The changes of energy metabolism indexes such as nsity lipoprotein, LDL), TNF- alpha, interleukin 6, VCAM-1, ICAM-1 positive cells, Real-time PCR, RT-PCR method were used to determine the expression of M RNA in each factor, and the inflammatory cascade in the rat model of phlegm heat cerebral ischemia model was investigated by the method of Real-time PCR, RT-PCR. The effect of reaction and energy metabolism and its neuroprotective mechanism. Results: 1. the changes of blood lipid and blood sugar in the model rats after cerebral ischemia reperfusion and the effect of TNU side on it were compared with that of the normal group. The triglyceride and blood sugar of the rats in the simple cerebral infarction group were significantly increased (P0.05); the blood sugar in the model group of phlegm heat Fu Fu model rats Compared with the sham operation group and the simple cerebral infarction group (P0.05), the total cholesterol, triglyceride and low density lipoprotein were significantly higher than the sham operation group (P0.05), and the TC, TG and LDL-C in the nimping group were significantly decreased (P0.05) compared with the 1 day group of phlegm heat, and the 3 day group of nimodipine was significantly different from the 3 days of nimodipine group (P0.05); the group was 1 days, 3 days, 7 days. TC, TG, LDL-C were significantly decreased (P0.05), and HDL-C had a significant increase (P0.05) compared with the phlegm heat group (P0.05); GLU in the nepin group and Zhan Fang Group 3 days, 7 days significantly reduced (P0.05).2. phlegm heat cerebral ischemia model rats after reperfusion nerve cell injury and the protective effect of Zhan Li Si Fang, the sham operation group did not appear to increase the score Compared with the sham operation group and the normal group, the nerve function score of the rats after the MCAO operation was significantly higher than that of the sham group and the sham group. The nerve function score of the rats in the 1 day, the 3 day and the 7 day was significantly higher than that of the sham group (P0.01), especially in the 1 day group, and the nerve function defect in the phlegm heat model group Compared with the simple cerebral infarction group, the nerve function score of the rats in the nimodipine group and the nimodipine group began to decrease after the administration, and the score decreased with the prolongation of the time of Administration (P0.05). The nimodipine group's nerve function score was slightly higher than the.3. phlegm heat syndrome rat brain tissue after the cerebral ischemia reperfusion. The changes of neuronal cell morphology and the effect of Zi Li Si Fang on the ischemic side brain cells of rats with phlegm heat and cerebral ischemia reperfusion. The results of HE staining showed that the structure of the hippocampal structure in the sham operation group was neatly arranged, the body, the nucleolus were clear, the coloration was uniform, and the neuron cells were closely arranged. In the model group, the cell structure of the hippocampus was loose and scattered, the neuron cells disappeared more, the nuclei of some nerve cells became smaller, and the cells in the cells were visible in different size vacuoles. The nuclei were deeply dyed and the nucleolus disappeared, and the lines were indistinct. Most of the cells were degenerated, and the cells of the deity were eosinophilic, and the nerve cells in the Zhan Fang Group and the nepin group were changed. There was a slight disorder in the arrangement of the cells. The peripheral nerve cells were slightly swelling, the cytoplasm, the membrane and the nucleus were still distinct, and the extent of the cells was clearly reduced. The expression of the inflammatory factors after the cerebral ischemia reperfusion in the.4. phlegm heat model rats and the effect of zhali Nu Si Fang on it were better than the nimodipine group, and the influence of zhali Nu Si Fang on it was 4.1 zhali Nu square The expression of TNF- alpha was less in the sham operation group than in the sham operation group. The number of positive cells in the simple cerebral infarction group increased significantly (P0.01) compared with the sham operation group (P0.01). The number of TNF- alpha positive cells decreased with the prolongation of time, and was still higher than the sham operation group. The number of TNF- alpha positive cells in the niping group and the Zhan Fang group compared with the phlegm heat group, the number of TNF- alpha positive cells was all The decrease (P0.01), with zari Nu side 1D, 3D decreased more obviously (P0.05), and the expression of TNF- alpha m RNA in the model group was significantly higher than that of the sham operation group (P0.01), and the expression of TNF- alpha m RNA decreased significantly (P0.01) in the nimping group and the Zai Fang Group, and the zari Zaru side group was lower than the nimodipine group. Nu Si Fang 1,7 days decreased significantly (P0.05).4.2 Zhan Nu side effect on IL-6 beta expression in simple cerebral infarction group, the number of IL-6 positive cells increased significantly (P0.05), and the time points in the zhali Nu Sifang group were also significantly different from those in the simple cerebral infarction group and the phlegm heat group (P0.05), and the group was compared with the niping group at 1 days and 3 days. There were significant differences (P0.05); the expression of IL-6 m RNA in the sham operation group was low, and the expression of IL-6 m RNA in the model group was significantly higher than that in the sham operation group (P0.01), and reached the peak at 3 days. The expression of IL-6 m RNA was significantly lower in the nimodipine group and the zhali Nu Fang group than in the model group (P0.01); 1 days, 3 days, and 7 days compared with the nimodipine group. Group, IL-6 m RNA expression decreased significantly (P0.05).4.3 zhalnu side effect on the expression of ICAM-1 in sham operation group less expression of ICAM-1 positive cells, pure cerebral infarction group, compared with the sham group, the expression of positive cells in the ischemic side brain tissue in the sham operation group increased significantly (P0.01), 1 days after the operation, the most, zhfang group and the nipping group rats ischemic side brain group The expression of positive cells in the fabric decreased significantly (P0.01) compared with that in the model group (P0.01). Compared with the neping group, the expression of positive cells decreased significantly at 1 days and 3 days. The expression of ICAM-1 m RNA in the model group increased significantly after 1 days of cerebral ischemia, and decreased in 3 days (P0.01); the expression of phlegm heat group was slightly more than that in the simple cerebral infarction group at 7 days; ligation prescription The expression of ICAM-1 m RNA in each group and nping group decreased significantly (P0.01, P0.05), and the expression of ICAM-1 m RNA in the group of Zhan Fang and nping 1 days, 3 days and 7 days was significantly lower (P0.05). The expression of VCAM-1 positive cells in the sham operation group was less than that in the sham operation group, and the simple cerebral infarction group and the sham operation group were less. The positive expression of VCAM-1 cells in each time point increased significantly (P0.05), and the positive expression of VCAM-1 cells in the Zhan Fang Group and nimodipine group decreased significantly (P0.05), and the positive expression of VCAM-1 cells in the 1 day group was significantly lower than that in the nimodipine group (P0.05), and the VCAM-1 m RNA had been expressed in a small amount in the sham operation group, and the model of VCAM-1 m RNA had been expressed in a small amount. Compared with the sham operation group at all time points, VCAM-1 m RNA increased significantly (P0.01), and the expression of VCAM-1m RNA in Zhan Fang Group and neping group was significantly decreased (P0.01, P0.05), compared with non medication group (P0.01, P0.05), and the expression of VCAM-1 m RNA was obviously decreased (P0.05) in the group of Zhan Fang 1 days, 3 days and 7 days. Conclusion: 1. phlegm heat Fu Fu model rats stress after cerebral ischemia reperfusion injury. The increase of blood sugar, TG, cholesterol, LDL, the decrease of HDL, the improvement of blood sugar and blood lipid levels in the rats after the use of the Chi Nu Si Fang, shows that the lipid metabolism in the post infarction rats has a promoting effect on the.2. phlegm heat and Fu Fu model rats with different degrees of injury in the neural cells after the cerebral ischemia injury and showing the lack of nerve function. The loss of symptoms, Zaru Si Fang can promote the recovery of damaged cells and improve the degree of nerve function defect in.3. rats. The expression of inflammatory factors in rats after cerebral ischemia reperfusion is obviously increased. The expression of zari Nu side can be obviously reduced after zari Nu Fang, which indicates that it may play the role of brain protection by inhibiting the inflammatory cascade reaction.

【學(xué)位授予單位】：寧夏醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R29

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