【摘要】：研究背景:腦卒中是一種具有高發(fā)病率、死亡率、致殘率的常見神經(jīng)系統(tǒng)疾病,其中約87%是缺血性卒中。盡早恢復缺血區(qū)腦灌注,挽救缺血半暗帶瀕死神經(jīng)元是腦梗死治療的重心和目標。多項動物實驗證實促進腦梗死后血管新生可減輕神經(jīng)功能缺損,縮小腦梗死體積。三七三醇皂苷(Panaxatriol Saponins,PTS),是從中藥三七中提取的有效成份,其中三七皂苷R1、人參皂苷Rg1、人參皂苷Re為PTS的主要組成成分,前期多項研究證實PTS對腦梗死大鼠具有保護作用,但具體機制不明。近期研究表明PTS三種主要成分均有促人臍靜脈內(nèi)皮細胞增殖作用,然而目前尚未有PTS在缺血腦損傷動物體內(nèi)促血管新生方面的報道。目的:(1)觀察PTS對大鼠腦缺血再灌注模型的保護作用;(2)明確PTS促進腦梗死后微血管新生改善腦灌注作用;(3)探討PTS促進腦梗死后血管新生相關(guān)機制。方法:取SPF級SD雄性大鼠,隨機分為(1)假手術(shù)組:腹腔注射生理鹽水,每日1次;(2)MCAO(大腦中動脈梗死)模型組:MCAO+等量生理鹽水,腹腔注射,每日1次;(3)PTS治療組:MCAO+PTS,腹腔注射PTS,50mg/kg/d。缺血再灌注后不同時間點進行神經(jīng)行為學評分及腦組織TTC染色評價PTS對大鼠腦梗死的治療作用;采用Micro-PET影像技術(shù)評價PTS對腦血流灌注的影響;RT-PCR和Western blot技術(shù)檢測不同時間點血管生長相關(guān)因子基因及蛋白表達的變化;免疫熒光染色法評價腦梗死缺血半暗帶區(qū)微血管密度的改變;為進一步評價PTS促血管內(nèi)皮細胞增殖作用,予MCAO大鼠Brdu腹腔注射,行Brdu與CD31免疫熒光共定位,評價新生血管內(nèi)皮細胞數(shù)目;為深入探討PTS促血管新生機制,采取環(huán)靶明(Shh通路特異性阻斷劑)聯(lián)合PTS予MCAO大腹腔注射,Western blot技術(shù)評價Shh信號通路關(guān)鍵蛋白的表達及其對下游血管生長因子的影響。結(jié)果:(1)PTS對MCAO大鼠的腦保護作用:TTC及Neuron染色結(jié)果表明,PTS能夠明顯減小缺血再灌注后3天、7天、14天時間點的大鼠腦梗死體積,與對照組比較有統(tǒng)計學意義;Longa神經(jīng)功能評分表明,PTS給藥3天開始能夠改善MCAO大鼠神經(jīng)行為學評分,并持續(xù)到14天,與對照組比較P0.05,有統(tǒng)計學意義。(2)PTS增加MCAO大鼠缺血側(cè)腦灌注:Micro-PET/CT掃描結(jié)果顯示,MCAO造模后1天18F-PDG腦攝取明顯減少,術(shù)后14天復查Micro-PET可見PTS治療組18F-PDG腦攝取量明顯增加,表明腦血流量明顯改善,經(jīng)與對照組比較P0.05,有統(tǒng)計學意義。(3)PTS增加缺血側(cè)皮層血管生長相關(guān)因子的mRNA表達水平:RT-PCR結(jié)果表明,MCAO術(shù)后VEGF的mRNA水平隨時間延長,呈持續(xù)增高樣趨勢,并且在第3天時間點PTS治療組與對照組之間產(chǎn)生差異(P0.05),治療組優(yōu)于對照組,并且這種效果可以延續(xù)到14天;Ang-1的mRNA水平表達趨勢同VEGF;VEGFR2、Tie-2、CD31的mRNA表達水平在MCAO術(shù)后持續(xù)增高,并且在第7天達到高峰,14天時有所下降,治療組與對照組從第3天開始產(chǎn)生差異,治療組升高的幅度大于對照組(P0.05);α-SMA的mRNA水平在MCAO術(shù)后的表達的趨勢等同于Ang-1,同樣在第3天開始,治療組與對照組間產(chǎn)生差異,治療組升高的幅度明顯大于對照組(P0.05)。(4)PTS上調(diào)缺血側(cè)大腦皮層VEGF、Ang-1及VEGR2、Tie-2蛋白表達:western blot免疫印跡結(jié)果顯示,MCAO術(shù)后大鼠大腦皮層VEGF蛋白水平在術(shù)后第7天達到高峰,第14天有下降趨勢,PTS與對照組比較在第3天,7天,14天時間點有統(tǒng)計學意義(P0.05);Ang-1的蛋白表達水平隨時間延長呈逐漸增加趨勢,且PTS與對照組比較在第3天,7天,14天時間點有統(tǒng)計學意義(P0.05);在術(shù)后7天時間點,PTS治療組能夠明顯上調(diào)VEGFR2、Tie-2蛋白表達水平,經(jīng)統(tǒng)計學處理優(yōu)于對照組(P0.05)。(5)PTS增加缺血側(cè)皮層微血管密度:研究結(jié)果表明缺血缺氧誘導病灶周圍大量的血管新生,而且微血管密度在腦梗死后7天達到高峰,14天時有所下降,PTS治療組在第7天和14天時間點CD31/α-SMA陽性的微血管密度明顯高于對照組(P0.05)。(6)PTS增加缺血側(cè)皮層血管內(nèi)皮細胞增殖:CD31與Brdu免疫熒光共染顯示MCAO術(shù)后存在血管內(nèi)皮細胞增殖,PTS治療組CD31與Brdu共定位細胞明顯多于對照組,兩組比較P0.05,有統(tǒng)計學意義。(7)PTS激活Shh信號通路,上調(diào)血管生長因子表達:術(shù)后7天westernblot檢測Shh通路主要信號蛋白(Shh、Smo、Ptch-1)表達,結(jié)果顯示,術(shù)后PTS能夠明顯上調(diào)Shh、Smo、Ptch-1表達,環(huán)靶明靶向阻斷Shh信號后明顯減少PTS誘導的VEG及Ang-1表達,表明PTS可能主要通過激活Shh信號通路促進腦梗死后血管新生。結(jié)論:研究結(jié)果表明,腦梗死后PTS能夠通過激活Shh信號通路,上調(diào)缺血側(cè)腦組織VEGF/VEGFR2和Ang-1/Tie-2系統(tǒng)的表達,增加腦微血管密度,改善缺血區(qū)腦灌注,進而減小腦梗死體積,改善神經(jīng)行為學評分。該實驗結(jié)果為PTS的臨床推廣應(yīng)用提供更多的理論依據(jù),為中醫(yī)藥治療腦梗死的研究提供新的思路和方法。
[Abstract]:BACKGROUND: Stroke is a common nervous system disease with high morbidity, mortality and disability, of which about 87% is ischemic stroke. Early recovery of cerebral perfusion in the ischemic region and rescue of dying neurons in the ischemic penumbra are the focus and goal of the treatment of cerebral infarction. Panaxatriol Saponins (PTS) is an effective component extracted from Panax notoginseng. Panaxatriol R1, ginsenoside Rg1 and ginsenoside Re are the main components of PTS. Previous studies have confirmed that PTS has protective effect on cerebral infarction rats, but the specific mechanism is unclear. Studies have shown that PTS can promote the proliferation of human umbilical vein endothelial cells, but there is no report on the role of PTS in promoting angiogenesis in ischemic brain injury. (3) To explore the mechanism of PTS promoting angiogenesis after cerebral infarction. Methods: SPF grade SD male rats were randomly divided into (1) sham operation group: intraperitoneal injection of normal saline once a day; (2) MCAO (middle cerebral artery infarction) model group: MCAO + normal saline, intraperitoneal injection once a day; (3) PTS treatment group: MCAO + PTS, intraperitoneal injection of PTS, 50mg / kg / d. Neurobehavioral score and TTC staining were performed at different time points after perfusion to evaluate the therapeutic effect of PTS on cerebral infarction in rats; Micro-PET imaging was used to evaluate the effect of PTS on cerebral blood flow perfusion; RT-PCR and Western blot were used to detect the gene and protein expression of angiogenesis-related factors at different time points; immunofluorescence staining was used to evaluate the effect of PTS on cerebral infarction in rats. To further evaluate the effect of PTS on proliferation of vascular endothelial cells, Brdu was injected intraperitoneally into MCAO rats and co-localized with CD31 immunofluorescence to evaluate the number of neovascular endothelial cells. Results: (1) Protective effect of PTS on the brain of MCAO rats: TTC and Neuron staining showed that PTS could significantly reduce cerebral infarction at 3, 7 and 14 days after reperfusion. Volume, compared with the control group had statistical significance; Longa neurological function score showed that PTS administration could improve the neurobehavioral score of MCAO rats from 3 days to 14 days, and lasted for 14 days, compared with the control group, P 0.05, with statistical significance. (2) PTS increased ischemic cerebral perfusion of MCAO rats: Micro-PET/CT scan showed that 1 day after MCAO modeling 18F-PDG brain. Micro PET showed that the brain uptake of 18F-PDG in PTS treatment group was significantly increased, indicating that cerebral blood flow was significantly improved, compared with the control group, P 0.05, with statistical significance. (3) PTS increased the expression of angiogenesis-related factors mRNA in ischemic cortex: RT-PCR results showed that the level of VEGF mRNA after MCAO with time The expression of Ang-1 mRNA was similar to that of VEGF, and the expression of VEGF R2, Tie-2, CD31 mRNA increased continuously after MCAO, and reached a peak on the 7th day. The expression of alpha-SMA mRNA was similar to that of Ang-1 after MCAO, and was significantly higher in the treatment group than in the control group (P 0.05). PTS up-regulated the expression of VEGF, Ang-1, VEGR2, Tie-2 protein in ischemic cerebral cortex: Western blot immunoblot showed that the level of VEGF protein in the cerebral cortex of rats after MCAO reached a peak on the 7th day after operation, and decreased on the 14th day. Compared with the control group, the expression of Ang-1 protein was statistically significant on the 3rd, 7th and 14th day (P 0.05). Compared with the control group, PTS increased the expression of VEGF R2 and Tie-2 on the 3rd day, 7th day and 14th day (P 0.05). At the 7th day after operation, PTS treatment group significantly increased the expression of VEGF R2 and Tie-2 protein, which was superior to the control group (P 0.05). (5) PTS increased the microvessel density of ischemic cortex: A study. The results showed that hypoxia-ischemia induced a large number of angiogenesis around the lesion, and the microvessel density reached a peak at 7 days after cerebral infarction, and decreased at 14 days. The CD31/alpha-SMA positive microvessel density in PTS treatment group was significantly higher than that in control group at 7 days and 14 days (P 0.05). (6) PTS increased the proliferation of vascular endothelial cells in ischemic cortex: CD31. The co-localization of CD31 and Brdu in PTS group was significantly higher than that in control group (P 0.05). (7) PTS activated Shh signaling pathway and up-regulated the expression of vascular growth factor. The main signal proteins of Shh signaling pathway (Shh, Smo, Ptch-1) were detected by Western blot 7 days after MCAO. The results showed that PTS could significantly up-regulate the expression of Shh, Smo and Ptch-1 after operation. Cyclotargeting could significantly reduce the expression of VEG and Ang-1 induced by PTS after blocking Shh signal, suggesting that PTS could promote angiogenesis after cerebral infarction mainly by activating Shh signal pathway. The results provide more theoretical basis for the clinical application of PTS and provide new ideas and methods for the study of TCM treatment of cerebral infarction. Method.
【學位授予單位】：南京中醫(yī)藥大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R743.3

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