本文選題：腦微血管內(nèi)皮細(xì)胞 + necroptosis　； 參考：《北京中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：近些年,國內(nèi)外學(xué)者發(fā)現(xiàn)了一種新的細(xì)胞死亡形式"Necroptosis",它是由死亡受體介導(dǎo)的,被一系列信號傳導(dǎo)通路所調(diào)控的caspase非依賴性的細(xì)胞死亡方式,同時具有壞死和凋亡的特征。這種細(xì)胞死亡形式最早在小鼠大腦中動脈缺血再灌注損傷模型中發(fā)現(xiàn),隨后多個實(shí)驗(yàn)證實(shí)necroptosis參與了缺血缺氧造成的腦組織損害。三七是我國著名的傳統(tǒng)中藥材,臨床廣泛應(yīng)用于缺血性腦卒中的治療。三七總皂苷(total saponins ofpanax notoginseseng,PNS)是中藥三七的主要藥效組分,前期研究發(fā)現(xiàn)三七總皂苷能明顯降低實(shí)驗(yàn)性腦缺血大鼠的神經(jīng)功能評分,減少腦梗死體積,減輕腦水腫,其發(fā)揮腦保護(hù)的作用與阻抑腦缺血后血管炎癥損傷、促進(jìn)血管修復(fù)和保護(hù)血管內(nèi)皮細(xì)胞密切相關(guān)。因此,在前期實(shí)驗(yàn)研究的基礎(chǔ)上,本實(shí)驗(yàn)利用缺氧缺糖復(fù)氧復(fù)糖法聯(lián)合caspase抑制劑z-VAD-FMK干預(yù)制備了缺血再灌注損傷腦微血管內(nèi)皮細(xì)胞necroptosis模型;觀察了三七總皂苷對缺血再灌注損傷誘導(dǎo)的腦微血管內(nèi)皮細(xì)胞發(fā)生necroptosis及其RIP1-RIP3-MLKL信號通路相關(guān)因子表達(dá)的影響,以深入揭示三七總皂昔在腦缺血再灌注損傷中發(fā)揮血管內(nèi)皮保護(hù)作用的分子機(jī)制。目的:探討三七總皂苷對缺血再灌注損傷誘導(dǎo)的腦微血管內(nèi)皮細(xì)胞necroptosis的影響,以及對RIP1-RIP3-MLKL信號轉(zhuǎn)導(dǎo)通路和線粒體損傷的調(diào)節(jié)作用,以揭示三七總皂苷在腦缺血再灌注損傷中發(fā)揮抗腦微血管內(nèi)皮細(xì)胞necroptosis的分子機(jī)制,為三七總皂苷治療缺血性腦中風(fēng)的藥理機(jī)制提供新的科學(xué)詮釋。方法:1.缺血再灌注損傷腦微血管內(nèi)皮細(xì)胞necroptosis模型的建立:利用原代培養(yǎng)的大鼠腦微血管內(nèi)皮細(xì)胞(brain microvascular endothelial cells,BMECs),首先采用缺氧缺糖復(fù)氧復(fù)糖的方法篩選了最佳損傷時間點(diǎn),制備擬缺血再灌注損傷(ischemia-reperfusion injury,IRI)模型。在此基礎(chǔ)上利用caspase抑制劑z-VAD-FMK進(jìn)行干預(yù),分別采用CCK-8檢測、透射電鏡觀察、Annexin V-FITC/PI雙染色法進(jìn)行流式細(xì)胞分析,觀察造模后細(xì)胞的死亡特征,建立necroptosis細(xì)胞模型。2.觀察三七總皂苷對缺血再灌注損傷腦微血管內(nèi)皮細(xì)胞necroptosis的影響:將培養(yǎng)傳至第3代的大鼠腦微血管內(nèi)皮細(xì)胞隨機(jī)分成4組:正常組、IRI+z-VAD-FMK組、IRI+z-VAD-FMK+PNS組和IRI+z-VAD-FMK+Nec-1組。除正常組外,其余三組均按照上述方法進(jìn)行造模。IRI+z-VAD-FMK+PNS組,細(xì)胞在造模前3h和造模過程中給藥,三七總皂苷的給藥濃度是22μg/ml。IRI+z-VAD-FMK+Nec-1組,細(xì)胞在造模前30min及造模過程中按10μmol/L濃度加入necroptosis特異性阻斷劑necstatin-1(Nec-1)。造模及處理結(jié)束后,采用CCK-8檢測各組細(xì)胞活性,透射電鏡觀察細(xì)胞超微結(jié)構(gòu)和形態(tài)變化,Annexin V-FITC/PI雙染色法利用流式細(xì)胞儀檢測細(xì)胞死亡方式。3.三七總皂苷對缺血再灌注損傷腦微血管內(nèi)皮細(xì)胞RIP1-RIP3-MLKL信號通路的影響:將傳至第3代的大鼠腦微血管內(nèi)皮細(xì)胞和7代以后的人腦微血管內(nèi)皮細(xì)胞隨機(jī)分成 4 組:正常組、IRI+z-VAD-FMK 組、IRI+z-VAD-FMK+PNS 組和 IRI+z-VAD-FMK+Nec-1組。給藥方法同以上。采用熒光定量PCR(RT-PCR)和免疫印跡(Western Blotting)法檢測RIP1-RIP3-MLKL信號通路中RIP1、RIP3、MLKL的mRNA及蛋白磷酸化水平。4.三七總皂苷對缺血再灌注損傷腦微血管內(nèi)皮細(xì)胞necroptosis線粒體膜電位變化的影響:細(xì)胞的分組同上,采用RT-PCR和Western Blotting法檢測各組細(xì)胞PGAM5、Drp1的mRNA及蛋白表達(dá)水平;通過JC-1染色,分別用流式細(xì)胞儀和熒光倒置相差顯微鏡觀察各組細(xì)胞線粒體膜電位的變化。結(jié)果:1.制備了擬缺血再灌注損傷腦微血管內(nèi)皮細(xì)胞necroptosis模型:缺氧缺糖2h復(fù)氧復(fù)糖8h時,OD值降低明顯(P0.01),細(xì)胞損傷明顯且損傷主要發(fā)生在再灌后4-8h,比較符合缺血再灌注損傷特點(diǎn),因此確定該時間點(diǎn)作為模擬缺血再灌注損傷的最佳時間點(diǎn)。加入z-VAD-FMK后細(xì)胞具有明顯壞死細(xì)胞特征,necroptosis特異性抑制劑Nec-1可顯著降低Q2象限細(xì)胞百分比率,提高細(xì)胞活性(P0.05),提示細(xì)胞在這一過程中發(fā)生了 necroptosis。2.三七總皂苷可減輕缺血再灌注損傷腦微血管內(nèi)皮細(xì)胞發(fā)生necroptosis:與IRI+z-VAD-FMK 組相比,IRI+z-VAD-FMK+PNS 組與 IRI+z-VAD-FMK+Nec-1 組細(xì)胞活性明顯上升(P0.01,P0.01),細(xì)胞結(jié)構(gòu)損傷有所改善,細(xì)胞核和細(xì)胞膜較完整,線粒體損傷改善;Q2象限細(xì)胞比率顯著降低(P0.05),Q3象限比率顯著升高(P0.01),具有統(tǒng)計(jì)學(xué)意義。3.三七總皂苷對缺血再灌注損傷腦微血管內(nèi)皮細(xì)胞RIP1-RIP3-MLKL信號通路有下調(diào)作用:造模后,RIP1-RIP3-MLKL信號轉(zhuǎn)導(dǎo)通路被激活,RIP1、RIP3、MLKLmRNA表達(dá)和蛋白磷酸化水平明顯升高,與IRI+z-VAD-FMK組相比較,三七總皂苷能夠降低三種信號因子蛋白磷酸化及mRNA的表達(dá),與Nec-1顯示出相似作用。4.三七總皂苷可降低缺血再灌注損傷腦微血管內(nèi)皮細(xì)胞necroptosis線粒體損傷:造模后,大鼠腦微血管內(nèi)皮細(xì)胞PGAM5、Drp1的mRNA和蛋白表達(dá)明顯升高,線粒體膜電位下降;與IRI+z-VAD-FMK組相比,IRI+z-VAD-FMK+PNS組和IRI+z-VAD-FMK+Nec-1組細(xì)胞PGAM5、Drp1mRNA和蛋白表達(dá)均下降,線粒體膜電位上升。結(jié)論:1.缺氧缺糖復(fù)氧復(fù)糖法聯(lián)合z-VAD-FMK干預(yù)后腦微血管內(nèi)皮細(xì)胞的死亡特征符合necroptosis的特點(diǎn),提示該方法可制備necroptosis模型。2.三七總皂苷可有效降低上述方法誘導(dǎo)的腦微血管內(nèi)皮細(xì)胞necroptosis的發(fā)生,其內(nèi)在機(jī)制與抑制RIP1-RIP3-MLKL信號通路活化,繼而下調(diào)下游PGAM5、Drp1的表達(dá),減輕線粒體損傷有關(guān),這可能是三七總皂苷在腦缺血再灌注損傷中發(fā)揮血管內(nèi)皮保護(hù)作用的分子機(jī)制之一。該結(jié)論為中藥三七的"通絡(luò)"作用提供了現(xiàn)代生物學(xué)基礎(chǔ)。
[Abstract]:In recent years, domestic and foreign scholars have discovered a new form of cell death "Necroptosis", which is a death receptor mediated, caspase non dependent cell death mode regulated by a series of signal transduction pathways, and has the characteristics of necrosis and apoptosis. This form of cell death was the earliest in the middle cerebral artery ischemia and reperfusion in mice. It was found in the damage model that the subsequent experiments confirmed that necroptosis was involved in the brain tissue damage caused by ischemia and hypoxia. 37 it was a famous traditional Chinese medicine in China, and was widely used in the treatment of ischemic stroke. 37 total saponins (total saponins ofPanax Notoginseseng, PNS) were the main components of Chinese medicine 37, the preliminary study It is found that 37 total saponins can obviously reduce the neurological function score of experimental cerebral ischemia rats, reduce the volume of cerebral infarction and reduce the brain edema. The effect of the total saponins can be closely related to the inhibition of vascular inflammation after cerebral ischemia, the promotion of vascular repair and the protection of vascular endothelial cells. The necroptosis model of cerebral microvascular endothelial cells damaged by ischemia-reperfusion injury was prefabricated by hypoxic and glucose deficient reoxygenation combined with caspase inhibitor z-VAD-FMK, and the effects of 37 total saponins on the expression of necroptosis and RIP1-RIP3-MLKL signaling pathway related factors in cerebral microvascular endothelial cells induced by ischemia-reperfusion injury were observed. To explore the molecular mechanism of vascular endothelium protection of 37 total soap shake in cerebral ischemia reperfusion injury. Objective: To explore the effect of 37 total saponins on the necroptosis of cerebral microvascular endothelial cells induced by ischemia-reperfusion injury, and the regulation of RIP1-RIP3-MLKL signal transduction pathway and mitochondrial damage to reveal three Seven total saponins play the molecular mechanism of anti cerebral microvascular endothelial cell necroptosis in cerebral ischemia reperfusion injury, and provide a new scientific interpretation for the pharmacological mechanism of 37 total saponins in the treatment of ischemic stroke. Method: the establishment of the necroptosis model of cerebral microvascular endothelial cells in 1. ischemia reperfusion injury: using the primary culture of rat brain microsphere Brain microvascular endothelial cells (BMECs), first of all, the optimal time point of injury time was screened by the method of hypoxia and glucose deficiency complex carbohydrate. The model of pseudo ischemia-reperfusion injury (ischemia-reperfusion injury, IRI) was prepared. On the basis of this, the caspase inhibitor z-VAD-FMK was used to intervene with CCK-8 detection and transmission, respectively. Electron microscope observation, Annexin V-FITC/PI double staining method for flow cytometry, observed the death characteristics of the cells after the model, and established the necroptosis cell model.2. to observe the effect of 37 total saponins on the cerebral microvascular endothelial cell necroptosis in the ischemic reperfusion injury: the cultured rat cerebral microvascular endothelial cells were divided into 4 groups randomly. The normal group, the IRI+z-VAD-FMK group, the IRI+z-VAD-FMK+PNS group and the IRI+z-VAD-FMK+Nec-1 group. Except the normal group, the other three groups were made the model.IRI+z-VAD-FMK+PNS group according to the above methods. The cells were given in the 3H and the mold making process before the model, and the concentration of the 37 total saponins was 22 Mu g/ ml.IRI+z-VAD-FMK+Nec-1 group. The cells were in the 30min and the model before making the mold. The necroptosis specific blocking agent necstatin-1 (Nec-1) was added to the concentration of 10 micron mol/L. After the model and treatment, the cell activity was detected by CCK-8, the ultrastructure and the morphological changes were observed by transmission electron microscopy. The Annexin V-FITC/PI double staining method was used to detect the cell death of.3. 37 total saponins by flow cytometry. The effect of perfusion injury on RIP1-RIP3-MLKL signal pathway of cerebral microvascular endothelial cells: third generations of rat brain microvascular endothelial cells and 7 generations of human brain microvascular endothelial cells were randomly divided into 4 groups: normal group, IRI+z-VAD-FMK group, IRI+z-VAD-FMK+PNS group and IRI+z-VAD-FMK+ Nec-1 group. PCR (RT-PCR) and immunoblotting (Western Blotting) assay were used to detect the effects of RIP1, RIP3, MLKL, and protein phosphorylation level.4. 37 total saponins on the changes in the necroptosis mitochondrial membrane potential of cerebral microvascular endothelial cells in the RIP1-RIP3-MLKL signaling pathway. The mRNA and protein expression levels of PGAM5 and Drp1 were measured in each group. The changes in mitochondrial membrane potential of each cell were observed by flow cytometry and fluorescence inversion phase contrast microscope by JC-1 staining. Results: 1. the necroptosis model of cerebral microvascular endothelial cells with ischemic reperfusion injury was prepared. The decrease of OD value was reduced when hypoxia and glucose deficient 2H reoxygenate 8h was reduced. Obviously (P0.01), the cell damage is obvious and the damage mainly occurs after reperfusion 4-8h, which is more in line with the characteristics of ischemia-reperfusion injury. Therefore, the time point is the best time point for simulating the ischemia reperfusion injury. After the addition of z-VAD-FMK, the cells have obvious necrotic cell characteristics, and the necroptosis specific inhibitor Nec-1 can significantly reduce the Q2 quadrant. The percentage of cells increased cell activity (P0.05), suggesting that necroptosis.2. 37 total saponins in this process could reduce the incidence of necroptosis: in cerebral microvascular endothelial cells from ischemia reperfusion injury and IRI+z-VAD-FMK group, and the cell activity of IRI+z-VAD-FMK+PNS group and IRI+ z-VAD-FMK+Nec-1 group increased significantly (P0.01, P0.01). Cell structure damage improved, nuclear and cell membrane were more complete, mitochondrial damage improved, Q2 quadrant cell ratio decreased significantly (P0.05), Q3 quadrant ratio increased significantly (P0.01), and.3. 37 total saponins had a downregulation effect on RIP1-RIP3-MLKL signaling pathway of cerebral microvascular intravascular cells after ischemia-reperfusion injury: after modeling, RI The P1-RIP3-MLKL signal transduction pathway was activated, and the expression of RIP1, RIP3, MLKLmRNA and protein phosphorylation increased significantly. Compared with the IRI+z-VAD-FMK group, 37 total saponins could reduce the phosphorylation of three signal factor proteins and the expression of mRNA. The similar effect of.4. 37 total saponins to Nec-1 could reduce the cerebral microvascular injury of ischemia reperfusion injury. Necroptosis mitochondrial damage in the skin cells: after the model, the rat brain microvascular endothelial cells PGAM5, Drp1 mRNA and protein expression significantly increased, the mitochondrial membrane potential decreased. Compared with the IRI+z-VAD-FMK group, IRI+z-VAD-FMK+PNS and IRI+z-VAD-FMK+Nec-1 group PGAM5, Drp1mRNA and protein expression decreased, mitochondrial membrane potential increased. Conclusion: 1. deficiency The characteristics of the death of cerebral microvascular endothelial cells after z-VAD-FMK intervention with oxygen glucose deficiency combined with carbohydrate and reoxygenation method are consistent with the characteristics of necroptosis. It is suggested that the preparation of necroptosis model.2. 37 total saponins can effectively reduce the occurrence of necroptosis in the cerebral microvascular endothelial cells induced by the above method, and its intrinsic mechanism and the suppression of RIP1-RIP3-MLKL signal The pathway activation, then down down downstream PGAM5, Drp1 expression, alleviates mitochondrial damage, which may be one of the molecular mechanisms of 37 total saponins playing the protective role of vascular endothelium in cerebral ischemia reperfusion injury. This conclusion provides a biological basis for the "collaterals" of Chinese medicine 37.

【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R285

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