發(fā)布時(shí)間：2018-01-03 06:28

  本文關(guān)鍵詞：人參皂甙Rd對(duì)成年大鼠缺血性腦卒中后血管發(fā)生的機(jī)制研究　出處：《第四軍醫(yī)大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 人參皂甙Rd 人臍靜脈微血管內(nèi)皮細(xì)胞 氧糖剝奪 血管內(nèi)皮細(xì)胞生長(zhǎng)因子 低氧誘導(dǎo)因子1-α 哺乳動(dòng)物雷帕霉素靶蛋白 蛋白激酶-B

【摘要】：缺血性腦卒中以其高發(fā)病率、高死亡率和高致殘率成為影響全球健康的首要?dú)⑹�。腦血流一旦被阻塞超過數(shù)分鐘,神經(jīng)細(xì)胞便發(fā)生不可逆的損傷和死亡。人參皂甙(Ginsenoside Rd,GSRd)是從三七、人參中提取的有效單體成分,本課題組前期的實(shí)驗(yàn)顯示GSRd除了可以促進(jìn)大鼠MCAO后梗死灶周圍的微血管發(fā)生,還可以促進(jìn)體外培養(yǎng)人臍靜脈微血管內(nèi)皮細(xì)胞(human umbilical vein microvascular endothelial cells,HUVECs)的管腔形成,并初步發(fā)現(xiàn)GSRd的作用與Hif-1α的激活相關(guān)。本課題擬應(yīng)用免疫組化、Western blot、人臍靜脈微血管內(nèi)皮細(xì)胞培養(yǎng)等技術(shù),分別在體內(nèi)和體外,進(jìn)一步探討GSRd的促血管發(fā)生作用機(jī)制是否與信號(hào)通路AKT-mTORHif-1α-VEGF的序列性激活相關(guān)。實(shí)驗(yàn)一人參皂甙Rd對(duì)成年大鼠腦梗死后血管發(fā)生的影響目的:進(jìn)一步驗(yàn)證GSRd對(duì)成年大鼠腦梗死后血管發(fā)生的影響。方法:160只成年雄性Sprague-Dawley大鼠,體重280~300g,采用MCAO模型,隨機(jī)分為4組:Sham+SA組,Sham+GSRd組,MCAO+SA組,MCAO+GSRd組。造模成功6 h后開始腹腔注射GSRd 10 mg/kg/d,連續(xù)7 d,MCAO對(duì)照組給予同體積生理鹽水,并分別在PSD 1、PSD 3、PSD 7、PSD 14和PSD 21留取大鼠腦標(biāo)本行冰凍切片,用RECA-1單克隆抗體分別標(biāo)記不同時(shí)間點(diǎn)大鼠的微血管,觀察缺血半暗帶區(qū)微血管的密度和分支點(diǎn)的變化。結(jié)果:各Sham組之間微血管密度和分支點(diǎn)無變化(P0.05,Sham+SA組vs.Sham+GSRd組);MCAO+SA組微血管的密度和分支點(diǎn)在PSD 1明顯減少(P0.05),在PSD 3、PSD 7和PSD 14時(shí)微血管的密度和分支點(diǎn)呈逐漸上升的趨勢(shì),并且在PSD14達(dá)到峰值,PSD 21有所下降(MCAO+SA組vs.Sham組);MCAO+GSRd組微血管密度和分支點(diǎn)在各時(shí)間點(diǎn)的變化趨勢(shì)和MCAO+SA組基本一致,但其微血管密度和分支點(diǎn)在每個(gè)時(shí)間點(diǎn)均高于MCAO+SA組(P0.05,MCAO+GSRd組vs.MCAO+SA組)。結(jié)論:GSRd可明顯促進(jìn)大鼠腦梗死后微血管的發(fā)生。實(shí)驗(yàn)二人參皂甙Rd對(duì)成年大鼠腦梗死后血管發(fā)生的機(jī)制研究目的:探索GSRd促進(jìn)血管生長(zhǎng)因子靶點(diǎn)是否包括Hif-1α的上游因子AKT和mTOR,進(jìn)而探討GSRd是否通過激活信號(hào)通路AKT-mTOR-Hif-1α-VEGF而發(fā)揮促血管發(fā)生的作用。方法:(1)成年雄性SD大鼠隨機(jī)分為4組:Sham+SA組,Sham+GSRd組,MCAO+SA組,MCAO+GSRd組。MCAO模型成功后6 h開始腹腔注射給藥GSRd 10 mg/kg,連續(xù)給藥3 d。在PSD 3,應(yīng)用Western blot檢測(cè)半暗帶區(qū)腦組織中的VEGF、Hif-1α、p-mTOR和p-AKT的蛋白表達(dá)水平。(2)成年雄性SD大鼠隨機(jī)分為3組:MCAO+SA,MCAO+GSRd,MCAO+GSRd+雷帕霉素。造模30 min前腹腔注射雷帕霉素250 ug/kg,后再連續(xù)給藥2 d,MCAO模型成功后6 h腹腔注射GSRd 10 mg/kg,連續(xù)3 d后,用Western blot檢測(cè)半暗帶區(qū)腦組織中的VEGF、Hif-1α、p-mTOR和p-AKT的蛋白表達(dá)變化,觀察雷帕霉素抑制mTOR的激活后,其下游因子表達(dá)的情況。(3)成年SD大鼠隨機(jī)分為3組:MCAO+SA組,MCAO+GSRd組,MCAO+GSRd+LY294002組。側(cè)腦室立體定位埋入腦室套管,待大鼠恢復(fù)5 d后行MCAO模型,每天經(jīng)腦室套管注射AKT抑制劑LY294002(10 m M,5 ul),腹腔注射GSRd10 mg/kg/d,兩者均連續(xù)3 d,用Western blot檢測(cè)半暗帶區(qū)腦組織中VEGF、Hif-1α、p-mTOR和p-AKT的蛋白表達(dá)變化。(4)分組同(3),大鼠連續(xù)用LY294002和GSRd,方法同(3),連續(xù)給藥7 d腦組織冰凍切片后,用RECA-1單克隆抗體免疫組化染色,觀察LY294002對(duì)大鼠微血管的影響。結(jié)果:(1)在PSD 3,Sham組之間VEGF、Hif-1α、p-mTOR和p-AKT的表達(dá)無差別(P0.05,Sham+SA組vs.Sham+GSRd組);MCAO后VEGF、Hif-1α、p-mTOR和p-AKT的表達(dá)均下降(P0.05,MCAO+SA組vs.Sham組);GSRd可明顯促進(jìn)VEGF、Hif-1α、p-mTOR和p-AKT的表達(dá)(P0.05,MCAO+GSRd組vs.MCAO+SA組)。(2)在加入mTOR特異性阻斷劑雷帕霉素后,mTOR的活化形式p-mTOR以及其下游Hif-1α和VEGF的表達(dá)均明顯降低(P0.05),而其上游p-AKT的表達(dá)不受影響(P0.05,MCAO+GSRd+雷帕霉素組vs.MCAO+GSRd組)。(3)在加入AKT抑制劑LY294002后,p-AKT、p-mTOR、Hif-1α和VEGF的表達(dá)均明顯降低(P0.05,MCAO+GSRd+LY294002組vs.MCAO+GSRd組);(4)LY294002可明顯減少半暗帶區(qū)微血管密度(P0.01,MCAO+GSRd+LY294002組vs.MCAO+GSRd組=426.56±48.38 vs.532.03±16.17),同時(shí)減少分支點(diǎn)(P0.01,MCAO+GSRd+LY294002組vs.MCAO+GSRd組=212.50±24.32 vs.330.47±20.44)。結(jié)論:在體內(nèi),GSRd促進(jìn)血管發(fā)生的作用可能與AKT-mTOR-Hif-1α-VEGF信號(hào)通路激活相關(guān)。實(shí)驗(yàn)三人參皂甙Rd對(duì)體外培養(yǎng)人臍靜脈微血管內(nèi)皮細(xì)胞(HUVECs)的作用目的:在體外培養(yǎng)的HUVECs中探討GSRd是否通過AKT-mTOR-Hif-1α-VEGF的激活而發(fā)揮促血管發(fā)生的作用。方法:(1)模型的建立。采用氧糖剝奪/再灌注損傷模型(oxygen-glucose deprivation/reperfusion,OGD)。體外培養(yǎng)HUVECs,實(shí)驗(yàn)分為5組:正常培養(yǎng)組、OGD組、OGD+GSRd 1 uM組、5 uM組、10 uM組。正常培養(yǎng)的HUVECs氧糖剝奪8 h,復(fù)氧12h后,吸取上清20 ul檢測(cè)LDH釋放量,向貼壁細(xì)胞的96孔板中加入CCK-8試劑測(cè)定細(xì)胞活性,貼壁細(xì)胞經(jīng)消化后采用流式細(xì)胞儀檢測(cè)細(xì)胞凋亡。(2)GSRd作用機(jī)制的探索。實(shí)驗(yàn)分為6組:正常組、OGD組、OGD+GSRd 1 uM組、OGD+GSRd 1 uM+2-ME2組、OGD+GSRd 1 uM+雷帕霉素組、OGD+GSRd 1 uM+LY294002組。OGD 8 h復(fù)氧12 h后,分別提取細(xì)胞總蛋白或核蛋白,通過Western blot檢測(cè)VEGF、p-mTOR、p-AKT或Hif-1α的表達(dá)。結(jié)果:(1)OGD后細(xì)胞存活明顯減少,細(xì)胞毒性和細(xì)胞凋亡明顯增加(P0.05,OGD組vs.正常培養(yǎng)組);1 uM和5 uM的GSRd干預(yù)后細(xì)胞存活均明顯提高,毒性顯著降低,凋亡明顯減少(P0.05,GSRd 1或5 uM組vs.OGD組),5 uM組和1 uM組之間無差別(P0.05),而10 uM的GSRd干預(yù)后細(xì)胞存活卻明顯降低、凋亡比例顯著上升(P0.05,OGD+GSRd 10 uM vs.OGD+GSRd 1 uM)。(2)正常培養(yǎng)的細(xì)胞可表達(dá)VEGF、Hif-1α、p-mTOR和p-AKT,OGD后VEGF、Hif-1α、p-mTOR和p-AKT的表達(dá)均明顯降低(P0.05,OGD組vs.正常培養(yǎng)組);1 uM GSRd干預(yù)后上述因子的表達(dá)明顯增高(P0.05,GSRd 1 uM組vs.OGD組);分別加入Hif-1α抑制劑2-ME2、mTOR抑制劑雷帕霉素和AKT抑制劑LY294002后,Hif-1α和VEGF的表達(dá)均被明顯抑制(P0.05,OGD+GSRd+抑制劑組vs.OGD+GSRd組),雷帕霉素可顯著抑制p-mTOR的表達(dá)但不能抑制其上游信號(hào)通路中p-AKT的表達(dá),LY294002則可同時(shí)抑制p-AKT及其下游p-mTOR、Hif-1α和VEGF的表達(dá)。結(jié)論:在體外,GSRd促進(jìn)血管發(fā)生的作用可能與AKT-mTOR-Hif-1α-VEGF信號(hào)通路的激活相關(guān)。
[Abstract]:Ischemic stroke with high incidence, high mortality and high disability rate becomes the most important global health killer. Once the cerebral blood flow is blocked for more than a few minutes, nerve cells will occur irreversible injury and death. Ginsenoside (Ginsenoside Rd, GSRd) is from 37, one of effective components of ginseng extract and our previous experiments showed that GSRd can promote the occurrence of microvascular around the infarcted area of rats with MCAO, but also can promote the in vitro cultured human umbilical vein endothelial cells (human umbilical vein microvascular endothelial cells, HUVECs) lumen formation, and initially found that interaction with Hif-1 alpha GSRd activation related to this. Subject to the application of immunohistochemistry, Western blot, human umbilical vein endothelial cell culture technique, respectively in vivo and in vitro, to further explore the GSRd angiogenesis mechanism and whether the letter The sequence of AKT-mTORHif-1 alpha -VEGF activation pathway. The effect of ginsenoside Rd on cerebral infarction in adult rats after angiogenesis Objective: to further verify the effect of GSRd on cerebral infarction in adult rats after vascular. Methods: 160 adult male Sprague-Dawley rats, weight 280~300g, using the MCAO model, were randomly divided into 4 group: Sham+SA group, Sham+GSRd group, MCAO+SA group, MCAO+GSRd group. The successful model of 6 h after intraperitoneal injection of GSRd 10 mg/kg/d, MCAO for 7 d, the control group was given the same volume of physiological saline, and in PSD 1, PSD 3, PSD 7, PSD 14 and PSD 21 specimens from rat brain specimens frozen sections marked microvessels in rats at different time points respectively with RECA-1 monoclonal antibody, observe the changes of the ischemic penumbra microvessel density and the branch point. Results: between the Sham group of microvessel density and branch points of no change (P0.05, Sham+SA group vs.Sham+GSR Group D); group MCAO+SA microvessel density and branch point in PSD 1 was significantly reduced (P0.05), PSD 3, PSD 7 and PSD 14 density and branch points of microvessels increased gradually, and reached a peak at PSD14, PSD 21 decreased (MCAO+SA group vs.Sham); group MCAO+GSRd microvessel density and branch points are basically the same at each time point and the change trend of MCAO+SA group, but the microvessel density and branch points at each time point were higher than MCAO+SA group (P0.05 group, MCAO+GSRd group vs.MCAO+SA). Conclusion: GSRd can significantly promote the angiogenesis after cerebral infarction in rats. Experiment two ginsenoside Rd the cerebral infarction in adult rats after vascular Study on the mechanism of the objective: To explore the GSRd promotes vascular endothelial growth factor alpha target including Hif-1 upstream factor AKT and mTOR, and investigate whether GSRd through activation of AKT-mTOR-Hif-1 signaling pathway and angiogenesis play a -VEGF. . methods: (1) adult male SD rats were randomly divided into 4 groups: Sham+SA group, Sham+GSRd group, MCAO+SA group, MCAO+GSRd group,.MCAO Model 6 h after intraperitoneal injection of GSRd 10 mg/kg, administered continuously for 3 d. in PSD 3, the application of Western blot detection of ischemic penumbra in VEGF, Hif-1 alpha, the expression level of p-mTOR and p-AKT protein. (2) adult male SD rats were randomly divided into 3 groups: MCAO+SA, MCAO+GSRd, MCAO+GSRd+ and rapamycin. Model 30 min intraperitoneal injection of rapamycin 250 ug/kg, after continuous administration of 2 D, after the model of MCAO 6 h by intraperitoneal injection of GSRd 10 mg/kg, 3 consecutive D, using Western blot to detect ischemic penumbra in VEGF, Hif-1 alpha, the expression of p-mTOR and p-AKT protein activation observed in rapamycin inhibits mTOR, expression of its downstream factors. (3) Adult SD rats were randomly divided into 3 groups: MCAO group +SA, MCAO+GSRd group, MCAO+GSRd+LY294002 group. 渚ц剳瀹ょ珛浣撳畾浣嶅煁鍏ヨ剳瀹ゅ綆，

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