人参皂甙Rd对成年大鼠缺血性脑卒中后血管发生的机制研究

发布时间：2018-01-03 06:28

  本文关键词：人参皂甙Rd对成年大鼠缺血性脑卒中后血管发生的机制研究　出处：《第四军医大学》2016年硕士论文　论文类型：学位论文

  更多相关文章： 人参皂甙Rd 人脐静脉微血管内皮细胞 氧糖剥夺 血管内皮细胞生长因子 低氧诱导因子1-α 哺乳动物雷帕霉素靶蛋白 蛋白激酶-B

【摘要】：缺血性脑卒中以其高发病率、高死亡率和高致残率成为影响全球健康的首要杀手。脑血流一旦被阻塞超过数分钟,神经细胞便发生不可逆的损伤和死亡。人参皂甙(Ginsenoside Rd,GSRd)是从三七、人参中提取的有效单体成分,本课题组前期的实验显示GSRd除了可以促进大鼠MCAO后梗死灶周围的微血管发生,还可以促进体外培养人脐静脉微血管内皮细胞(human umbilical vein microvascular endothelial cells,HUVECs)的管腔形成,并初步发现GSRd的作用与Hif-1α的激活相关。本课题拟应用免疫组化、Western blot、人脐静脉微血管内皮细胞培养等技术,分别在体内和体外,进一步探讨GSRd的促血管发生作用机制是否与信号通路AKT-mTORHif-1α-VEGF的序列性激活相关。实验一人参皂甙Rd对成年大鼠脑梗死后血管发生的影响目的:进一步验证GSRd对成年大鼠脑梗死后血管发生的影响。方法:160只成年雄性Sprague-Dawley大鼠,体重280~300g,采用MCAO模型,随机分为4组:Sham+SA组,Sham+GSRd组,MCAO+SA组,MCAO+GSRd组。造模成功6 h后开始腹腔注射GSRd 10 mg/kg/d,连续7 d,MCAO对照组给予同体积生理盐水,并分别在PSD 1、PSD 3、PSD 7、PSD 14和PSD 21留取大鼠脑标本行冰冻切片,用RECA-1单克隆抗体分别标记不同时间点大鼠的微血管,观察缺血半暗带区微血管的密度和分支点的变化。结果:各Sham组之间微血管密度和分支点无变化(P0.05,Sham+SA组vs.Sham+GSRd组);MCAO+SA组微血管的密度和分支点在PSD 1明显减少(P0.05),在PSD 3、PSD 7和PSD 14时微血管的密度和分支点呈逐渐上升的趋势,并且在PSD14达到峰值,PSD 21有所下降(MCAO+SA组vs.Sham组);MCAO+GSRd组微血管密度和分支点在各时间点的变化趋势和MCAO+SA组基本一致,但其微血管密度和分支点在每个时间点均高于MCAO+SA组(P0.05,MCAO+GSRd组vs.MCAO+SA组)。结论:GSRd可明显促进大鼠脑梗死后微血管的发生。实验二人参皂甙Rd对成年大鼠脑梗死后血管发生的机制研究目的:探索GSRd促进血管生长因子靶点是否包括Hif-1α的上游因子AKT和mTOR,进而探讨GSRd是否通过激活信号通路AKT-mTOR-Hif-1α-VEGF而发挥促血管发生的作用。方法:(1)成年雄性SD大鼠随机分为4组:Sham+SA组,Sham+GSRd组,MCAO+SA组,MCAO+GSRd组。MCAO模型成功后6 h开始腹腔注射给药GSRd 10 mg/kg,连续给药3 d。在PSD 3,应用Western blot检测半暗带区脑组织中的VEGF、Hif-1α、p-mTOR和p-AKT的蛋白表达水平。(2)成年雄性SD大鼠随机分为3组: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,后再连续给药2 d,MCAO模型成功后6 h腹腔注射GSRd 10 mg/kg,连续3 d后,用Western blot检测半暗带区脑组织中的VEGF、Hif-1α、p-mTOR和p-AKT的蛋白表达变化,观察雷帕霉素抑制mTOR的激活后,其下游因子表达的情况。(3)成年SD大鼠随机分为3组:MCAO+SA组,MCAO+GSRd组,MCAO+GSRd+LY294002组。侧脑室立体定位埋入脑室套管,待大鼠恢复5 d后行MCAO模型,每天经脑室套管注射AKT抑制剂LY294002(10 m M,5 ul),腹腔注射GSRd10 mg/kg/d,两者均连续3 d,用Western blot检测半暗带区脑组织中VEGF、Hif-1α、p-mTOR和p-AKT的蛋白表达变化。(4)分组同(3),大鼠连续用LY294002和GSRd,方法同(3),连续给药7 d脑组织冰冻切片后,用RECA-1单克隆抗体免疫组化染色,观察LY294002对大鼠微血管的影响。结果:(1)在PSD 3,Sham组之间VEGF、Hif-1α、p-mTOR和p-AKT的表达无差别(P0.05,Sham+SA组vs.Sham+GSRd组);MCAO后VEGF、Hif-1α、p-mTOR和p-AKT的表达均下降(P0.05,MCAO+SA组vs.Sham组);GSRd可明显促进VEGF、Hif-1α、p-mTOR和p-AKT的表达(P0.05,MCAO+GSRd组vs.MCAO+SA组)。(2)在加入mTOR特异性阻断剂雷帕霉素后,mTOR的活化形式p-mTOR以及其下游Hif-1α和VEGF的表达均明显降低(P0.05),而其上游p-AKT的表达不受影响(P0.05,MCAO+GSRd+雷帕霉素组vs.MCAO+GSRd组)。(3)在加入AKT抑制剂LY294002后,p-AKT、p-mTOR、Hif-1α和VEGF的表达均明显降低(P0.05,MCAO+GSRd+LY294002组vs.MCAO+GSRd组);(4)LY294002可明显减少半暗带区微血管密度(P0.01,MCAO+GSRd+LY294002组vs.MCAO+GSRd组=426.56±48.38 vs.532.03±16.17),同时减少分支点(P0.01,MCAO+GSRd+LY294002组vs.MCAO+GSRd组=212.50±24.32 vs.330.47±20.44)。结论:在体内,GSRd促进血管发生的作用可能与AKT-mTOR-Hif-1α-VEGF信号通路激活相关。实验三人参皂甙Rd对体外培养人脐静脉微血管内皮细胞(HUVECs)的作用目的:在体外培养的HUVECs中探讨GSRd是否通过AKT-mTOR-Hif-1α-VEGF的激活而发挥促血管发生的作用。方法:(1)模型的建立。采用氧糖剥夺/再灌注损伤模型(oxygen-glucose deprivation/reperfusion,OGD)。体外培养HUVECs,实验分为5组:正常培养组、OGD组、OGD+GSRd 1 uM组、5 uM组、10 uM组。正常培养的HUVECs氧糖剥夺8 h,复氧12h后,吸取上清20 ul检测LDH释放量,向贴壁细胞的96孔板中加入CCK-8试剂测定细胞活性,贴壁细胞经消化后采用流式细胞仪检测细胞凋亡。(2)GSRd作用机制的探索。实验分为6组:正常组、OGD组、OGD+GSRd 1 uM组、OGD+GSRd 1 uM+2-ME2组、OGD+GSRd 1 uM+雷帕霉素组、OGD+GSRd 1 uM+LY294002组。OGD 8 h复氧12 h后,分别提取细胞总蛋白或核蛋白,通过Western blot检测VEGF、p-mTOR、p-AKT或Hif-1α的表达。结果:(1)OGD后细胞存活明显减少,细胞毒性和细胞凋亡明显增加(P0.05,OGD组vs.正常培养组);1 uM和5 uM的GSRd干预后细胞存活均明显提高,毒性显著降低,凋亡明显减少(P0.05,GSRd 1或5 uM组vs.OGD组),5 uM组和1 uM组之间无差别(P0.05),而10 uM的GSRd干预后细胞存活却明显降低、凋亡比例显著上升(P0.05,OGD+GSRd 10 uM vs.OGD+GSRd 1 uM)。(2)正常培养的细胞可表达VEGF、Hif-1α、p-mTOR和p-AKT,OGD后VEGF、Hif-1α、p-mTOR和p-AKT的表达均明显降低(P0.05,OGD组vs.正常培养组);1 uM GSRd干预后上述因子的表达明显增高(P0.05,GSRd 1 uM组vs.OGD组);分别加入Hif-1α抑制剂2-ME2、mTOR抑制剂雷帕霉素和AKT抑制剂LY294002后,Hif-1α和VEGF的表达均被明显抑制(P0.05,OGD+GSRd+抑制剂组vs.OGD+GSRd组),雷帕霉素可显著抑制p-mTOR的表达但不能抑制其上游信号通路中p-AKT的表达,LY294002则可同时抑制p-AKT及其下游p-mTOR、Hif-1α和VEGF的表达。结论:在体外,GSRd促进血管发生的作用可能与AKT-mTOR-Hif-1α-VEGF信号通路的激活相关。
[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�楃�，

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