抗血小板药物对高血压大鼠微循环及血管重构的作用和炎症内皮微粒对内皮通透性的作用研究

发布时间：2018-01-14 18:18

本文关键词：抗血小板药物对高血压大鼠微循环及血管重构的作用和炎症内皮微粒对内皮通透性的作用研究　出处：《北京协和医学院》2015年硕士论文　论文类型：学位论文

【摘要】：目的：第一部分：探讨抗血小板药物氯吡格雷对自发性高血压大鼠(SHR)微循环及血管重构的作用。第二部分：探讨炎症因子C反应蛋白刺激人脐静脉内皮细胞产生的内皮微粒对体外培养单层内皮细胞通透性的作用。方法：第一部分：SPF级雄性13周龄SHR随机分为溶剂对照组、氯吡格雷组、奥美拉唑组及两药混合组,每组5只,分别灌胃1 mL 0.5%羧甲基纤维素钠(CMC-Na)、氯吡格雷溶液(10mg/kg).奥美拉唑溶液(20mg/kg)及两药混合溶液,每日一次。连续四周后,首先用高功率激光多普勒血流探测仪(LDF)检测大鼠脑皮质、耳廓及趾血流量、血细胞聚集度和血流速度,用血流成像仪(LDPI)观察脑血流分布状态；取溶剂对照组和氯吡格雷组大鼠的脑和主动脉固定,利用HE染色观察主动脉管壁厚度、管壁面积、管腔面积及管壁平滑肌、利用HE染色观察脑微血管数及利用免疫组化方法检测脑皮质血管内皮细胞CD31表达量等血管重构和微血管稀疏情况。采用SPSS17.0统计学软件进行统计学分析和比较。第二部分：体外培养人脐带静脉内皮细胞株(HUVEC),以倒置显微镜观察细胞生长过程并绘制细胞生长曲线。HUVEC经不同浓度C反应蛋白(0、25、50、75μg/mL)作用12 h,收集细胞培养液以制备内皮微粒。利用流式细胞仪检测CD62E+内皮微粒的百分率；利用Transwell系统检测内皮微粒对HUVEC通透性的影响；利用免疫荧光染色观察不同浓度内皮微粒对HUVEC肌动蛋白F-actin的表达影响。结果：第一部分：氯吡格雷组高血压大鼠的脑皮质、耳廓和趾血流量显著高于溶剂对照组(P值分别0.05、0.01和0.05)；氯吡格雷组高血压大鼠的耳廓和趾血流速度显著高于溶剂对照组(P值分别0.001和0.05)；氯吡格雷组高血压大鼠脑血流分布曲线与溶剂对照组相比整体右移,其脑高血流量(800-1500 PU)面积百分比显著高于溶剂对照组(P0.01)；本实验结果中,奥美拉唑在对微循环作用中并未显示对氯吡格雷改善微循环的减效作用。氯吡格雷组大鼠主动脉管壁厚度及面积明显低于溶剂对照组(P0.05),血管平滑肌增殖较溶剂对照组有所改善,排列相对连续工整,管腔面积与管壁面积比值明显高于溶剂对照组(P0.05),氯吡格雷组大鼠脑皮质内微血管数明显高于溶剂对照组(P0.05),血管CD31表达量明显高于溶剂对照组(P0.05)。第二部分：细胞接种8h后完全贴壁,6d后细胞融合可达80-90%,其中2-4d为细胞对数生长期,数目明显增多,体积增大,胞浆丰富,其他时间则变化不明显；与对照组相比,梯度浓度C反应蛋白(25、50、75μg/mL)可以刺激HUVEC产生更高的CD62E+内皮微粒,其中50和75 μg/mL两个浓度有显著差异(P值分别0.01和0.05)；与对照组相比,不同浓度内皮微粒(2500、5000、10000个/mL)使HUVEC通透性在15、30、60、90 min时刻显著增高(P值均分别0.01、0.05和0.001)；与对照组相比,随着刺激HUVEC的内皮微粒浓度升高,F-actin由细胞周围和细胞连接处向细胞内迁移,致密外周束逐渐减少,应力纤维和中央短纤维逐渐增粗增多。。结论：第一部分：氯吡格雷能够明显改善高血压大鼠体表和脑微循环,其机制可能与改善血管重构及有效增加微血管数量相关,有利于高血压预后和降低心脑血管并发症的发生。第二部分：在炎症因子C反应蛋白刺激下内皮细胞产生了特定表型的内皮微粒,以这种内皮微粒再作用于正常内皮细胞,可导致正常内皮细胞的细胞通透性发生显著变化,这种改变会对进一步导致内皮细胞损伤起重要作用。
[Abstract]:Objective: the first part: To investigate the drug clopidogrel in spontaneously hypertensive rats (SHR) microcirculation and vascular remodeling. The second part: endothelial microparticles of cytokine C reactive protein in cultured human umbilical vein endothelial cells from cultured monolayer endothelial cells permeability effect on in vitro. Methods: the first part: SPF male 13 weeks of age SHR were randomly divided into solvent control group, clopidogrel group, omeprazole group and the two drug combination group, 5 rats in each group were intragastric administration of 1 mL of 0.5% sodium carboxymethyl cellulose (CMC-Na), clopidogrel solution (10mg/kg). Omeprazole solution (20mg/kg) and the two drugs mixed solution, once a day. Four weeks, with the first high power laser Doppler blood flow detector (LDF) to detect the cerebral cortex of rats, ear and foot blood flow, blood cell aggregation and blood flow velocity, flow imaging (LDPI) to observe the distribution of cerebral blood flow. State; take the solvent control group and clopidogrel group rat brain and aorta fixation, staining of aortic wall thickness by HE, wall area, lumen area and tube wall smooth muscle, using HE staining to observe the cerebral microvessel count and by immunohistochemistry expression of skin cell CD31 content of vascular remodeling and microvascular rarefaction situation detection of cerebral blood vessel. And statistical analysis was conducted using SPSS17.0 statistical software. The second part: the in vitro cultured human umbilical vein endothelial cells (HUVEC), with inverted microscope to observe the cell growth process and cell growth curves of.HUVEC with different concentration of C reactive protein (0,25,50,75 g/mL) 12 h, the cell culture medium was collected in the preparation of endothelial microparticles. Using the percentage of detection of CD62E+ endothelial microparticles by flow cytometry; using Transwell system to detect endothelial microparticles effect on HUVEC permeability by immune; Fluorescent staining to observe the expression of different concentrations of HUVEC F-actin endothelial microparticles actin effect. Results: the first part: clopidogrel group hypertensive rats cerebral cortex, auricle and toe blood flow was significantly higher than that of the solvent control group (P = 0.05,0.01 and 0.05); the auricle and toe blood flow velocity high blood pressure in rats with clopidogrel group was significantly higher than that of the solvent control group (P = 0.001 and 0.05); clopidogrel group hypertensive rats with cerebral blood flow distribution curve and the solvent control group compared to the overall shift, the cerebral blood flow (800-1500 PU) high percentage was significantly higher than that of the solvent control group (P0.01); the experimental results, did not show the effects of omeprazole in improving microcirculation of clopidogrel on the microcirculation in rat aorta. The clopidogrel group was significantly lower than the wall thickness and area of solvent control group (P0.05), the proliferation of vascular smooth muscle compared with solvent control Group improved continuous arrangement relatively neat, lumen area and wall area ratio was significantly higher than that of the solvent control group (P0.05), the cerebral cortex of rats in the clopidogrel group was significantly higher than that of microvessels in solvent control group (P0.05), vascular expression of CD31 was significantly higher than that of the solvent control group (P0.05). The second part: after inoculation with 8h cells adherenced after cell fusion, 6D up to 80-90%, which 2-4d cells in logarithmic growth phase, significantly increased the number, size, abundant cytoplasm, the rest of the time did not change significantly; compared with the control group, the concentration gradient of C reactive protein (25,50,75 g/mL) HUVEC CD62E+ can stimulate endothelial microparticles higher, among them 50 and 75 g/mL two concentration had significant difference (P = 0.01 and 0.05); compared with the control group, different concentration of endothelial microparticles (2500500010000 /mL) so that the permeability of HUVEC was significantly higher in the 15,30,60,90 time min (P value were 0 .01,0.05 and 0.001); compared with the control group, with the increase of HUVEC concentration stimulated endothelial microparticles, F-actin from around the cells and cell junction migration into cells, dense peripheral beam gradually reduced, stress fibers and central short fibers gradually thickening increased. Conclusion: the first part: clopidogrel can significantly improve the surface of hypertensive rats and the cerebral microcirculation, and its mechanism may improve vascular remodeling and increase the microvascular number related to hypertension, prognosis and reduce cardiovascular complications. The second part: the endothelial cells induced by pricking endothelial microparticles in inflammatory cytokines C reactive protein had a specific phenotype, in this role in normal endothelial microparticles and endothelial cells. May cause normal endothelial cell permeability change, this change will lead to further damage of endothelial cells plays an important role.

【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R544.1

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