【作者】 贺权威；

【导师】 胡波； 梅元武；

【作者基本信息】 华中科技大学 ， 神经病学， 2013， 博士

【摘要】 目的：研究miRNA-150调节大鼠脑梗塞后的血管新生及机制研究。方法：制作SD大鼠大脑中动脉梗塞模型,应用实时荧光定量PCR检测脑梗塞后1,3,7天时大脑和血清中的miR-150的浓度；原代大鼠脑微血管内皮细胞4-5代后,应用糖氧剥夺模拟体外缺血模型,应用实时荧光定量PCR检测糖氧剥夺2,4,6小时后检测脑微血管内皮细胞miR-150的表达。通过转染(?)miR-150mimic和inhibitor上下调脑微血管内皮细胞的表达,用基质胶实验检测内皮管腔形成能力,用MTT法或ki-67免疫荧光染色法检测细胞增殖能力,用划痕试验检测细胞迁移能力,并建立大鼠永久性脑缺血模型,侧脑室注射miR-150inhibitor或negative inhibitor,观察神经行为学评分,HE切片观察梗塞周边脑血管密度,用FITC标记的葡聚糖检测皮层血流灌注；脑微血管内皮细胞转染miR-150inhibitor或negative inhibitor,应用实时荧光定量PCR的方法检测c-myb和VEGF的mRNA水平,用Western blotting检测上述分子的蛋白水平水平改变；用慢病毒载体的siRNA技术和加特异性VEGF抗体的方法,分别下调c-Myb的表达和抑制VEGF的功能,观察miR-150在脑微血管内皮细胞管腔形成、增殖和迁移的作用是否依赖于c-myb和VEGF的作用。结果：1)大鼠脑梗塞1,3,7天后脑组织和血清中miR-150的表达水平下降,于3天最低；脑微血管内皮细胞糖氧剥夺2,4,6小时后,其miR-150的表达水平下降,以4小时为最低。2)miR-150mimic后抑制脑微血管内皮细胞管腔形成,细胞增殖,细胞迁移；而miR-150inhibitor促进脑微血管内皮细胞管腔形成,细胞增殖,细胞迁移。SD大鼠永久性脑缺血模型后侧脑室注射miR-150inhibitor增加其梗塞周边区血管密度,和血流灌注。3)miR-150inhibitor显著增加脑微血管内皮细胞c-myb及VEGF的mRNA和蛋白水平。分别下调c-myb及VEGF的功能后,miR-150对脑微血管内皮细胞的管腔形成能力,增殖能力,迁移能力被逆转。结论：脑梗塞抑制miR-10的表达。miR-150可以调节脑梗塞后脑微血管内皮细胞内皮血管新生,这种调节可能依赖于miR-150对c-myb和VEGF的抑制作用。miR-150可能成为脑缺血的治疗靶点。目的：研究脑梗塞后星形胶质细胞对脑微血管内皮细胞血管新生的作用及可能的机制。方法：原代大鼠脑微血管内皮细胞4-5代后,应用糖氧剥夺模拟缺血。用基质胶实验检测血管新生情况,用ki-67免疫荧光方法检测细胞增殖,用划痕实验检测细胞迁移能力；并用Elisa的方法检测培养基中Shh的表达；特异性的阻滞剂Cyclopamine,检测星形胶质细胞促进脑微血管内皮细胞血管新生是否与Shh有关。应用RT-PCR的方法检测RhoA, ROCK的mRNA水平,用免疫印迹法(Western blotting)检测上述因子的蛋白水平。用慢病毒转染的方法下调RhoA的表达水平,用Y27632抑制ROCK后,星形胶质细胞对脑微血管内皮细胞管腔形成、细胞增殖和迁移的能力的促进作用能否被逆转。结果：1)OGD处理后,星形胶质细胞分泌Shh分泌增加,与原代培养的脑微血管内皮细胞共培养可促进其OGD处理后的管腔形成、增殖和迁移能力,且其作用可被Shh的阻滞剂Cyclopamine部分逆转；2)星形胶质细胞与脑微血管内皮细胞共培养增加脑微血管内皮细胞的RhoA, ROCK的mRNA和蛋白的表达,这种增加也可以被Shh的阻滞剂Cyclopamine部分逆转。3)慢病毒转染的方法下调RhoA的表达及Y27632抑制ROCK后,星形胶质细胞对脑微血管内皮细胞管腔形成、增殖和迁移能力的促进作用可被逆转。结论：星形胶质细胞可以通过分泌Shh蛋白促进糖氧剥夺后脑微血管内皮细胞的血管新生。更多还原

【Abstract】 Objective:to explore the effect of miR-150on the angiogenesis and explored the underlying molecular basis after ischemic stroke in rat.Methods:SD rats were subjected ischemic stroke by the middle cerebral artery occlusion (MCAO) for1,3and7days respectively, and then the expression of miR-150in the brain and in the serum were detected by RT-PCR. The primary rat brain microvascular endothelial cells (BMEVCs) were cultured and were subjected oxygen glucose deprivation (OGD) for2,4, and6hours, then the expression of miR-150in the brain and in the serum were also detected by RT-PCR. The up-or down-regulation of miR-150were obtained by miR-150mimic or miR-150inhibitor transfection; the tube formation, proliferation and migration of BMEVCs were respectively detected by matrigel assay, by MTT assay and quantified by anti-Ki-67positive immunofluorescence and by scratch test. SD rat subjected MCAO were tansfected with miR-150inhibitor by daily intracerebroventricular injection, and the density of microvascules and blood flow in the peri-infarction regions were calculated by FITC-labeled dextran. The mRNA and protein expression of c-myb and VEGF in BMEVCs of miR-150inhibitor or negative inhibitor transfected were detecte by RT-PCR and western blotting. The down-regulation of c-myb or VEGF was achieved by lentiviral transfection or special antibody, and the role of c-myb and VEGF on miR-150mediated angiogensis were detected.Results:We found that:1) the expression of miR-150in the brain and serum were down-regulated after1-,3-,7d-ischemia, and the expression of miR-150in BMEVCs was also down-regulated after2-,4-,6h-OGD.2) The tube formation, proliferation and migration of BMEVCs were decrease by miR-150mime trasfection, while the tube formation, proliferation and migration of BMEVCs were increase by miR-150inhibitor trasfection. The density of microvascules and blood flow in the peri-infarction regions were increased with miR-150inhibitor transfection by intracerebroventricular injection after MCAO.3) The mRNA and protein expression of c-myb and VEGF in BMEVCs were up-regulated by miR-150transfection. The increase of tube formation, proliferation, and migration of BMEVCs were reversed by the down-regulation of c-myb or VEGF.Conclusions:the expression of miR-150is down-regulated after ischemia. MiR-150could mediate angiogensis by modulating c-myb and VEGF after ischemia in rat. MiR-150could be a potential target of the treatment for cerebral ischemia. Objective:to investigate the role of astrocytes in the angiogenesis of brain microvascular endothelial cells after oxygen glucose deprivation and to explore the underlying molecular basis.Methods:SD rat brain microvascular endothelial cells (BMVECs) and astrocytes were primary cultured. Oxygen glucose deprivation (OGD) was used as the ischemia model in vitro. The proliferation, migration and tube formation in BMECs were detected by anti-ki-67immunofluorescence, scratch test and matrigel assay respectively. The concentration of Shh in the medium was detected by Elisa. Cyclopamine (a Shh antagonist) was added to exam the role of Shh in angiogenesis. The expression mRNA and protein of RhoA and ROCK were detected by RT-PCR and west blotting. Lentiviral transfection of RhoA and ROCK inhibitor, Y27632were used respectively to exam the effect of RhoA and ROCK on angiogenesis.Results:We found that1) the treatment of oxygen-glucose deprivation increase astrocytic secretion of Shh; co-cultured astrocytes with BMVECs increase the tube formation, proliferation and migration after OGD; the increase was reversed by Cyclopamine.2) Co-cultured astrocytes with BMVECs increase the mRNA and protein expression of RhoA and ROCK, and the increase was reversed by Cyclopamine.3) The increase of the tube formation, proliferation and migration after OGD were respectively reversed by Lentiviral transfection of RhoA and ROCK inhibitor, Y27632.Conclusions:astrocytes may promote the angiogenesis in BMVECs after OGD by the secretion of Shh.更多还原