【作者】 李波；

【导师】 严金川；

【作者基本信息】 江苏大学 ， 内科学（专业学位）， 2017， 硕士

【摘要】 目的 探讨CD137-CD137L信号是否通过激活细胞自噬影响内皮细胞管腔形成及动脉环血管新生。方法1.构建C57BL/6J小鼠胸主动脉动脉环模型,在倒置相差显微镜下观察:(1)CD137-CD137L信号对动脉环出芽长度的影响;(2)干预自噬对CD137-CD137L信号介导的动脉环出芽长度的影响;2.基质胶上培养人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs),倒置相差显微镜下动态观察:(1)CD137-CD137L信号对内皮细胞管腔形成能力的影响;(2)干预自噬对CD137-CD137L信号介导的内皮细胞管腔形成能力的影响;3.运用蛋白质免疫印记(Western blot)技术检测干预CD137-CD137L信号对自噬关键蛋白分子LC3 II/I、P62表达的影响;4.利用自噬双荧光(mRFP-GFP-LC3)标记的腺病毒感染内皮细胞,在荧光显微镜下观察干预CD137-CD137L信号对胞浆自噬小体(黄色荧光)生成情况以及自噬小体与溶酶体融合(红色荧光)情况的影响;5.运用Transwell细胞迁移试验,检测干预自噬后CD137-CD137L信号介导的内皮细胞迁移能力的变化;6.采用CCK-8细胞增殖试剂盒,检测干预自噬后CD137-CD137L信号介导的内皮细胞增殖能力的变化。结果1.激活CD137-CD137L信号,HUVECs管腔形成总长度增加(P<0.05 vs对照组)、动脉环出芽总长度平均值较对照组显著增加(P<0.01 vs对照组);抑制CD137-CD137L信号HUVECs管腔形成总长度减少(P<0.05 vs刺激组)、动脉环出芽总长度平均值显著减少(P<0.01 vs刺激组);2.激动CD137-CD137L信号内皮细胞自噬关键分子LC3II蛋白表达增加,且P62蛋白表达减少较对照组(P均<0.05 vs对照组),同时自噬小体和自噬小体溶酶体均显著增加(P均<0.01 vs对照组);而抑制CD137-CD137L信号,HUVECs LC3II蛋白表达下调、P62蛋白相对表达上调(P均<0.05 vs刺激组),同时自噬小体和自噬小体溶酶体均显著减少(P均<0.01 vs刺激组);3.3-MA(5mM)抑制内皮细胞自噬后,激动CD137-CD137L信号,HUVECs管腔形成总长度减少(P<0.05)且动脉环出芽长度平均值显著减少(P<0.01);抑制自噬后激活CD137-CD137L信号,内皮细胞迁移及增殖能力均显著增加(P均<0.01)。结论CD137-CD137L信号可能通过激活细胞自噬介导内皮细胞管腔形成及动脉环血管新生。更多还原

【Abstract】 ObjectivesTo explore whether the CD137-CD137L signal influences the endothelial cell tube formation and sprouts of aortic ring via activating endothelial autophagy.Methods1.Constructed the ex-vivo model of C57BL/6J mice to observe the following indicators under the inverted phase contrast microscope:(1)The effect of CD137-CD137L on the sprouts of aortic ring;(2)The effect of CD137-CD137L on the sprouts of aortic ring after intervention of autophagy process.2.To culture the human umbilical vein endothelial cells(HUVECs)on gelled basement membrane extract to observe the following indicators under the inverted phase contrast microscope:(1)The effect of CD137-CD137L on endothelial cell tube formation;(2)The effect of CD137-CD137L on the endothelial cell tube formation after intervention of autophagy process.3.Autophagy markers LC3 Ⅱ/Ⅰ and P62 proteins were detected by applying Western blot after the intervention of the CD137-CD137L signal.4.Autophagy was detected by using mRFP-GFP-LC3 adenovirus.Using mRFP-GFP-LC3 adenovirus to transfect the endothelial cells and observe the number of autophagosomes and autolysosomes under fluorescence microscopy by intervention of the CD137-CD137L singal.5.Transwell assay was adopted to measure the number of migratory cells mediated by the CD137-CD137L signal after intervention of autophagy process.6.CCK-8 kits was used to detected the CD137-CD137L signal mediated endothelial cell proliferation ability after intervention of autophagy process.Results1.Compared with the control group,the total tube length was increased(P<0.05)and the mean length of sprouts was significantly increased(P<0.01)in the agonist-CD137 group;Compared with the agonist-CD 137 group,the total tube length of HUVECs and the mean length of sprouts in the anti-CD137 group was significantly decreased(P<0.01).2.Compared with the control group,the relative expression of LC3 Ⅱ protein expression was increased(P<0.05),the relative expression of autophagy substrate protein P62 was decreased(P<0.05)and the number of autophagosomes and autolysosomes were both obviously increased in the agonist-CD 137 group(P<0.01)when compared with the control group.Compared with the agonist-CD137 group,the relative expression of LC3 II protein was decreased(P<0.05),relative expression of P62 protein was increased(P<0.05)in the anti-CD137 group.Compared with the agonist-CD137 group,the number of autophagosomes and autolysosomes were both obviously decreased in the anti-CD 137 group(P<0.01).3.Applied 3-MA(5mM)to inhibited autophagy before activating the CD137-CD137L signal,the total tube length of HUVECs was decreased(P<0.05)and the mean length of sprouts were significantly decreased(P<0.01)in 3-MA group when compared with the agonist-CD137 group;The proliferation and migration ability of endothelial cells were both sharply decreased in 3-MA group(P<0.01)when compared with the agonist-CD137 group.ConclusionCD137-CD137L signal may influence te endothelial tube formation and aortic ring angiogenesis via activating endothelial autophagy.更多还原