CD137-CD137L信号通过激活自噬影响内皮细胞管腔形成以及动脉环血管新生

发布时间：2018-03-25 18:40

  本文选题：CD137　切入点：自噬　出处：《江苏大学》2017年硕士论文

【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(P0.05 vs对照组)、动脉环出芽总长度平均值较对照组显著增加(P0.01 vs对照组);抑制CD137-CD137L信号HUVECs管腔形成总长度减少(P0.05 vs刺激组)、动脉环出芽总长度平均值显著减少(P0.01 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(P0.05)且动脉环出芽长度平均值显著减少(P0.01);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。
[Abstract]:Objective to investigate whether CD137-CD137L signal affects endothelial cell lumen formation and angiogenesis by activating autophagy. Methods 1. A model of thoracic aortic rings in C57BL/6J mice was established. Observation on the effect of CD137-CD137L signal on the sprouting length of arterial ring under inverted phase contrast microscope) intervention of autophagy on the length of CD137-CD137L signaling mediated arterial ring sprouting. 2. Cultured human umbilical vein endothelial cells on matrix glue, inverted phase. Dynamic observation of the effect of CD137-CD137L signal on Endothelial Cell Endothelial Cell Endothelial Cell cavities under differential microscope. The effect of autophagy on the Endothelial Cell Endothelial Cell Endothelial Cell Endothelial Cell Endothelial formation mediated by CD137-CD137L signal. The effect of protein Immunoimprint Western blot technique on the Endothelial Cell Endothelial cavity formation ability. The effect of signal on the expression of LC3 II / Igni P62, a key protein molecule in autophagy. (4) Adenovirus-labeled adenovirus was used to infect endothelial cells, which was labeled with mRFP-GFP-LC3. The effects of interference with CD137-CD137L signal on the formation of cytoplasmic autophagy (yellow fluorescence) and the fusion of autophagy and lysosome (red fluorescence) were observed under fluorescence microscope. The Transwell cell migration test was used. To detect the changes of endothelial cell migration mediated by CD137-CD137L signal after intervention of autophagy. The CCK-8 cell proliferation kit was used. The changes of endothelial cell proliferation mediated by CD137-CD137L signal after intervention were detected. Results 1.Activation of CD137-CD137L signal increased the total length of lumen formation of HUVECs (P0.05 vs control group), and the mean of total length of arterial ring bud increased significantly (P0.01 vs control group). 2. In control group, the total length of HUVECs lumen formation was decreased in CD137-CD137L signal inhibition group (P0.05) vs stimulation group (P 0.05), and the mean of total sprouting length of arterial ring decreased significantly (P 0.01 vs stimulation group) 2. The expression of LC3II protein, a key molecule of autophagy, was increased in activated CD137-CD137L signal endothelial cells. The expression of P62 protein was lower than that of control group (P 0.05 vs control group, P 0.05 vs control group), and that of autophagy and autophagy lysosomes were significantly increased (P 0.01 vs control group, respectively), while the down-regulation of P62 protein expression was down-regulated by inhibiting CD137-CD137L signal expression in HUVECs and upregulating the relative expression of P62 protein. P 0.05 vs stimulation group, while autophagy and autophagy lysosomes decreased significantly (P 0.01 vs stimulation group 3.3-MA-5mm) inhibited endothelial cell autophagy. The total length of lumen formation of HUVECs was reduced by P0.05) and the mean sprouting length of arterial ring was significantly reduced by P0.01C, and the activation of CD137-CD137L signal was inhibited after autophagy. Both migration and proliferation of endothelial cells increased significantly (P < 0.01). Conclusion CD137-CD137L signal may mediate endothelial cell lumen formation and arterial ring angiogenesis by activation of autophagy.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54

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