【作者】 陈晓；

【导师】 李曦；

【作者基本信息】 山东大学 ， 遗传学， 2021， 硕士

【摘要】 动脉粥样硬化作为一种慢性炎症性疾病,主要病理特征为脂质成分在动脉内膜沉积最终在血管壁形成粥样斑块,经常伴随有平滑肌细胞和纤维成分异常增生,研究表明,一氧化氮分子在保护心血管功能,预防粥样硬化发挥着较为重要作用。在本研究中,血管内皮细胞通过蓝光敏感蛋白视黑素Melanopsin(OPN4)和血红蛋白α(Hb α)响应蓝光刺激,促进一氧化氮的释放,光照(450μW/cm2)内皮细胞后,提高了 eNOS的表达。短程(180分钟)或长程(2小时/天,12-14周)蓝光照射可提高动脉粥样硬化模型(ApoE-/-)小鼠血清一氧化氮水平。长程光照后粥样硬化斑块有一定改善,其中巨噬细胞指标CD14+和CD68+降低,平滑肌细胞指标α-SMA+增加。[研究目的]本课题主要研究血管内皮细胞对蓝光光刺激的反应以及其导致内源性一氧化氮(NO)产生的分子机制。[研究方法]1、利用免疫荧光等技术检测血管内皮细胞是否表达蓝光光敏感蛋白;2、通过不同光照强度的单色蓝光(455 nm)和单色红光(635 nm)系统照射三种血管内皮细胞:HUVECs(人脐静脉内皮细胞)、HAECs(人主动脉内皮细胞)和TIMEs(端粒酶永生化人微血管内皮细胞),采用改良的Griess reagent法检测培养基中总一氧化氮水平;3、利用Western Blot和qPCR技术检测不同强度蓝光照射后,eNOS和ACE2在HUVECs细胞中的表达情况;4、使用FLUO-3AM探针检测蓝光刺激HUVECs后钙离子内流情况;5、使用DAF-FM DA探针检测蓝光刺激HUVECs后一氧化氮的产生;6、选用单色蓝光(455 nm,300μw/cm2)照射HUVECs后,通过EdU掺入、划痕愈合实验分别对细胞的增殖和迁移能力进行检测;7、短程及长程的蓝光照射野生型和动脉粥样硬化模型小鼠,检测小鼠血清中一氧化氮水平的变化,并分析长程蓝光照射后ApoE-/-小鼠斑块形成情况。[实验结果]1、内皮细胞表达光敏蛋白OPN4和Hb α;2、三种内皮细胞经不同强度蓝光(0、300、350、450、550 uW/cm2)照射120分钟后,培养基中的一氧化氮含量明显增加;单色红光(0、300、350、450、550μW/cm2)照射后一氧化氮的含量变化趋势不明显;3、Western Blot以及qPCR结果显示HUVECs在不同强度的蓝光刺激后,eNOS和ACE2表达增加,在450μW/cm2增加最明显;4、DAF-FM DA探针检测NO生成,结果显示450 μW/cm2的蓝光照射可显著提高内皮细胞一氧化氮释放量;5、蓝光(455 nm,300μW/cm2)促进内皮细胞增殖和迁移;6、短程或长程蓝光照射野生型和动脉粥样硬化模型小鼠都可以提高血清NO水平,长程蓝光照射后可一定程度改善斑块炎性浸润程度。[结论]本研究发现血管内皮细胞可以表达光敏感蛋白OPN4和Hbα,在此基础上,蓝光照射血管内皮细胞后通过增加内皮型一氧化氮合酶(eNOS)和血管紧张素转换酶2(ACE2)的表达诱导内源性一氧化氮的释放,并且可以改善ApoE-/-小鼠粥样斑块指标。更多还原

【Abstract】 As a chronic inflammatory disease,atherosclerosis is mainly characterized by the deposition of lipid components and inflammatory substances in the intima of the artery to form atherosclerotic plaques,accompanied by the proliferation of smooth muscle cells and fibrous components.Nitric oxide plays an important role in protecting cardiovascular function and preventing atherosclerotic diseases.In this study,vascular endothelial cells responded to blue light stimulation through the blue light sensitive protein Melanopsin(OPN4)and hemoglobin α(Hb α)and induced nitric oxide release.Irradiation(450μW/cm2)on endothelial cells increased the expression of endothelial nitric oxide synthase(eNOS).Further,Short-term(180 minutes)or long-term(2 hours/day,12-14 weeks)blue light stimulation increased serum nitric oxide levels in atherosclerotic model(ApoE-/-)mice.After long-term light exposure,atherosclerotic plaque was improved to some extent,in which macrophage index CD14+and CD68+decreased,smooth muscle cell index α-SMA+increased.ObjectiveTo investigate endothelial cells response to blue light stimulation and the molecular mechanism that leads to endogenous nitric oxide release.Methods1、Immunofluorescence techniques were used to detect the expression of blue light sensitive protein in vascular endothelial cells;2、Three types of endothelial cells:HUVECs((human umbilical vein endothelial cells),HAECs(human aortic endothelial cells)and TIMEs(telomerase immortalized human microvascular endothelial cells)were irradiated by monochromatic blue light(455 nm)and monochromatic red light(635 nm)systems with different light intensities.The level of total nitric oxide in culture medium was detected by modified Griess reagent method;3、Western Blot and qPCR techniques were used to detect the expression of eNOS and ACE2 in HUVECs after different intensities of blue light irradiation;4、Detection of calcium influx after blue light stimulation of HUVECs by calcium ion probe(FLUO-3AM);5、Detection of nitric oxide production after blue light stimulation of HUVECs by nitric oxide probe(DAF-FM DA);6、After stimulating HUVECs with monochromatic blue light(455 nm,300μw/cm2),the proliferation and migration ability were detected by EdU incorporation and wound healing in HUVECs;7、Wild-type(WT)and atherosclerotic model(ApoE-/-)mice were irradiated with short-term and long-term blue light,The changes of serum nitric oxide levels in mice were detected,and the plaques of ApoE-/-mice after long-term irradiation were analyzed.Results1、Endothelial cells expressed light sensitive proteins OPN4 and Hb α;2、The content of nitric oxide in the culture medium increased significantly after three types of endothelial cells were irradiated with different intensities of blue light(0、300、350、450 and 550μW/cm2)for 120 min,while the change trend of nitric oxide content was not obvious after irradiation with monochromatic red light(0、300、350、450 and 550μW/cm2);3、Western Blot and qPCR showed that the expression of eNOS and ACE2 increased after different intensities of blue light stimulation in HUVECs,especially at the intensity of 450μW/cm2;4、Using fluorescent probe(DAF-FM DA),and the results showed that 450 μW/cm2 blue light irradiation significantly increased the release of nitric oxide from endothelial cells;5、Blue light(455 nm,300 μW/cm2)accelerated the proliferation and migration of endothelial cells;6、Short-term or long-term blue light irradiation for wild-type and atherosclerotic model mice increased the level of serum nitric oxide,and long-term blue light irradiation could improve the degree of plaque inflammatory infiltration to a certain extent.ConclusionsIn this study,we found that vascular endothelial cells expressed blue light sensitive proteins OPN4 and Hb α.On this basis,blue light irradiated vascular endothelial cells induced the release of endogenous nitric oxide by increasing the expression of endothelial nitric oxide synthase(eNOS)and angiotensin converting enzyme 2(ACE2),and improved the index of atherosclerotic plate in ApoE-/-mice.更多还原