【作者】 曾鹏；

【导师】 邓春玉；

【作者基本信息】 华南理工大学 ， 临床医学， 2022， 硕士

【摘要】 目的:血管平滑肌细胞（Vascular smooth muscle cells,VSMCs）衰老广泛参与高血压、动脉粥样硬化等年龄相关性心血管疾病的进展,增加心血管事件发生率,并与钙调控紊乱密切相关。钙感受器STIM1介导钙池操纵性钙内流（store-operated calcium entry,SOCE）通过维持细胞内钙平衡,对血管收缩、重塑及修复起重要调控作用,然而STIM1在血管衰老中的作用尚不清楚。因此,本研究在氧化应激诱导的衰老VSMCs模型及自然衰老的动物模型中,观察STIM1是否参与主动脉平滑肌细胞衰老进程,及其相关作用机制。方法:使用200μM过氧化氢溶液（H₂O₂）处理人主动脉平滑肌细胞（Human aorta vascular smooth muscle cells,HA-VSMCs）2h构建氧化应激衰老VSMCs模型;C57BL/6分别饲养至3月龄、12月龄、18月龄,建立自然衰老动物模型;Cre-lox P基因重组技术构建平滑肌特异性STIM1敲除小鼠(smc STIM1^-/-)模型;采用衰老相关β-半乳糖苷酶染色检测衰老VSMCs阳性率;Western blot检测细胞或组织中钙池操纵性钙通道（store-operated calcium channel,SOC）相关蛋白STIM1、STIM2、ORAI1表达水平、衰老相关标记蛋白蛋白p53、p21、p16及自噬相关蛋白p62、LC3B等表达水平;激光共聚焦测钙技术检测单个细胞的SOCE的钙信号变化,微血管张力测定技术检测收缩剂诱导的及SOC介导的血管收缩功能;转录组高通量测序技术检测STIM1敲除对主动脉平滑肌转录水平的变化。透视电镜检测STIM1敲除对小鼠主动脉平滑肌超微结构的影响;m RFP-GFP-LC3串联荧光蛋白系统检测细胞自噬流变化;流式细胞仪检测细胞内活性氧（reactive oxygen species,ROS）水平。结果:（1）在H₂O₂诱导的衰老VSMCs模型上,衰老相关蛋白p21、p16表达上调,衰老相关β-半乳糖苷酶染色阳性细胞率增加,伴STIM1及ORAI1蛋白表达下调,SOCE水平降低;（2）老龄小鼠（12月龄、18月龄）主动脉组织SOC相关蛋白STIM1和ORAI1表达下调,以及SOC通道介导的血管收缩反应下降（3）smc STIM1^-/-小鼠主动脉组织中STIM1水平下降,而STIM2及ORAI1蛋白表达无明显差异,SOC介导的收缩反应几乎消失,不同浓度的Phe诱导的血管收缩无明显差异,然而在硝苯地平预处理后,有或无细胞外钙条件下血管收缩几乎完全消失;（4）smc STIM1^-/-小鼠主动脉衰老相关蛋白p53、p21及p16均上调,sh STIM1下调细胞STIM1表达,并伴随着衰老相关蛋白p53、p21及p16表达以及ROS水平的增加;（5）smc STIM1^-/-小鼠主动脉组织转录组高通量测序显示下调基因富集在自噬与线粒体自噬中;（6）smc STIM1^-/-小鼠主动脉组织,超微结构下线粒体肿胀、嵴消失,线粒体面积及长度显著增加,线粒体分裂相关蛋白FIS1显著下调,线粒体融合蛋白MFN2表达上调,自噬相关蛋白PINK1、Parkin表达下调,p62表达上调;（7）sh STIM1下调细胞STIM1表达后,自噬相关蛋白p62、LC3BII表达上调,进一步使用HCQ处理后,sh STIM1+HCQ组LC3BII表达量不高于sh NC+HCQ组。（8）m RFP-GFP-LC3串联荧光蛋白系统检测,提示氧化应激衰老VSMCs模型自噬小体数量显著增多,而自噬溶酶体数量增加不变;在衰老VSMCs模型中过表达STIM1导致自噬溶酶体数量显著增多,并比自噬小体数量多,HCQ处理可逆转该过程。（9）过表达STIM1能够减少衰老VSMCs p21、p16表达以及ROS水平上调,以及衰老相关β-半乳糖苷酶染色阳性细胞率增加,且HCQ处理后被逆转。结论:（1）在氧化应激衰老VSMCs模型及老龄小鼠主动脉组织中STIM1表达下调及SOCE功能受损;（2）组织和细胞水平下调STIM1加速VSMCs衰老以及促进ROS的生成;（3）STIM1下调可抑制VSMCs自噬及线粒体自噬功能,过表达STIM1通过改善衰老VSMCs模型的自噬功能障碍,减少氧化应激并且缓解VSMCs衰老。更多还原

【Abstract】 Objective:Vascular smooth muscle cells（VSMCs）senescence affects normal vascular function,and is widely involved in the progression of age-related cardiovascular diseases such as hypertension and atherosclerosis,ultimately leading to an increase in the incidence of cardiovascular events.The calcium sensor STIM1 and the ORAI1 channel mediate store-operated calcium entry（SOCE）,maintain intracellular calcium balance,and play an important role in vasoconstriction,remodeling,and repair.However,the role of STIM1 in vascular senescence remains unclear.Therefore,this study investigated whether STIM1 is involved in the aging process of aortic smooth muscle cells in oxidative stress-induced senescent cell models and natural aging animal models,and explored its related mechanisms.Methods:Human aorta vascular smooth muscle cells（HA-VSMCs）were treated with200μM Hydrogen peroxide solution（H₂O₂）to construct oxidative stress senescent cell models;C57BL/6 were raised to 3 months,12 months,and 18 months,respectively.A natural aging animal model was established;Cre-lox P technology was used to construct smc STIM1^-/-model;senescence-relatedβ-galactosidase staining was used to detect senescent VSMCs;Western blot was used to detect the expression of SOC-related proteins STIM1,STIM2,ORAI1,senescence-related proteins p53,p21,p16,and autophagy-related proteins p62,LC3B,etc.;Laser confocal microscopy was used to detect the calcium signal changes of SOCE in single cells;Microvascular tonometry was used to detect constrictor-induced and SOC-mediated vasoconstriction;Transcriptome high-throughput sequencing was used to detect the effect of STIM1 knockout on aortic smooth muscle on Transcription level.The effect of STIM1knockout on the ultrastructure of mouse aortic smooth muscle was detected by fluoroscopy electron microscope;the changes of autophagy flux were detected by m RFP-GFP-LC3 dual fluorescence system;the reactive oxygen species（ROS）were detected by flow cytometry.Results:（1）In the H₂O₂-induced senescent VSMCs model,the expression of p21 and p16 was up-regulated,and the rate of senescence-relatedβ-galactosidase staining positive cells was up-regulated,together with the down-regulation of STIM1 and ORAI1 protein expression,and the impaired SOCE function;（2）The expression of SOC-related proteins STIM1 and ORAI1 in aortic tissue of aged mice（12 months old and 18 months old）is reduced,and the vasoconstriction response mediated by SOC channels is decreased（3）STIM1 in aortic tissue of smc STIM1^-/-mice levels decreased,while there was no significant difference in STIM2 and ORAI1 protein expression,SOC-mediated contractile response almost disappeared,and there was no significant difference in vasoconstriction induced by different concentrations of Phe,however,under nifedipine pretreatment with or without extracellular calcium conditions,vasoconstriction almost completely disappeared;（4）Aortic senescence-related proteins p53,p21 and p16 of smc STIM1^-/-mice were all up-regulated,while sh STIM1 down-regulated the expression of STIM1 in VSMCs,accompanied by the increase in the expression of senescence-related proteins p53,p21 and p16 and the level of ROS.（5）High-throughput sequencing of transcriptome of smc STIM1^-/-mouse aortic tissue transcriptome showed that reduced genes were enriched in autophagy and mitophagy;（6）smc STIM1^-/-mouse aortic tissue,ultrastructure The mitochondria swelled,the cristae disappeared,the mitochondrial area and length increased significantly,the mitochondrial fission-related protein FIS1 was significantly down-regulated,the mitochondrial fusion protein MFN2 was up-regulated,the autophagy-related proteins PINK1 and Parkin were down-regulated,and the expression of p62 was significantly up-regulated;（7）sh STIM1down-regulated cells After the expression of STIM1,the autophagy-related proteins p62 and LC3BII were significantly up-regulated.After further treatment with HCQ,the expression of LC3BII in the sh STIM1 group was not significantly higher than that in the sh NC group.（8）The detection of m RFP-GFP-LC3 dual fluorescence system showed that the number of autophagosomes increased significantly in the senescent VSMCs model,while the number of autolysosomes was not significantly increased;overexpression of STIM1 in the senescent cell model resulted in autolysosomes.The number of autophagosomes was significantly increased and higher than that of autophagosomes,and HCQ treatment was able to reverse this process.（9）Overexpression of STIM1 can reduce the expression of p21 and p16 and the up-regulation of ROS levels in the senescent VSMCs model,as well as the rate of senescence-relatedβ-galactosidase positive cells,which were reversed after HCQ treatment.Conclusions:（1）STIM1 expression is down-regulated and SOCE function is impaired in oxidative stress senescent cell model and aortic tissue of aged mice;（2）Down-regulation of STIM1 in vivo and in vitro accelerate the senescence of VSMCs and promotes the generation of ROS;（2）Down-regulation of STIM1 inhibits autophagy and mitophagy in VSMCs,and overexpression of STIM1 improves senescence Autophagy dysfunction and alleviates senescence in senescent VSMCs.更多还原