【作者】 王宁；

【导师】 孙英贤；

【作者基本信息】 中国医科大学 ， 内科学， 2020， 博士

【摘要】 目的:目前,心血管相关疾病（cardiovascular disease,CVD）仍是人类健康的首要威胁。血管重构（vascular remodeling）是CVD基础病理生理学改变。血管平滑肌细胞（Vascular smooth muscle cells,VSMCs）增殖、迁移以及合成分泌在血管重构中发挥关键作用,进而导致包括高血压、动脉粥样硬化和血管内再狭窄等多种疾病^[1,2]。作为血管壁的主要细胞构成,VSMCs生理条件下表现为高度分化状态,即“收缩型”表型（也被称为“静止型”）,维持正常的血管收缩舒张功能。收缩型VSMCs具有相当低的增殖和迁移能力。不同于高度分化的骨骼肌或心肌,VSMCs高度保留了细胞的可塑性,病理条件下其表型可能发生转换。VSMCs由收缩表型向合成分泌表型转化时,VSMCs表现出异常的增殖、迁移、细胞外基质（extracellular matrix,ECM）分泌活性,从而导致血管重构诱发疾病。因此,明确VSMCs增殖、迁移和分泌机制对抑制血管重构、防治CVD具有重要临床意义。肾素-血管紧张素系统（renin angiotension system,RAS）是血管功能最主要的调节系统。目前认为,RAS系统的主要生物学效应由血管紧张素Ⅱ（Angiotensin Ⅱ,Ang-Ⅱ）产生。大量文献表明Ang-Ⅱ作为一种有力的VSMCs表型转化促进因子,可以有效的促进VSMCs由“静止”状态向增殖、迁移和分泌能力活跃状态转化,从而导致血管重构^[3,4]。自噬是溶酶体介导的细胞内物质动态循环过程。细胞通过自噬消化自身受损胞质物质作为“原料”以更新胞内细胞器或生成自身代谢所需物质。自噬被越来越多的实验证实为VSMCs发生表型转化的其中一个重要机制。Ang-Ⅱ是自噬的诱导因素已被大量实验证实。其作用机制是激活AT1受体、NADPH氧化酶和线粒体KATP通道。Septins家族传统上作为GTP结合蛋白一直以来在细胞动力学和微丝形成能力方面被广泛讨论。随着科学研究的深入,多项其他领域的生物学效应也被揭示,这些效应包括膜动力学、细胞骨架重组、囊泡运输和肿瘤发生^[5]。Septin4是Septins家族内一个亚型,具有GTP酶活性。人类Septin4基因编码两种主要蛋白异构体:Sept4＿i1（H5/PNUTL2）和Sept4＿i2/ARTS。Septin4被认为是细胞骨架的重要组成被广泛表达于真核细胞内,并与多重要的生理活动相关^[6],因而其功能发生改变可导致多种病理现象发生,包括小肠干细胞生存、小鼠精子活力、肝脏纤维化、亨廷顿病（Huntington’s disease,HD）等。我们的团队新近研究结果显示,Septin4作为一个重要调控因素参与了氧化应激导致的血管内皮细胞损伤,而其作用机制则是通过与凋亡相关蛋白PARP1相互作用^[7]。而作为与VSMCs无论是从功能上还是结构上都密切联系的VSMCs,目前尚未有文献报道Septin4是否对VSMCs的病理生理具有调控作用。我们旨在对Septin4在Ang-Ⅱ诱导的血管平滑肌细胞增殖和迁移过程中是否发挥效应进行验证,并探讨其机制是否与自噬相关。研究方法:1、复制小鼠血管重构模型。选取8周龄的雄性C57B6小鼠12只。随机选取6只作为Ang-Ⅱ干预组,其余6只为对照组。诱导麻醉后在干预组小鼠背部皮下植入微量注射泵,泵内预充Ang-Ⅱ,以1.5mg/kg/天持续输注小鼠体内。对照组小鼠植入的微量注射泵内预充生理盐水。14天后处死全部小鼠。提取小鼠主动脉组织。对小鼠主动脉组织行HE染色检测主动脉血管壁（内膜-中膜）厚度,以及Masson染色检测主动脉血管壁内胶原纤维含量评估纤维化程度。2、提取小鼠主动脉组织蛋白,通过Western blot检测Ang-Ⅱ干预组和对照组血管组织内Septin4蛋白表达含量,对比两组差异。3、培养人主动脉平滑肌细胞（Human aortic smooth muscle cells,HASMCs）,分别用梯度浓度为0M、10^-8M、10^-7M、10^-6M、10^-5M的Ang-Ⅱ处理细胞48h。通过CCK-8实验检测细胞增殖活力,通过transwell实验检测细胞迁移能力。提取5组细胞内总蛋白,用Western blot检测Septin4蛋白表达水平,对比组间差异。4、通过质粒转染在HASMCs内建立Septin4过表达模型,细胞分为两组:Flag-Septin4（过表达Septin4）和Flag-control（对照）组。分别用梯度浓度为0M、10^-8M、10^-7M、10^-6M、10^-5M的Ang-Ⅱ处理细胞48h。通过CCK-8实验检测细胞增殖活力。将细胞分为Flag-Septin4、Flag-Septin4+Ang-Ⅱ、Flag-control、Flag-control+Ang-Ⅱ四组,通过transwell实验检测细胞迁移能力,对比4组细胞迁移数目的差异。5、对Flag-Septin4、Flag-Septin4+Ang-Ⅱ、Flag-control、Flag-control+Ang-Ⅱ 4组HASMCs提取蛋白,通过Western blot检测相关蛋白表达:增殖能力标志物PCNA,合成能力标志物col-1,迁移能力标志物MMP2,自噬流标志物p62和LC3（Ⅱ和I）,对比4组蛋白含量差异。6、将HASMCs分为6组:Flag-Septin4、Flag-Septin4+Ang-Ⅱ、Flag-Septin4+CQ（氯喹）、Flag-Septin4+Ang-Ⅱ+CQ、Flag-control、Flag-control+Ang-Ⅱ)。通过CCK-8实验检测细胞增殖活力。以及transwell实验检测细胞迁移能力。收集细胞提取总蛋白,通过Western blot检测相关蛋白表达:PCNA,col-I,MMP2,p62和LC3（Ⅱ和I）,对比6组蛋白含量差异。7、将Flag-Septin4组HASMCs提取总蛋白,免疫沉淀目标蛋白Septin4及其相互作用蛋白后通过凝胶电泳分离,染色后切取含差异蛋白复合物的胶片行蛋白质谱分析。结果:1、14天后,HE染色显示Ang-Ⅱ干预组小鼠主动脉血管壁内膜-中膜厚度显著高于对照组。Masson染色显示Ang-Ⅱ干预组小鼠主动脉血管壁胶原蛋白含量显著高于对照组,血管纤维化水平增高。2、Western blot法显示Ang-Ⅱ干预组小鼠主动脉血管壁内Septin4蛋白含量显著高于对照组,约为对照组3倍。3、加入Ang-Ⅱ后,HASMCs迁移数目增多,并且细胞迁移数目随着Ang-Ⅱ干预浓度的增强而增加。加入10^-5M Ang-Ⅱ的细胞迁移数目显著增多,较未加药组增加超过1倍。加入Ang-Ⅱ后,HASMCs活力升高,并且细胞活力随着Ang-Ⅱ干预浓度的增强而增加,加入10^-5M Ang-Ⅱ的细胞活力显著增多,较未加药组增加超过1/3。免疫组化结果显示,加入Ang-Ⅱ后HASMCs内Septin4蛋白含量增加,并且二者之间存在量-效关系,Ang-Ⅱ浓度越高,细胞内Septin4蛋白表达水平越高。加入10^-5M Ang-Ⅱ的细胞Septin4蛋白表达水平显著增多,是未加药组的3倍以上。4、CCK-8结果显示,与Flag-control组类似,Flag-Septin4组HASMCs加入Ang-Ⅱ后细胞活力增加,并且细胞活力随着Ang-Ⅱ干预浓度的增强而增加,加入10^-5 M Ang-Ⅱ的细胞活力显著增多。Flag-Septin4和Flag-control组细胞相比,没有加入Ang-Ⅱ时,Flag-Septin4组细胞活力轻度低于Flag-control,但这种差异不具有统计学意义。而在分别加入10^-8 M、10^-7 M、10^-6 M、10^-5 M的Ang-Ⅱ后,Flag-Septin4组细胞活力均显著低于相同Ang-Ⅱ浓度干预下的Flag-control组。综合以上实验结果,我们从中选取10^-6 M作为最适Ang-Ⅱ浓度用于以后干预组实验。Transwell结果显示,与Flag-control组类似,Flag-Septin4组HASMCs加入Ang-Ⅱ后细胞迁移数目显著增加。Flag-control组HASMCs加入Ang-Ⅱ后细胞迁移数量约是未加药组的3倍,而Flag-Septin4组细胞加入Ang-Ⅱ后细胞迁移数量约是未加药组的2倍,在分别加入10^-6 M的Ang-Ⅱ后,Flag-Septin4组细胞迁移数量显著低于Flag-control组。5、Westerm blot结果显示,Flag-control组HASMCs加入Ang-Ⅱ后细胞增殖标志物PCNA蛋白表达显著增加,Flag-Septin4组加入Ang-Ⅱ后PCNA表达也显著增加。在分别加入Ang-Ⅱ后Flag-Septin4组PCNA蛋白表达显著低于Flag-control组。Flag-control组HASMCs加入Ang-Ⅱ后细胞合成分泌的col-1蛋白表达显著增加。Flag-Septin4组加入Ang-Ⅱ后col-1表达也显著增加。在分别加入Ang-Ⅱ后Flag-Septin4组col-1蛋白表达显著低于Flag-control组,约为Flag-control的3/5。Flag-control组HASMCs加入Ang-Ⅱ后细胞迁移标志物MMP2蛋白表达显著增加,超过未加药组1.5倍以上。Flag-Septin4组加入Ang-Ⅱ后MMP2表达也显著增加,但超过未加药组不到0.5倍。而在分别加入Ang-Ⅱ后Flag-Septin4组MMP2蛋白表达显著低于Flag-control组,约为Flag-control的2/3。在没有加入Ang-Ⅱ时Flag-Septin4组p62蛋白显著高于Flag-control组,是Flag-control组2倍以上。加入Ang-Ⅱ后Flag-Septin4组p62蛋白表达也显著高于Flag-control组,约为Flag-control组2倍。Flag-control组HASMCs加入Ang-Ⅱ后细胞自噬标志物之一LC3-Ⅱ/GAPDH比值显著升高,约为未加药的2倍。加入Ang-Ⅱ后,Flag-Septin4组LC3-Ⅱ/GAPDH比值显著低于Flag-control组,不到Flag-control组1/2。进一步对比LC3-Ⅱ/I我们发现Flag-Septin4+Ang-Ⅱ组LC3-Ⅱ/I比值显著低于Flag-+control+Ang-Ⅱ组。6、CCK-8结果显示,在没有加入Ang-Ⅱ时,Flag-control、Flag-Septin4、Flag-Septin4+CQ组细胞活力呈梯度下降趋势,其中Flag-Septin4+CQ细胞活力显著低于Flag-control组。而在加入Ang-Ⅱ后,Flag-control、Flag-Septin4、Flag-Septin4+CQ组细胞活力仍然呈梯度下降趋势,其中Flag-Septin4+Ang-Ⅱ和Flag-Septin4+Ang-Ⅱ+CQ细胞活力均显著小于Flag-control+Ang-Ⅱ组,并且二者间Flag-Septin4+Ang-Ⅱ+CQ细胞活力显著小于Flag-Septin4+Ang-Ⅱ组。Transwell结果显示,在没有加入Ang-Ⅱ时,Flag-control、Flag-Septin4、Flag-Septin4+CQ组细胞迁移数量呈梯度下降趋势,其中Flag-Septin4+CQ细胞迁移数量显著低于Flag-control组。而在加入Ang-Ⅱ后,Flag-control、Flag-Septin4、Flag-Septin4+CQ组细胞迁移数量仍然呈梯度下降趋势,其中Flag-Septin4+Ang-Ⅱ和Flag-Septin4+Ang-Ⅱ+CQ细胞迁移数量均显著小于Flag-control+Ang-Ⅱ组。二者间Flag-Septin4+Ang-Ⅱ+CQ细胞迁移数量显著小于Flag-Septin4+Ang-Ⅱ组。Flag-control组HASMCs加入Ang-Ⅱ后,细胞增殖标志物PCNA蛋白表达显著升高。在加入Ang-Ⅱ后,相较Flag-control组,Flag-Septin4和Flag-Septin4+CQ的PCNA表达显著减低,二者表达均少于Flag-control组1/2。Flag-control组HASMCs加入Ang-Ⅱ后,细胞合成col-1蛋白表达显著升高,约为未加药组2倍。相较于Flag-control组,Flag-Septin4和Flag-Septin4+CQ的col-1表达显著减低,其中Flag-Septin4+Ang-Ⅱ+CQ少于Flag-control+Ang-Ⅱ组1/2。Flag-control组HASMCs加入Ang-Ⅱ后,细胞迁移标志物MMP2蛋白表达显著升高,较未加药组增加1/2。加入Ang-Ⅱ后,相较与Flag-control组,Flag-Septin4和Flag-Septin4+CQ的MMP2表达显著减低,其中Flag-Septin4+Ang-Ⅱ+CQ约为Flag-control+Ang-Ⅱ组的1/3。加入Ang-Ⅱ前后,Flag-Septin4组p62蛋白均显著高于Flag-control组。Flag-Septin4+CQ组p62显著高于Flag-Septin4。类似地,Flag-Septin4+Ang-Ⅱ+CQ组p62显著高于Flag-Septin4+Ang-Ⅱ组。加入Ang-Ⅱ前后,Flag-Septin4组LC3-Ⅱ/GAPDH比值均显著低于Flag-control组。加入CQ后,Flag-Septin4+CQ组LC3-Ⅱ/GAPDH比值显著高于Flag-Septin4组。类似地,Flag-Septin4+Ang-Ⅱ+CQ组LC3-Ⅱ/GAPDH比值显著高于Flag-Septin4+Ang-Ⅱ组。进一步对比LC3-Ⅱ/I我们发现Flag-Septin4+Ang-Ⅱ+CQ组LC3-Ⅱ/I比值显著高于Flag-Septin4+Ang-Ⅱ组7、通过蛋白质谱（mass spectrometry,MS）分析发现14种与Septin4结合的蛋白。通过文献查阅,包含（TCP-1）的分子伴侣（CCT）复合体中的CCT2（氨基酸覆盖率19.14%）可能介导了Septin4对自噬的抑制效应。结论:1、Septin4通过抑制自噬削弱了Ang-Ⅱ对VSMCs增殖和迁移能力的增强效应,进而影响血管重构;2、Septin4可能通过与CCT2结合抑制VSMCs自噬。更多还原

【Abstract】 Objective:Vascular smooth muscle cells（VSMCs）proliferation and migration play a fundamental role during the process of hypertensive angiopathy.Angiotensin-Ⅱ（Ang-Ⅱ）is one of the robust phenotype-modulating agents,which changes VSMCs to efficiently proliferate and migrate.The mechanism of the proliferation and migration is not well understood yet.Autophagy is the major intracellular process for lysosome-mediated dynamic recycling system.The purpose of autophagy is to eliminate the damaged cytoplasmic material and produce new building blocks and energy for cellular renovation and homeostasis.Recent studies have approved that autophagy is a critical determinant in the changes of VSMC proliferative and migratory properties.Septin4,as a member of GTP binding protein family,is widely expressed in the eukaryotic cells and considered to be an essential component of the cytoskeleton which is involved in many important physiological processes.But it is still nounknown yet that if Septin4 plays a role in the cardiovascular diseases.We designed experiments to explore if Septin4 was involved in VSMCs proliferation and migration induced by Ang-Ⅱ.Furthermore,we discussed the mechanism whether it was related to autopahgy activity.Methods:1.We used mice as animal model.Osmotic pumps with Ang-Ⅱ（1.5mg/kg/day）or 0.9%NaCl as control were implanted subcutaneously.The aortas of the mice were harvested after 14 days,which were further stained by hematoxylin-eosin（HE）and Masson protocols.2.After aorta tissue was lysed,Septin4 protein amount was evaluated by Western blot assay.3.Human aortic smooth muscle cells（HASMCs）were cultured and Ang-Ⅱ at concentration gradient as0M,10^-8M,10^-7M,10^-6M,10^-5M were added and incubated for48 hours.CCK-8 and transwell tests were conducted in order to evaluate the proliferative and migratory activity.Septin4 protein amount was evaluated by western blot assay.At the same time,the most appropriate treating concentration of Ang-Ⅱ was screened.4.Overexpressing Septin4 was performed by plasmid transfection.Ang-Ⅱ at concentration gradient as0M,10^-8M,10^-7M,10^-6M,10^-5M were added and incubated for48 hours.CCK-8 assay was conducted to evaluate the proliferative activity.We used10-6 M Ang-Ⅱ as the best treating concentration to incubate and perform transwell test to examine the migratory activity.5.We set 4 groups as follows:Flag-control+0 M Ang-Ⅱ,Flag-control+10^-6 M Ang-Ⅱ,Flag-Septin4+Ang-Ⅱ+0 M Ang-Ⅱ,Flag-Septin4+10^-6 M Ang-Ⅱ.PCNA,col-I,MMP2,p62,LC3 protein amount was evaluated by Western blot assay.6.We designed six groups as follows,Flag-control,Flag-control+Ang-Ⅱ,Flag-control+Ang-Ⅱ+chloroquine（CQ）,Flag-Septin4,Flag-Septin4+Ang-Ⅱ,Flag-Septin4+Ang-Ⅱ+CQ.PCNA,col-I,MMP2,p62,LC3 protein was calculated through western blot assay.Besides,CCK-8 and transwell tests were performed to evaluate the proliferative and migratory activity.7.Mass spectrometry（MS）was conducted to test protein binding to Septin4.Results:1.Ang-Ⅱ increased Septin4 expression,the intima-media thickness and collagen content in mice aortas.2.With the increase of Ang-Ⅱ concentration,Septin4 expression was increased,as well as the capacity of VSMCs proliferation and migration.3.Stimulation effects of Ang-Ⅱ on proliferation and migration were alleviated by Septin4overexpression.4.Stimulation effects of Ang-Ⅱ on autophagy were inhibited by Septin4 overexpression.5.Ang-Ⅱ stimulated proliferation and migration in Septin4 overexpressing VSMCs were further inhibited by autophagy inhibitor.6.Mass spectrometry（MS）showed 14 protein binding to Septin4.By literature analysis,we speculated that binding to CCT2 may be the mechanism by which Septin4 modulated autophagy.Conclusion:Septin4 was involved in the VSMCs switching to a proliferative and migratory state stimulated by Ang-Ⅱ through modulating autophagic activity and the mechanism may be binding to CCT2.更多还原