【作者】 吴琪；

【导师】 王志维；

【作者基本信息】 武汉大学 ， 外科学， 2022， 博士

【摘要】 第一部分GLUT10与冠状动脉内膜增生相关性的研究目的:研究GLUT10在冠状动脉再狭窄后的表达变化,并分析其与冠状动脉再狭窄发病的相关性。方法:于武汉大学人民医院心血管外科临床标本库,选取接受心脏移植手术且冠状动狭窄患者的冠脉组织标本(Restenostic arteries group),以及接受心脏移植手术且冠状动脉正常的冠状动脉标本(Normal arteries group),所有患者均经冠脉CTA评估冠脉狭窄程度。记录患者的基本信息,包括年龄,性别,有无高血压糖尿病等。Western-Blot检测冠脉组织中GLUT10在蛋白水平的表达变化。冠脉组织切片免疫组化,分析GLUT10与VSMCs表型转化相关蛋白指标在冠脉再狭窄与正常冠脉组织标本中的表达变化;通过配对样本相关性分析,评估GLUT10与VSMCs表型转化的相关性。随后,筛选GEO数据库中动脉狭窄相关基因芯片,分析GLUT10在狭窄动脉中的表达变化,在m RNA水平探讨GLUT10表达与VSMCs表型转化蛋白指标的相关性。构建GLUT10全敲转基因小鼠,通过颈动脉结扎模型,观察GLUT10敲低后对于内膜增生进展的实际影响。结果:本研究共纳入27例正常冠状动脉组织标本,33例再狭窄组织标本。对两组患者临床相关数据统计分析,除发生冠状动脉狭窄外,与正常冠脉组患者相比,冠脉再狭窄组各项基本情况无显著差异(P>0.05)。Western-Blot发现,GLUT10表达在冠状动脉狭窄患者显著降低,且差异有统计学意义(P<0.05)。对60例冠脉标本行免疫组化染色,GLUT10表达趋势与Western-Blot结果一致;与正常冠脉相比,α-SMA,P21表达在狭窄冠状动脉表达下调;PCNA,MMP2,PDGF-BB表达显著升高;通过配对样本相关性分析表明,GLUT10与α-SMA表达呈正相关(r=0.6085,P<0.05);GLUT10与P21表达呈正相关(r=0.5256,P<0.05);GLUT10与PCNA表达呈负相关(r=-0.4749;P<0.05);与MMP2表达呈负相关(r=-0.4808,P<0.05);与PDGF-BB表达呈负相关(r=-0.4884,P<0.05)。同时,通过对GEO数据库基因表达芯片(GSE100927)分析发现,再狭窄冠脉GLUT10表达在m RNA水平显著降;同时,对(GSE100927)及(GSE24495)中GLUT10与VSMCs表型转化相关蛋白指标进行相关性分析发现,GLUT10与VSMCs收缩表型相关蛋白指标表达呈正相关;与合成型相关蛋白指标表达呈负相关。结论:冠状动脉再狭窄后,GLUT10表达在蛋白水平较正常冠状动脉显著降低;GLUT10表达下调促进VSMCs向合成型转化;同时,GLUT10敲除后促进内膜增生进展,因此,GLUT10表达水平降低会促进内膜增生进展。第二部分GLUT10通过调控VSMCs表型转化参与内膜增生过程目的:研究GLUT10定位于动脉内何种细胞成分,在体特异性干预特定细胞成分GLUT10表达后,对VSMCs表型转换以及内膜增生发展的影响。方法:收集到的人体冠脉标本组织切片行GLUT10与α-SMA免疫荧光双染,确定GLUT10蛋白是否主要表达于VSMCs。构建小鼠颈动脉结扎后0天,7天,14天,28天动物模型,观察GLUT10表达在内膜增生进展过程中的时间变化趋势。PDGF-BB在不同时间点刺激RSMCs,RT-PCR和Western-Blot观察GLUT10在m RNA水平以及蛋白水平的表达变化。将携带SM22α启动子的慢病毒与F-127凝胶结合,实现小鼠颈动脉原位GLUT10过表达与敲低;免疫荧光以及RT-PCR评估慢病毒转染效率;EVG染色检测小鼠颈动脉中膜面积与内膜面积之比;免疫荧光分析在小鼠颈动脉结扎模型中,VSMCs在GLUT10表达干预后α-SMA,SM22α,CNN1,Cyclin D1,PCNA,MMP2,MMP9的表达变化情况。腺病毒干预RSMCs中GLUT10的表达,Western-Blot验证腺病毒干预效果;细胞迁移实验检测RSMCs在GLUT10表达干预后的迁移能力;凝胶收缩实验评估GLUT10干预后RSMCs收缩能力变化。结果:GLUT10与α-SMA免疫荧光双染,发现GLUT10主要表达于冠状动脉中膜VSMCs;且随着冠状动脉狭窄程度的加重,GLUT10表达也逐渐降低。GLUT10在小鼠颈动脉内膜增生模型中的表达呈现先升高后降低的趋势,在第7天GLUT10表达升至最高,与造模0天相比差异有统计学意义(P<0.05);随后GLUT10表达逐渐降低,并在28天表达最低,相较于0天造模组,差异有统计学意义(P<0.05)。在体外实验中,GLUT10的表达与体内实验的趋势基本一致,呈现先升高后降低的变化;RSMCs在PDGF-BB刺激36小时后,GLUT10表达最高,在PDGF-BB刺激72小时后GLUT10表达降至最低,与PDGF-BB处理0小时相比,差异具有统计学意义(P<0.05)。慢病毒特异性敲低小鼠颈动脉VSMCs中GLUT10,并构建小鼠颈动脉结扎模型后,小鼠颈动脉增生内膜面积与中膜面积之比较对照组显著增高,且差异有统计学意义(P<0.05);相较于对照组,小鼠颈动脉α-SMA,SM22α,CNN1三种VSMCs收缩型蛋白标志物表达显著降低,差异有统计学意义(P<0.05);而Cyclin D1,PCNA,MMP2,MMP9四种VSMCs合成型标志蛋白表达显著升高,差异有统计学意义(P<0.05)。同时腺病毒敲低RSMCs中GLUT10后,RSMCs迁移能力较对照组显著增强,收缩能力显著降低,且差异均具有统计学意义。通过慢病毒在小鼠颈动脉VSMCs中过表达GLUT10,构建小鼠颈动脉结扎模型,发现小鼠颈动脉增生内膜面积与中膜面积之比较对照组显著降低,且差异有统计学意义(P<0.05);相较于对照组,小鼠颈动脉α-SMA,SM22α,CNN1三种VSMCs收缩表型指标表达显著升高,差异有统计学意义(P<0.05);而Cyclin D1,PCNA,MMP2,MMP9四种VSMCs合成型标志蛋白表达显著下调,差异有统计学意义(P<0.05)。在RSMCs中过表达GLUT10联合PDGF-BB刺激后,Ad-OE-GLUT10+PDGF-BB组较Ad-GFP+PDGF-BB组迁移能力显著减弱,细胞收缩能力增强,且差异均具有统计学意义(P<0.05)。而在不结合PDGF-BB处理时,SMC-OE-GLUT10组与SMC-GFP组表达无显著差异。结论:GLUT10主要定位于冠脉VSMCs,并且在内膜增生过程中的表达呈现出先升高后降低的趋势。体内或体外敲低GLUT10可能通过促进VSMCs向合成型分化加速内膜增生的发生发展,而VSMCs特异性过表达GLUT10则会在一定程度上逆转这一过程。第三部分GLUT10通过影响VSMCs线粒体功能稳态调控VSMCs表型转化目的:阐明GLUT10通过何种具体机制调控VSMCs表型转化进而影响内膜增生进展。方法:Tom20与GLUT10免疫荧光双染,共聚焦显微镜观察PDGF-BB在不同时间点处理后,GLUT10在RSMCs内线粒体转位情况;提取接受PDGF-BB在不同时间点刺激后RSMCs中的线粒体,Western-Blot进一步验证GLUT10在线粒体中表达水平的变化;RT-PCR以及Western-Blot检测GLUT10敲低后线粒体分裂融合相关蛋白DRP1,p DRP1ser616,Fis1,MFN1,MFN2,OPA1在m RNA及蛋白水平的表达变化。Tom20与KDEL免疫荧光双染,共聚焦显微镜观察敲低GLUT10后,MAM的形成情况。Mitotracker对线粒体染色,评估GLUT10敲低后线粒体形态变化。RT-PCR检测线粒体氧化呼吸链相关蛋白ND1,ND2,ND3,ND4,ND4L,ND5,CYTB,COX1,COX2,COX3,ATP6,ATP8以及TFAM表达变化情况。ATP以及OCR检测试剂盒检测GLUT10敲低后,RSMCs中ATP及OCR的变化趋势。GLUT10敲低后,将线粒体移植入线粒体功能被抑制的RSMCs中,确定GLUT10敲低后线粒体功能对RSMCs表型转化的影响,Western-Blot分析线粒体移植后VSMCs表型转化相关指标;细胞迁移实验以及胶原收缩实验,分析RSMCs迁移能力和收缩能力改变。结果:GLUT10在PDGF-BB刺激36小时后,主要定位于线粒体;在PDGF-BB处理72小时后,GLUT10在线粒体的定位减少;与Western-Blot检测RSMCs线粒体蛋白的趋势基本一致,且两组间比较有统计学意义(P<0.05)。敲除RSMCs中GLUT10后,通过RT-PCR检测,发现线粒体分裂相关蛋白Fis1表达显著升高,线粒体融合蛋白MFN2表达显著降低,且具有统计学意义(P<0.05);然而OPA1,DRP1,MFN1表达则无显著性差异。通过Western-Blot发现,p DRP1Ser616/Drp1在GLUT10敲低后显著升高(P<0.05),Fis1,MFN1,MFN2及OPA1的表达变化与RT-PCR趋势一致。在GLUT10敲低后,共聚焦显微镜观察发现MAM形成增多,同时线粒体分裂增强。RSMCs中GLUT10敲低后,mt DNA编码的线粒体氧化呼吸链复合体Ⅰ组成蛋白,ND1,ND2表达显著降低;线粒体氧化呼吸链复合体Ⅲ组成蛋白,CYTB表达显著降低;线粒体氧化呼吸链复合体Ⅳ组成蛋白,COX1,COX2,COX3表达显著降低;线粒体氧化呼吸链复合体Ⅴ组成蛋白,ATP6,ATP8表达显著降低,且差异均具有统计学意义(P<0.05)。同时,RSMCs中ATP含量在GLUT10敲低后显著降低(P<0.05);OCR速率亦显著降低,且差异具有统计学意义(P<0.05)。结论:GLUT10对于线粒体功能稳态的调控具有极为重要的作用,并极可能通过影响线粒体功能调控VSMCs表型转化。第四部分GLUT10通过促进VSMCs中mt DNA去甲基化影响线粒体功能稳态调控VSMCs表型转化目的:阐明GLUT10调控线粒体功能稳态的作用机制,及对于内膜增生进展的影响。方法:气相色谱分析GLUT10表达干预后线粒体内DHA的摄入速率变化;AA检测试剂盒检测GLUT10敲低后线粒体内AA含量的变化。Dot-blot检测不同浓度AA处理后mt DNA的甲基化水平;Dot-blot检测正常冠脉和再狭窄冠脉标本中mt DNA甲基化水平变化;在RSMCs中敲低GLUT10后,提取mt DNA,通过Dot-blot检测5-hm C水平变化;MSP检测mt DNA上D-loop区甲基化水平的变化情况。提取RSMCs线粒体蛋白,通过Western-Blot检测线粒体TET1,TET2,TET3蛋白表达变化。合成线粒体靶向的纳米材料,扫描电镜分析合成纳米材料性状,检测纳米材料电势,粒径,细胞凋亡流式检测纳米材料处理细胞后对细胞功能状态的影响。为了检测纳米颗粒的线粒体靶向性,通过FITC标记纳米颗粒处理RSMCs,并采用Mitotracker进行染色,共聚焦显微镜下观察线粒体对纳米颗粒的摄取情况,评估纳米颗粒的线粒体靶向性。纳米颗粒装载Bobcat339,靶向抑制线粒体内TET家族蛋白活性,Dot-blot检测其对mt DNA甲基化的实际效果。在RSMCs中过表达GLUT10,在PDGF-BB的处理下,纳米材料抑制TET家族功能后,通过Western-Blot分析RSMCs表型转化的相关指标。细胞迁移实验以及胶原收缩实验,分析RSMCs迁移能力和收缩能力改变。结果:GLUT10敲低后抑制线粒体内DHA转运,较对照组差异有统计学意义(P<0.05),线粒体内AA含量明显降低,较对照组差异有统计学意义(P<0.05)。再狭窄冠脉mt DNA去甲基化水平较正常冠脉显著降低;GLUT10敲低后,mt DNA甲基化水平降低,且mt DNA上D-loop区甲基化水平显著升高。线粒体内TET2,TET3蛋白在GLUT10敲低后表达显著降低,较对照组差异有统计学意义(P<0.05),通过扫描电镜可见纳米颗粒成功合成,同时纳米材料粒径检测电势为负,细胞亲和力强;细胞流式发现细胞纳米颗粒摄入性强;凋亡流式表明纳米材料处理细胞后对细胞的正常功能状态无明显影响;共聚焦显微镜观察表明线粒体靶向纳米材料成功富集于线粒体。与此同时,在PDGF-BB处理下,过表达GLUT10部分逆转PDGF-BB诱导的VSMCs向合成型转化;而通过纳米材料抑制TET家族蛋白功能后,此逆转效应被显著削弱。结论:GLUT10低表达通过抑制线粒体DHA的转运限制AA的合成,进而下调TET2及TET3蛋白表达水平,上调mt DNA中D-loop区甲基化水平,导致线粒体功能紊乱,平滑肌细胞向合成型转化。第五部分MA通过激活AMPK/Drp1轴促进线粒体融合维持VSMCs收缩表型抑制内膜增生进展目的:探明MA调控VSMCs线粒体分裂融合对内膜增生进展的实际效果与具体作用机制。方法:组织贴壁法培养RSMCs;MA浓度梯度处理RSMCs,摸索MA的最佳作用浓度。PDGF-BB诱导RSMCs由收缩型向合成型转化,RT-PCR、Western-Blot检测PDGF-BB联合或不联合MA处理对RSMCs增殖、迁移相关指标MMP2与PCNA的表达的影响。划痕实验与Transwell细胞迁移实验分析PDGF-BB联合或不联合MA处理对RSMCs迁移能力的影响。免疫荧光、Western-Blot检测PDGF-BB联合或不联合MA处理对RSMCs收缩力相关蛋白的表达影响。细胞流式分析PDGF-BB联合或不联合MA处理后ROS水平的影响。RSMCs在Mitotracker染色后于共聚焦显微镜下观察线粒体形态变化;Western-Blot分析线粒体动力学相关指标MFN1、MFN2、OPA1、Fis1、Drp1、Drp1Ser616在蛋白水平的表达变化。AMPK特异性抑制剂Compound C与PDGF-BB或MA联合处理评估MA是否经由AMPK信号通路发挥作用。构建小鼠颈动脉结扎模型,给与或不给与MA腹腔注射,EVG染色评估增生内膜与中膜面积,评估MA对内膜增生过程的实际效果。免疫组化染色分析小鼠颈动脉内膜增生标本中线粒体分裂融合以及VSMCs表型转化相关指标的表达变化。结果:MA抑制了PDGF-BB诱导的RSMCs增殖、迁移,部分逆转了PDGF-BB诱导RSMCs向合成型转化的趋势;MA通过抑制线粒体分裂,减轻RSMCs活性氧水平,维持RSMCs收缩表型。MA可能通过激活AMPK功能参与线粒体分裂融合的调控;MA减轻小鼠颈动脉增生内膜面积,降低增生内膜面积与中膜面积之比。结论:MA通过激活AMPK/Drp1轴促进线粒体融合,促使VSMCs向收缩型转化,抑制增生内膜形成。更多还原

【Abstract】 Part Ⅰ Correlation Between GLUT10 and coronary neointimal formationObjective : To investigate the correlation between GLUT10 expression level and coronary artery restenosis.Method : Human coronary artery tissue samples of patients who underwent transplantation were selected and stained using immunohistochemistry.the degree of coronary stenosis was evaluated by CTA.Basic information of all patients including age,sex,history of hypertension,diabetes.The GLUT10 expression at protein level was examined by Western-blot.Immunohistochemical analysis of coronary artery tissues were performed to analysis the expression change of GLUT10,α-SMA,P21,MMP2,PDGF-BB between normal coronary artery and restenostic coronary artery.The expression correlation between GLUT10 and α-SMA,P21,MMP2,PDGF-BB in coronary artery tissue samples was evaluated by correlation analysis.Subsequently,Genechips from the GEO database were used to examinate GLUT10 expression in m RNA level in restenotic arteries.Correlations between GLUT10 levels and the m RNA expression of phenotype transformation-related genes were explored.In order to examine the function of GLUT10 in neointimal formation,GLUT10-knockout mice were constrcted.Result:A total of 27 normal coronary artery tissue specimens and 33 restenosis tissue specimens were included in this study.Western-Blot showed that GLUT10 expression was significantly lower in patients with coronary artery stenosis,and the difference was statistically significant(P<0.05)).Immunohistochemical staining of 60 coronary artery specimens showed a trend of GLUT10 expression consistent with Western-Blot results.GLUT10 was positively correlated with α-SMA expression(r=0.6085,P<0.05);GLUT10 was positively correlated with P21 expression(r=0.5256,P<0.05);GLUT10 was negatively correlated with PCNA expression(r=-0.4749;P<0.05);MMP2 expression(r=-0.4808,P<0.05);PDGF-BB expression was negatively correlated(r=-0.4884,P<0.05).Meanwhile,analysis of the GEO database gene expression microarray(GSE100927)revealed that GLUT10 expression in restenotic coronary arteries was significantly decreased at the m RNA level;Meanwhile,correlation analysis of GLUT10 and VSMCs phenotype transformation-related protein indicators in(GSE100927)and(GSE24495)revealed that GLUT10 and VSMCs The correlation between GLUT10 and VSMCs phenotypic transformation-related protein indicators in(GSE100927)and(GSE24495)was found to be positive and negative.Conclusion: After coronary restenosis,GLUT10 expression was significantly lower compared to normal coronary arteries at protein level;downregulation of GLUT10 expression promoted the VSMCs differentiation;meanwhile,GLUT10 knockdown promoted the progression of neointimal hyperplasia.Part Ⅱ GLUT10 is involves in neointimal hyperplasia by regulating the phenotypic transformation of VSMCsObjective : To investigate the localization of GLUT10 in coronary artery,and exploring the effect of GLUT10 on VSMCs phenotypic transformation,and analysis the neointimal formation progression after GLUT10 specific knockdown or overexpression in VSMCs.Method: The mouse carotide artery were harvested at 0,7,14 and 28 days after carotid artery ligation to observe the GLUT10 expression during the progression of neointimal formation.RT-PCR and Western-Blot were performed to observe the changes in GLUT10 expression at the m RNA level as well as at the protein level.The lentivirus carrying SM22α promoter was combined with F-127 gel to achieve in situ GLUT10 overexpression and knockdown in mouse carotid arteries;immunofluorescence and RT-PCR were used to assess the transfection efficiency of the lentivirus;EVG staining was performed to detect the ratio of intima area to the meida area in mouse carotid arteries;immunofluorescence analysis of VSMCs in a mouse carotid artery ligation model after GLUT10 expression intervention in α-SMA,SM22α,CNN1,Cyclin D1,PCNA,MMP2,MMP9 expression changes in a mouse carotid artery ligation model.The effect of adenovirus intervention on GLUT10 expression in RSMCs was analyzed by Western-Blot;cell migration assay was performed to evaluate the migration ability of RSMCs after GLUT10 expression knockdown or over-expression;gel contraction assay was performed to assess the change in contractility of RSMCs after GLUT10 knockdown or over-expression.Result:The expression of GLUT10 in the mouse carotid artery neointimal formation model showed an increasing and then decreasing trend,compared to day 0,the highest expression of GLUT10 on day 7,the difference was statistically significant(P<0.05));then GLUT10 expression gradually decreased and was lowest on day 28,compared with the day 0 group(P<0.05)).In the experiments in vitro,the trend of GLUT10 expression was showed same trends with in vivo experiments;RSMCs showed the highest GLUT10 expression after 36 h stimulation of PDGF-BB and the lowest expression GLUT10 after 72 h stimulation of PDGF-BB,and the difference was statistically significant compared with 0h of PDGF-BB treatment(P<0.05)).After lentivirus knockdown GLUT10 expression in mouse carotid VSMCs and then,construction of a mouse carotid artery ligation model,the ratio of intima area to media area of mouse carotid arteries were significantly higher compared to the control group,and the difference was statistically significant(P<0.05);compared to the control group,the expression of three VSMCs contractile protein markers,α-SMA,SM22αand CNN1 was significantly lower in mouse carotid arteries compared with the control group,the expression of three VSMCs contractile protein markers,namelyα-SMA,SM22α and CNN1 was significantly lower(P<0.05),while the expression of four VSMCs synthetic markers,namely Cyclin D1,PCNA,MMP2 and MMP9,was significantly higher(P<0.05).Meanwhile,after adenoviral knockdown the GLUT10 expression in RSMCs,the migration ability of RSMCs was significantly enhanced and the contractility was significantly decreased compared with the control group,and all differences were statistically significant.In the mouse carotid artery ligation model constructed by lentiviral overexpression of GLUT10 in mouse carotid VSMCs,it was found that the ratio of intima area to media area of mouse carotid arteries were significantly lower compared to the control group,and the differences were statistically significant(P<0.05).Compared with the control group,the expression of three VSMCs,namely α-SMA,SM22α and CNN1,was significantly increased,and the difference was statistically significant(P<0.05),while the expression of four VSMCs,Cyclin D1,PCNA,MMP2 and MMP9,was significantly down-regulated,and the difference was statistically significant(P<0.05).After overexpression of GLUT10 combined with PDGF-BB stimulation in RSMCs,the Ad-OE-GLUT10+PDGF-BB group showed significantly reduced migratory capacity and enhanced cell contraction compared to the Ad-GFP+PDGF-BB group,and all differences were statistically significant(P<0.05).In contrast,there was no significant difference in expression between the SMC-OE-GLUT10 group and the SMC-GFP group when not combined with PDGF-BB treatment.Conclusion: GLUT10 is mainly localized in coronary VSMCs,and its expression tends to increase and then decrease during neointimal formation.Knockdown of GLUT10 in vivo or in vitro may accelerate the development of neointimal hyperplasia by promoting the differentiation of VSMCs,whereas VSMCs-specific overexpression of GLUT10 reverses this process to some extent.Part Ⅲ GLUT10 regulates VSMCs phenotypic transformation by influencing mitochondrial functional homeostasisObjective:To elucidate the specific mechanisms by which GLUT10 regulates the phenotypic transformation of VSMCs and thereby influences the progression of neointimal formation.Method : Tom20 and GLUT10 immunofluorescence double-staining,confocal microscopy to observe mitochondrial translocation of GLUT10 in RSMCs after PDGF-BB treatment at different time points;extraction of mitochondria in RSMCs after receiving PDGF-BB stimulation at different time points,Western-Blot to further verify the expression level of GLUT10 in mitochondria,The mitochondrial dynamic-related proteins DRP1,p DRP1ser616,Fis1,MFN1,MFN2 and OPA1 were detected at the m RNA and protein levels after GLUT10 knockdown by RT-PCR and Western-Blot.Mitotracker staining of mitochondria was performed to assess the changes in mitochondrial morphology after GLUT10 knockdown.RT-PCR was performed to detect changes in the expression of ND1,ND2,ND3,ND4,ND4 L,ND5,CYTB,COX1,COX2,COX3,ATP6,ATP8 and TFAM,mitochondrial oxidative respiratory chain-related proteins.ATP and OCR assay kits were used to detect the trend of ATP and OCR changes in RSMCs after GLUT10 knockdown.After GLUT10 knockdown,mitochondria were transplanted into RSMCs to determine the effect of mitochondrial function on the phenotypic transformation of RSMCs after GLUT10 knockdown.Result : GLUT10 was mainly localized in mitochondria after 36 h of PDGF-BB stimulation;after 72 h of PDGF-BB treatment,the localization of GLUT10 in mitochondria was reduced;the trend was consistent with the Western-Blot assay of mitochondrial proteins in RSMCs,and the comparison between the two groups was statistically significant(P<0.05).After knockdown of GLUT10 in RSMCs,a significant increase in the expression of Fis1,a mitochondrial division-related protein,and a statistically significant decrease in the expression of MFN2,a mitochondrial fusion protein,were detected by RT-PCR(P<0.05);however,there was no significant difference in the expression of OPA1,DRP1,and MFN1.Western-Blot revealed that p DRP1Ser616/Drp1 was significantly increased after GLUT10 knockdown(P<0.05)),and the expression changes of Fis1,MFN1,MFN2 and OPA1 were consistent with the RT-PCR trend.The expression of mt DNA-encoded mitochondrial oxidative respiratory chain complex I component proteins,ND1 and ND2,was significantly reduced after GLUT10 knockdown in RSMCs;the expression of mitochondrial oxidative respiratory chain complex III component protein,CYTB,was significantly reduced;and the expression of mitochondrial oxidative respiratory chain complex IV component protein,COX1,was significantly reduced after GLUT10 knockdown in RSMCs.The expression of COX1,COX2 and COX3,the component proteins of mitochondrial oxidative respiratory chain complex IV,and the expression of ATP6 and ATP8,the component proteins of mitochondrial oxidative respiratory chain complex V,were significantly reduced,and the differences were statistically significant(P<0.05).Meanwhile,ATP content in RSMCs was significantly reduced after GLUT10 knockdown(P<0.05);OCR rate was also significantly reduced,and the difference was statistically significant(P<0.05).Conclusion: GLUT10 plays an important role in the regulation of mitochondrial functional homeostasis,and is likely to regulate the phenotypic transformation of VSMCs by affecting mitochondrial function.Part Ⅳ GLUT10 affects mitochondrial functional homeostasis by promoting mt DNA demethylation and regulates VSMCs phenotypic transformationObjective : To elucidate the mechanism of action of GLUT10 in regulating mitochondrial functional homeostasis and its impact on the progression of neointimal formation.Method : Gas chromatography analysis of changes in the rate of DHA uptake in mitochondria after GLUT10 expression intervention;vitamin C assay kit to detect the concentration of vitamin C levels in mitochondria after GLUT10 knockdown.methylation levels of mt DNA after treatment with different concentrations of ascorbic acid by Dot-blot;mt DNA methylation in normal and restenotic coronary specimens by Dot-blot;After GLUT10 knocked down in RSMCs,mt DNA was extracted and the changes in 5-hm C levels were detected by Dot-blot;the changes in methylation levels of the D-loop region on mt DNA were detected by MSP.Mitochondrial proteins of RSMCs were extracted and changes in mitochondrial TET1,TET2 and TET3 protein expression were detected by Western-Blot.Mitochondria-targeted nanomaterials were synthesized and the traits of the synthesized nanomaterials were analysed by scanning electron microscopy to detect nanomaterial potential,particle size;Flow cytometry was used to detect the apoptosis rate of RSMCs after nanomaterial treatment on the functional state of the cells.To detect the mitochondrial targeting of nanoparticles,the mitochondrial targeting of nanoparticles was assessed by FITC labelling of nanoparticles,treatment of RSMCs and staining with Mitotracker and confocal microscopy to observe the uptake of nanoparticles by mitochondria.Nanoparticles were loaded with Bobcat339 to target inhibit TET family protein activity in mitochondria;Dot-blot was used to detect their actual effect on mt DNA methylation.After overexpression of GLUT10 in RSMCs and inhibition of TET family function by nanomaterials under PDGF-BB treatment,VSMCs were analysed by Western-Blot for relevant indicators of phenotypic transformation.Cell migration assays as well as collagen contraction assays were performed to evaluate the migratory capacity and contractility of VSMCs.Result : GLUT10 knockdown inhibited mitochondrial DHA transport,statistically significant differences compared to control group(P<0.05),and mitochondrial AA levels were significantly reduced compared to controls(P<0.05).The level of mt DNA demethylation was significantly lower in restenotic coronary arteries compared to normal coronary arteries;GLUT10 knockdown resulted in lower mt DNA methylation levels and significantly higher methylation levels in the D-loop region on mt DNA.The expression of TET2 and TET3 proteins in mitochondria was significantly reduced after GLUT10 knockdown,and the difference was statistically significant compared to the control group(P<0.05).Successful synthesis of nanoparticles was seen by scanning electron microscopy,the nanomaterial particle size was detected with negative electrical potential;flow cytometry revealed strong uptake of nanoparticles;RSMCs apoptosis rate after nanomaterial treatment had no significant difference;Confocal microscopy showed that the mitochondria-targeted nanomaterials were successfully enriched in the mitochondria.Meanwhile,overexpression of GLUT10 partially reversed the PDGF-BB-induced transformation of VSMCs to a synthetic phenotype under PDGF-BB treatment.Conclusion: GLUT10 knockdown limits AA synthesis by inhibiting mitochondrial DHA transport,which in turn downregulates TET2 and TET3 protein expression levels and upregulates methylation levels in the D-loop region of mt DNA,leading to mitochondrial dysfunction and then promotes VSMCs differentiation.Part Ⅴ Mangiferin maintains the contractile phenotype of VSMCs by activating the AMPK/Drp1 axis to promote mitochondrial fusion and inhibit the progression of neointimal formationObjective : To investigate the actual effect and specific mechanism of action of mangostin in regulating mitochondrial fusion in VSMCs on the progression of neointimal hyperplasia.Method : The best concentration of mangiferin was determined by the gradient treatment of mangiferin,MMP2 and PCNA were detected by RT-PCR and Western-Blot.The effect of PDGF-BB treatment combine with or without mangiferin on the migration ability of VSMCs was analyzed by scratch assay and Transwell cell migration assay.Immunofluorescence and Western-Blot assays were performed to detect the effect of contractility-related proteins in RSMCs after administrated PDGF-BB combine with or without the treatment of mangiferin.flow cytometry was used to analysis the ROS levels after PDGF-BB treament and combined wirh or without mangiferin treatment.VSMCs were observed under confocal microscopy for mitochondrial morphological changes after Mitotracker staining;Western-Blot analysis of mitochondrial dynamic related indicators MFN1,MFN2,OPA1,Fis1,Drp1.Compound C,a specific inhibitor of AMPK,was combined with PDGF-BB or mangiferin to assess whether mangiferin acts via the AMPK signalling pathway.A mouse carotid artery ligation model was constructed with or without mangiferin intraperitoneal injection,EVG staining was performed to assess the actual effect of mangiferin on the neointimal hyperplasia process.Immunohistochemical staining was performed to analyse the expression of mitochondrial fusion and VSMCs phenotypic transformation related genes in mouse carotid artery.Result : Mangiferin inhibited PDGF-BB-induced proliferation and migration of RSMCs and partially reversed the PDGF-BB-induced conversion of VSMCs to the synthetic phenotype;Mangiferin maintained the contractile phenotype of VSMCs by inhibiting mitochondrial fission and attenuating the level of ROS in VSMCs.mangiferin may be involved in the regulation of mitochondrial fission and fusion through activation of AMPK function;mangiferin attenuated the neonatal intimal area and reduced the ratio of intima area/media area in mouse carotid artery.Conclusion: Mangiferin promotes mitochondrial fusion by activating the AMPK/Drp1 axis,which inhibited VSMCs dedifferentiation and inhibits neointimal formation.更多还原