【作者】 吴文胜；

【导师】 刘闺男；

【作者基本信息】 中国医科大学 ， 内科学， 2007， 博士

【摘要】 目的动脉粥样硬化(AS)是冠心病、脑血管意外等心脑血管疾病的病理基础。动脉粥样硬化的发病机理非常复杂,曾有多种学说从不同角度进行探讨,但至今尚未完全明了。目前发现,在各种因素的作用下发生粥样硬化的动脉中,平滑肌细胞表型的改变、增殖和迁移是病理过程的重要环节,转变为合成型后的平滑肌细胞可分泌多种生长因子,使自身和周围细胞大量增殖,并最终导致动脉管腔狭窄,粥样斑块和纤维帽的形成。何种因素启动了平滑肌细胞的表型转化目前仍不十分清楚,可能与多种生长因子、代谢产物等因素有关。研究发现,在平滑肌细胞由收缩型转化为合成型的过程中,一些细胞器,如核糖体、粗面内质网、高尔基体以及线粒体等大量增加,线粒体的增加尤为显著,猜测可能与细胞生长、分裂,以及分泌都需要氧化磷酸化提供大量的能量有关。但线粒体在动脉粥样硬化的形成及平滑肌细胞表型转化过程中的确切作用还不清楚。线粒体在功能改变时,先是线粒体DNA的表达发生转变,这种转变受核基因和线粒体基因共同调控,与其有关的基因很多,目前认为线粒体转录因子A(mtTFA)是线粒体DNA的转录和复制的关键因子,核基因通过调节mtTFA而调控线粒体的功能,启动线粒体内氧化磷酸化过程相关因子的复制、转录和翻译。mtTFA受核呼吸因子(NRF)的调节,有研究表明在寒冷或缺氧的条件下,NRF表达显著增加,同时上调mtTFA,增加线粒体DNA复制,进而增加氧化磷酸化提供能量,以维持细胞的功能。近年来有研究表明,过氧化物酶体增生激活受体γ协同刺激因子(PGC-1)参与细胞内多种物质的代谢过程,可以与多种核受体结合,调节细胞功能。为了能明确线粒体功能在动脉粥样硬化病理发生中的作用,了解其调控的可能机制,我们应用动物模型实验和平滑肌细胞培养实验来探讨可能的病理机制。方法一、血管平滑肌细胞表型改变与线粒体功能调节的动物实验研究1、动物模型建立雄性健康大耳白兔38只。动物模型建立及分组:将实验兔随机分为4组。正常对照组(C)8只,喂饲基础饲料,实验组(AS模型组)30只,每组10只,喂饲含2%胆固醇,8%猪油,4%蛋黄粉高脂饲料。2、血液生化指标的测定实验开始及第4、8、12周末由兔耳缘静脉抽血测血清总胆固醇(TC),甘油三脂(TG),高密度脂蛋白胆固醇(HDL-C)浓度,低密度脂蛋白胆固醇(LDL-C)浓度。指标测定由辽宁中医学院检验科全自动生化分析仪完成。3、标本取材实验开始时及第4、8、12周末,分别取实验组8只,取出主动脉,将主动脉纵行剪开,选择主动脉弓处取材。根据不同实验技术和检测指标,取材标本应用不同的方法处理和保存。4、主动脉血管壁形态学观察取己用福尔马林固定的主动脉弓标本经石蜡包埋、切片,HE染色镜检观察主动脉动脉病理变化。5、PGC-1、NRF-1、mtTFA及SMemb的mRNA水平测定(RT-PCR法)总RNA提取,用紫外分光光度计检测核酸OD值,将原液核酸配成1μg/μl浓度。逆转录反应:PCR仪按94℃2min,94℃40s,57℃40s,72℃1min的条件循环35次,完成PCR反应。6、PGC-1、NRF-1、mtTFA及SMemb的蛋白水平测定(Western-blot法)将主动脉新鲜组织块在生理盐水中漂洗后,放入EP管中—70℃保存;实验时,加入相应体积的裂解液,将样品剪碎后匀浆,离心收集上清;取上清10μl,检测蛋白浓度(测OD值);按说明书进行蛋白电泳和转膜。扫描仪扫描印记膜。二、平滑肌细胞增殖的干预实验1、细胞培养人动脉平滑肌细胞加入培养基置于37℃,5%CO2细胞培养箱中培养,每两天换一次液,当细胞长满瓶底80%左右时,用0.1%胰蛋白酶消化传代,第3～10代细胞用于实验。2、实验分为四组(1)正常对照组予以常规培养基。(2)oxLDL组,常规培养基中加入oxLDL使其终浓度达到10μg/ml作用24小时(3)棕榈酸组,在(2)组方法的基础上,培养基中加入棕榈酸使其终浓度达到0.75 mmol/l作用24小时。(4)叠氮钠组,在(2)组方法的基础上,培养基中加入叠氮钠使其终浓度达到32mmol/L作用24小时。3、各组PGC-1、NRF-1及mtTFA的蛋白表达水平检测,应用Western-blot方法。4、线粒体提取及功能测定线粒体提取功能测定,按试剂盒说明进行操作。5、MTT染色法检测细胞活性酶联免疫检测仪下选择波长490nm测定各孔的吸光度值(OD值)。细胞增殖率(%)=(实验组OD值-对照组OD值)/对照组OD值×100%。6、增殖细胞核抗原PCNA免疫组化检测SABC法免疫组化检测培养细胞之增殖细胞核抗原PCNA。结果一、平滑肌细胞表型改变与线粒体调控相关因子的检测结果1、实验动物一般状态实验过程中各组动物同期体重增加无显著性差异(P＞0.05)。2、不同时间的血脂测定结果随着喂饲高脂饲料的时间延长,动物血清中血脂含量水平呈上升趋势,其中胆固醇、低密度脂蛋白水平浓度增加显著。3、不同时期动脉内膜与中膜的厚度及内膜与中膜比值内膜在4W、8W、12W逐渐增厚,中膜在8W、12W增厚,内膜/中膜逐渐增加。4、PGC-1、NRF-1、mtTFA、SMemb的RT-PCR结果PGC-1的mRNA表达在4W、8W、12W与喂饲高脂食物前比较差异有显著性(p＜0.01),随着时间的延长,其表达呈现上升趋势(p＜0.01)。NRF-1的mRNA表达在4W,与喂高脂食物前比较差异显著性(p＜0.01),8W以后其表达趋于平稳,8W和12W与4W比较差异亦有显著性。而8W与12W比较差异无显著性(p＞0.05)。mtTFA的mRNA表达与NRF-1趋于同步升高。SMemb的mRNA表达随着时间的延长,其表达呈现上升趋势,4个时间段比较,差异均有显著性(p＜0.01)。5、PGC-1、NRF-1、mtTFA、SMemb的Westernblot测定结果PGC-1的蛋白表达4W、8W、12W与0W比较差异有显著性,其中4W与8W、12W比较差异有显著性(p＜0.01),而8W、12W比较差异无显著性(p＞0.05)。NRF-1的蛋白表达在4W与0W比较差异有显著性(p＜0.01),8W、12W与0W、4W差异有显著性(p＜0.01)。而8W与12W比较差异无显著性(p＞0.05)。mtTFA的蛋白表达在4W与0W比较差异显著性(p＜0.05),8W、12W与0W、4W差异有显著性(p＜0.05)。而8W与12W比较差异无显著性(p＞0.05)。SMemb蛋白表达随时间延长亦呈现上升趋势,与其mRNA水平改变一致。二、平滑肌细胞增殖干预与线粒体功能改变的检测结果1、不同干预因素作用下,PGC-1/NRF-1/mtTFA蛋白水平改变Ox-LDL组PGC-1、NRF-1、mtTFA蛋白表达增加(p＜0.05),棕榈酸组与ox-LDL组比较PGC-1、NRF-1、蛋白表达减少(p＜0.05),叠氮钠组与ox-LDL组比较PGC-1、NRF-1、mtTFA蛋白表达是增加的(p＜0.05)。2、不同干预因素作用下,线粒体细胞色素C氧化酶活性改变Ox-LDL组与对照组比较明显升高(p＜0.05),棕榈酸组与oxLDL组比较COX活性降低(p＜0.05),叠氮钠组与oxLDL组比较明显降低(p＜0.01)。3、不同干预因素作用下,细胞增殖活性的改变oxLDL促进细胞增殖(p＜0.05),棕榈酸组抑制细胞增殖(p＜0.05),应用叠氮钠后可以抑制细胞增殖(p＜0.01)。4、不同干预因素下平滑肌细胞PCNA的检测结果Ox-LDL组与对照组比较,PCNA蛋白表达明显增强(p＜0.05),棕榈酸组、叠氮钠组PCNA蛋白表达均明显减弱,与ox-LDL组比较差异有显著性(p＜0.01)。结论动脉中膜平滑肌细胞表型由收缩型变为合成型在粥样硬化的形成中起着重要的作用。1、在本实验中平滑肌细胞表型转变过程中,NRF-1的表达也在逐渐升高,与mtTFA成明显正相关。二者的表达增高和相互作用,共同调节线粒体功能,在平滑肌细胞表型改变中起着重要作用。2、随着平滑肌表型的改变,合成型平滑肌细胞不断增加,PGC-1也随着增加,并且与NRF-1成正相关,针对PGC-1的干预同样下调了NRF-1,说明二者存在内在的联系。在动脉粥样硬化过程中可能是PGC-1启动了NRF-1,导致NRF-1增加,进而启动了mtTFA从而导致mtDNA转录、复制加强,平滑肌细胞线粒体增加。3、由此推测平滑肌细胞表型改变过程中,PGC-NRF-mtTFA途径存在并起着重要作用。更多还原

【Abstract】 Atherosclerosis plays a important role in coronary artery disease and cerebrovascular disease,though its mechanism is not known exactly up to now. Historically,vascular smooth muscle cells（VSMC） were thought that local factors caused the normally contractile cells of the vessel media to modulate to a synthetic state,and these cells migrated and proliferated in the vessel intima,ultimately to generate an atheromatous plaque.But it is not known that what factors are involved in VSMC phenotypes convert.synthetic state of VSMC contains a large number of cellular organelles such as ribosomes,rough endoplasmic reticulum,Golgi complexes,and mitochondria in its cytoplasm.In particular,an enhancement of cell growth and division is accompanied by an increase in mitochondria,because under such circumstances cells,need to generate high energy through oxidative phosphorylation.Thus,mammalian mitochondria must enhance the transcription and replication of mitochondrial DNA （mtDNA） to respond to the increased cellular ATP demands and mitogenesis.mitochondrial transcription factor A（mtTFA） is critical to transcription and replication of mitochondrial DNA（mtDNA）,and is regulated by nuclear gene,such as nuclear respiratory factor-1 and peroxisome proliferator-activated receptor gamma coactivator 1（PGC-1）,which play an important role in binding to or coactivating with a number of transcription factors involved in mitochondrial biogenesis.To investigate the relationship between mitochondria function and VSMC phenotype, PGC-1,NRF and mtTFA of VSMC,in vivo and in vitro,were evaluated in this article.Methods1.Anminal motile study of vascular smooth muscle cells phenotype alternation and adjust of mitochondria function Model groupsAS model:38Japan white rabbit were subject to two group randomly:control group（n=8） fed with common diet,model group（n=30）was randomly divided into three group（n=10） fed with common diet and 2%Cholesterol 8%、pork fat、4% eggyolk,we collect blood,aorta respective,when before experiment,4 week,8 week,12 week.（1） Blood lipid assay:Enzymatic colorimetic test was principle.Serum lipid concentrations were detected（2） Reverse transcription-PCR technique:Total RNA was isolated using Trizol reagent,following the manufacture instructions,synthesis of single stranded cDNA according to "First strand cDNA synthesis Kit".Then Polymerase chain was react GAPDH was used as an internal control.The primers were synthesized by Takara Technologies,PCR products were phoresed in 2%agarose gels in TBE buffer stained in 0.5%μg/ml ethidium bromide and photographed.（3） Western blotting:tissue lysates were prepared in lysis buffer.Whole lysates were collected and then mixed with sample buffer,each were subjected to SDS -polyacrylamide gel electrophoresis,and the proteins were then transferred to a polyvinylidene difluoride membrane using a Semi-Dry Transblot,and then it was blocked by incubation with skim milk for 2h at room temperature.The blockde membrane was subsequently incubated with first antibody for 12h at 4℃after washing with TTBs,the membrane was incubated for 2h at room temperature with alkaline phosphatase-conjugated secondary antibody IgC,bands of protein on the membrane were visualized with NBT/BCIP kit. 2.The relationship between vascular smooth muscle cells proliferation and mitochondria function（1） cell culture human vascular smooth muscle cells were incubated with ox-LDL、palmitate、NAN₃ for 24h.（2） Activity of cells:The growing state of cultured endothelial cells were examined by MTT.（3） Immunocytochemistry examination:All cells were fixed with pure acetone. SP method was used to detect the expression of PNCA.（4） Western blotting:Cell lysates were prepared in lysis buffer.The experimental process is similar to 1.3（5） Mitochondria extraction:performanced according to the instruction of kit（6） cytochrome c oxidase activity,performanced according to the instruction of kit3.statical AnalysisAll sata were expressed as mean±standard deviation,and SPSS11.5 software was employed to analyzed the data.Statistical evaluation was performed using one- way ANOVA and correlation analysis.P＜0.05 was considered significant.Result1.The detection of the marker of vascular smooth muscle cells phenotype and regulative factors of mitochondria function.（1） Animal conditionsIn the experiment,there was no difference in weight between experimental groups （0.85±0.10）and the control（1.0±0.22）（P＞0.05）.（2） The results of blood lipid assayThe blood lipid had an increasing tendency with the time of feeding high-fat food going.The levels of TC and LDL increased significantly（p＜0.05）.（3） The results of RT-PCR for detecting mRNA of PGC-1、NRF-1、mtTFA、SMembThere was a significant difference among the level of PGC-1、NRF-1 and mtTFA after 4W、8W and 12W（p＜0.05）.they were upregulated with long time of high fat feeding.The expression of mtTFA had a consistant increasing with that of NRF-1 （p＜0.05）.As the maker of synthetic cells,A high level of SMemb was found after 8Ws which presented increasing synthetic cells（p＜0.05）（4） the result of western blot test for detection the protein of PGC-1、NRF-1、mtTFA、SMembSignificant difference were seen among the protein level of PGC-1 after 4W、8W and 12W（p＜0.05）,the same result can be found in NRF-1 and mtTFA（p＜0.05）.they increased as time of high fat feeding gone.There had a consistant increasing of protein of PGC-1,NRF-1 and mtTFA.With synthetic cells increased,A high level of SMemb was found（p＜0.05）2.The result of interfering proliferation of VSMC and the change of mitochondria function.（1） The protein level of PGC-1,NRF and mtTFA under various interfere factors.when ox-LDL existed,protein level of PGC-1,NRF-1 and mtTFA increased （p＜0.05）,but palmitate was given,protein level of them decreased（p＜0.05）.In another group,NAN₃ was given,the increasing proteins of PGC-1,NRF-1 and mtTFA could be found（p＜0.05）.（2） The result of cytochrome c oxidase activity test Under the condition of ox-LDL given,the cytochrome c oxidase activity has enhanced （p＜0.05）,but in palmitate group and NAN₃ group,the activity of cytochrome c oxidase were supressed（p＜0.05）.（3） The result of proliferation activity of VSMCOx-LDL can promote the activity of proliferation（p＜0.05）,while palmitate and NAN₃ has a reverse result（p＜0.05）.（4） The result of detection for PCNACompared with the control,PCNA protein revealed high level in ox-LDL group （p＜0.05）,but low level in palmitate and NAN₃ grou（p＜0.01）ConclusionGenerally,the change of VSMC Phenotype from contractile cells to synthetic cells contributes to atherosclerotic process.1.In our study,the expression of NRF-1 was up-regulated with synthetic cells increasing,the same finding was seen in expression of mtTFA.This suggested that there were definite relationship between NRF-1 and mtTFA,and that coactivation of them controlled the function of mitochondria and transform of VSMC phenotype.2.Positive correlation between PGC-1 and NRF-1 was found in VSMC proliferation.Furthermore,interfering expression of PGC-1 might down-regulate the expression of NRF-1.so it can be supposed that in atherosclerotic process,some factors switch on the increasing expression of PGC-1,which up-regulate some receptors, especially NRF-1 in nucleus,then activate mtTFA.3.So the pathway of PGC-NRF-mtTFA may play critical roles in VSMC proliferation and transform of phenotype that contribute to atherosclerotic.更多还原