【作者】 李清贤；

【导师】 廖玉华；

【作者基本信息】 华中科技大学 ， 内科学， 2008， 博士

【摘要】 第一部分斑块纤维帽厚度与血浆炎症标志物对冠心病患者不稳定斑块的预测阈值研究背景急性冠脉综合症的发病与动脉粥样硬化斑块的不稳定性密切相关,而斑块纤维帽厚度是决定斑块稳定性的关键因素之一,通常所指的易损斑块即为纤维帽厚度小于65μm;血管内光学相干断层成像技术为我们提供了高分辨率（¹0μm）,获得组织原位横截面图像的检测手段,光学相干断层成像技术的出现是我们检测不稳定斑块纤维帽厚度成为可能。临床心血管事件与某些炎症标记物水平同样密不可分;大量研究证实,外周血中炎症标志物水平具有预测心血管事件的价值;然而,斑块纤维帽厚度与血浆炎症标志物对冠心病患者易损斑块的预测阈值大小及其相关性尚未见报道。目的评价斑块纤维帽厚度与血浆炎症因子的相关性,初步探讨两者预测冠心病患者不稳定斑块的阈值。方法及结果将急性心肌梗死、不稳定性心绞痛、稳定性心绞痛及胸痛综合症患者纳入研究对象,进行对照研究。冠状动脉粥样硬化斑块纤维帽厚度采用血管内光学相干断层成像技术进行检测;通过酶联免疫吸附试验检测入选者血浆中超敏C反应蛋白、白介素-18及肿瘤坏死因子α血浆表达水平。随着冠脉事件风险增加,冠脉纤维帽厚度变薄,同样,患者血浆超敏C反应蛋白、白介素-18及肿瘤坏死因子α水平亦显著增加。冠状动脉纤维帽厚度与血浆炎症因子水平显著相关;当冠状动脉粥样硬化斑块纤维帽厚度小于65μm时,上述血浆炎症因子表达水平分别超出下述阈值水平:hs-CRP 0.75 mg/L,IL-18 75μg/L and TNF-α35μg/L,两者之间显著相关。结论由上可知,hs-CRP 0.75 mg/L,IL-18 75μg/L and TNF-α35μg/L可作为有效地甄别阈值用来预测动脉粥样硬化斑块的稳定性;为今后临床实践提供了一种简单有效地甄别不稳定斑块的无创性诊疗工具。第二部分冠心病患者炎症因子表达水平变化的相关性研究背景许多研究发现炎症反应不但在动脉粥样硬化的发生发展过程中起到了十分重要的作用,而且炎症反应在动脉粥样硬化斑块的不稳定过程中扮演了非常关键的角色,炎症同时也是动脉血栓形成各阶段的特征,是斑块形成和急性破裂而导致动脉闭塞和梗死的关键病理生理学机制,这些发现使得炎症因子与冠心病关系开始日益受到人们的重视。因此,明确冠心病患者血浆炎症因子水平及冠心病与炎症因子的相关性可能有助于危险冠心病患者的早期甄别。目的探讨冠心病患者不同临床阶段,白细胞介素-10（IL-10）,白细胞介素-17（IL-17）,白细胞介素-18（IL-18）、超敏C反应蛋白（hs-CRP）及肿瘤坏死因子-α（TNF-α）在血浆中的表达水平的变化,并初步明确两者的相关性。方法将急性心肌梗死、不稳定性心绞痛、稳定性心绞痛及胸痛综合症患者纳入研究对象,进行对照研究。采用酶联免疫双抗体夹心法（ELISA）对160例冠心病人(急性心梗50例,不稳定心绞痛68例,稳定性心绞痛42例和40例胸痛综合症患者血浆中IL-10、IL-17、IL-18、hs-CRP及TNF-α的水平进行检测。结果随着冠心病临床事件风险增加,冠心病患者血浆IL-17、IL-18、hs-CRP及TNF-α的水平均显著增加,IL-10血浆水平在冠心病患者均表达增加,与胸痛综合症组相比具有显著统计学差异,但并未表现出随临床事件风险增加而表达上升的趋势,在稳定型心绞痛组其表达量反高于不稳定心绞痛组,提示在稳定斑块向不稳定斑块进展的时候,可能有其他机制抑制IL-10的表达,但其具体机制尚待进一步明确。结论IL-10、IL-17、IL-18、hs-CRP及TNF-α等炎症因子可能共同参与了动脉粥样硬化发生、发展和演变的过程,其血浆水平可作为判断冠心病患者病情,筛选高危患者的指标之一。第三部分C-myc及PCNA对动脉粥样硬化斑块中血管平滑肌细胞增殖的影响背景动脉粥样硬化由脂质条纹进展至纤维酯性病变的过程中,内膜中的平滑肌细胞分裂,中膜的部分平滑肌细胞迁移至内膜;同时,平滑肌细胞分泌大量细胞外基质,促进细胞外基质在进展期动脉粥样硬化斑块处积聚。另外,平滑肌细胞的凋亡也参与了动脉粥样斑块复杂的发展过程。斑块纤维帽变薄即是平滑肌细胞增殖与凋亡失衡的结果。如何抑制血管平滑肌细胞增殖对延缓动脉粥样硬化进展意义重大。川芎是常用治疗动脉粥样硬化中药复方的主要成分,研究认为,川芎主要活性成分——川芎嗪可能具有抑制平滑肌细胞增殖的作用,但具体机制不明。C-myc原癌基因的表达产物c-myc蛋白作为一个“获能因子”可促进与VSMC增殖相关基因的开放,产生大量生长因子样物质,使VSMC进入增殖状态。细胞增殖核抗原（proliferating cell nuclearantigen,PCNA）是DNA复制、细胞周期及分裂过程中信号传导的关键一环,当VSMC细胞增殖旺盛时,其基因表达迅速增加。因此,川芎嗪抑制平滑肌细胞增殖的机制及C-myc及PCNA在平滑肌细胞增殖过程中的作用亟待阐明。目的观察c-myc和PCNA的反义寡核苷酸对原代培养VSMC增殖的抑制作用。同时对不同浓度川芎嗪抑制VSMC增殖的作用进行了研究,阐明其分子生物学作用基础。方法选择4-8代培养的鼠胸主动脉血管平滑肌细胞作为研究对象:分对照组、PCNA ODNs组和c-myc ODNs组进行对照研究,ODNs工作浓度均为1:50。选择细胞计数、MTT法、³H-TdR掺入法探讨ODNs对VSMC增殖的抑制作用;并以免疫组织化学法检测PCNA和c-myc ODNs转入VSMC及抑制相应PCNA和c-myc基因表达的情况。在观察川芎嗪作用中:分对照组（Ⅰ组）、川芎嗪50μg/ml（Ⅱ组）、100μg/ml（Ⅲ组）、200μg/ml（Ⅳ组）;采用细胞计数、VSMC对³H-TdR的摄取量和MTT法测OD₅₇₀值三种方法,探讨川芎嗪对VSMC生长的影响,通过检测PCNA（细胞增殖核抗原）和C-myc二种基因的表达来探讨川芎嗪抑制VSMC增殖的作用机理。结果细胞计数法、MTT法和³H-TdR摄取量检测法均显示PCNA和c-myc ODNs对VSMC的增殖有抑制作用,与对照组相比较均有显著性差异（p＜0.05）;免疫组织化学法显示PCNA和c-myc ODNs成功地转入了VSMC中并明显抑制相应基因的表达,与对照组相比较差异显著（P＜0.05）;细胞计数、³H-TdR摄取量检测及MTT法均显示川芎嗪对VSMC的增殖有抑制作用,且呈剂量依赖性,200μg/ml川芎嗪作用72小时对VSMC的增殖抑制作用最强,组间相比较均具有显著意义（P＜0.05）;川芎嗪抑制VSMC增殖过程中PCNA、C-myc的表达明显降低,且与川芎嗪的作用浓度相关,组间相比较均具有显著性差异（P＜0.05）。结论PCNA和c-myc基因在VSMC的增殖过程中起重要作用,PCNA和c-mycODNs导入VSMC后可成功抑制相应基因的表达,从而抑制VSMC的增殖;川芎嗪对鼠原代培养的VSMC的增殖确有明显抑制作用,并呈剂量依赖性;川芎嗪是通过影响与VSMC增殖有密切关系的基因PCNA、C-myc的表达发挥该作用;为深入挖掘祖国医学宝库治疗AS提供了理论依据,也为临床应用提供了重要依据更多还原

【Abstract】 PartⅠStudy the threshold of the thickness of fibroma and the level of serum inflammation markers in CAD patients with unstable plaquesBackground There are robust correlations between acute coronary syndrome and the stability of atherosclerotic plaque.However,the thickness of cap fibroatheroma is the critical factor affects the stability of atherosclerotic plaque.A vulnerable plaque was defined as the thin cap fibroatheroma(TCFA,＜65μm).Intravascular optical coherence tomography(OCT) provides high-resolution(~10μm),cross-sectional images of tissue in situ.The resolution of OCT is appropriate for measuring the cap thickness of a vulnerable plaque.There are robust correlations between clinical cardiovascular events and the leverls of serum inflammatory markers.Data from large-scale population-based studies have demonstrated that increased circulating levels of numerous markers of inflammation predict future cardiovascular events.The cut-off value and the correlation of the thickness of cap fibroatheroma and the levels of inflammatory markers are still need to be clarified.Aims To evaluate the correlation between fibrous cap thickness and levels of plasma inflammatory factors,and search those thresholds for predicting vulnerable plaques in patients with coronary artery diseases.Methods and results A comparative study was performed in patients with acute myocardial infarct(AMI),unstable angina pectoris(UAP),stable angina pectoris(SAP) and chest pain syndrome(CPS).Intravascular optical coherence tomography was used to measure the fibrous cap thickness of coronary artery atherosclerotic plaques.Enzyme linked immunosorbent assay was used to detect plasma levels of highly sensitive C-reacting proteins(hs-CRP),interleukin 18(IL-18) and tumor necrosis factor alpha(TNF-α).With increases of coronary artery event risks,plaque fibrous cap thickness decreased and plasma hs-CRP,IL-18 and TNF-αlevels increased considerably(P＜0.05).There were significant correlations between fibrous cap thickness and plasma levels of inflammatory factors (P＜0.05).Cap thickness＜65βm correlated with threshold levels of plasma inflammatory factors as follows:hs-CRP 0.75 mg/L,IL-18 75μg/L and TNF-α35μg/L.Conclusion Results show that cut-off value of hs-CRP≥0.75mg/L,IL-18＜75μg/L and TNF-α≥35μg/L could be the threshold to predict instability of atherosclerotic plaques. It may provide an easy way to identify vulnerable patients in future clinical practice.PartⅡCorrelation between inflammatory factors and different state of coronary artery diseaseBackground Abundant data link hypercholesterolaemia to atherogenesis.However, only recently have we appreciated that inflammatory mechanisms couple dyslipidaemia to atheroma formation.Leukocyte recruitment and expression of pro-inflammatory cytokines characterize early atherogenesis,and malfunction of inflammatory mediators mutes atheroma formation.Moreover,inflammatory pathways promote thrombosis,a late and dreaded complication of atherosclerosis responsible for myocardial infarctions and most strokes.Dependend these data,the new appreciation of the correlation between inflammation and coronary artery disease has been paid close attention to.Therefore, unrevalling the levels of serum inflammatory factors and identifying the correlation between inflammatory factors and different state of coronary artery disease may eventually furnish the discrimination of dangerous CAD patients.Objective To detecte the diversity of serum levels of interleukin-10(IL-10), intedeukin-17(IL-17),interleukin-18(IL-18),C reactive protein(CRP) and TNF-αin the process of coronary artery disease(CAD).and to identify the correlation between inflammatory factors and different state of CAD.Methods The levels of plasma IL-10,IL-17,IL-18,hs-CRP and TNF-αin 160 patients(AMI50,UAP68,and SAP42) with CAD and 40 CPS were determined by enzyme linked immunoadsorbent assay(ELISA).Results The plasma levels of IL-17,IL-18,hs-CRP and TNF-αin patients with CAD wre increased significantly than that in CPS patients(P＜0.05).The concentration of IL-10 in patients with CAD was increased significantly than that in CPS patients(P＜0.05), however,the serum level of IL-10 in UAP patients was lower than that in SAP group,there may be some mechanism need to be unrevlled to clarify this phenomenon.Conclutiou The findings suggest that IL-10,IL-17,IL-18,hs-CRP and TNF-αmay co-participate the pathogenesis and progression of CAD.The serum levels of IL-10,IL-17, IL-18,hs-CRP and TNF-αwill contribute to the assement of the pathogenetic condition with CAD.The levels of these inflammatory markers could be one of the references in discriminating the dangerous patients.PartⅢEffects of the c-myc and PCNA on the multiplication of VSMCBackground Smooth muscle cells in the intima divide,other smooth muscle cells that migrate into the intima from the media.Smooth muscle cells can then divide and elaborate extracellular matrix,promoting extracellular matrix accumulation in the growing atherosclerotic plaque.In this manner,the fatty streak can evolve into a fibrofatty lesion.In addition to smooth muscle cell replication,death of these cells can also participate in the complication of the atherosclerotic plaque.Thin cap fibroatheroma in the growing atherosclerotic plaque probably results from a tug-of-war between cell replication and cell death.Thus,inhibiting the multiplication of VSMC may be important to retard the development of atherosclerosis.Chuanxiong is the major component of general complex prescription of traditional Chinese herbal in treating atherosclerosis.Some data indicated that chuanxiongzine,the major active ingredient,may inhibit the proliferation of VSMC, however,the mechanism of this effectiveness is still unkown.C-myc protein as the expression product of c-myc proto-oncogene has been shown to be a "compentence factor", which could promote the related gene patency,brings about a great deal of GH-like substance,precipitate VSMC into vegetative state.PCNA is the critical link of signal conduction during the process of DNA duplication,cell cycle and fission.The VSMC gene expression would increase quickly,when the cell proliferate vigorously.Therefore,the mechanism of chuanxiongzine in inhibiting the proliferation of VSMC and the role of c-myc and PCNA in this process need to be unrevelled.Objective To study the depressive effect of the antisense oligonuceotides(ASODN) of c-myc and proliferating cell nuclear antigen(PCNA) on the proliferation of VSMC. Detecting the inhibitory effect of chuanxiongzine on vascular smooth muscle cell(VSMC) proliferation and exploring its molecular biology basis.Methods Taking the VSMC obtained from rat aorta thoracalis cultured 4～8 generation as research object.The objects were divided into three groups to carry out control study: control group;PCNA ASODN group and c-myc ASODN group.The ASODNs’ working concentration all were 1:50.The depressive effect of ASODN on VSMC proliferation was investigated by cell counting,MTT and ~3H-TdR incorporation assay;PCNA and c-myc expression were detected by immunohistochemical method after transferring PCNA and c-myc ASODN into VSMC.Taking the VSMC obtained from rat aorta thoracalis cultured 4～8 generation as research object.The objects were divided into four groups to carry out control study:(Ⅰ) control group,(Ⅱ) chuanxiongzine(50μg/ml) group,(Ⅲ) chuanxiongzine(100μg/ml) group and(Ⅳ) chuanxiongzine(200μg/ml) group.The inhibitory effect of chuanxiongzine on VSMC proliferation was investigated by cell counting,MTT and ~3H-TdR incorporation assay.In order to illuminate the molecular biology mechanism of chuanxiongzine,the expression of proliferating cell nuclear antigen (PCNA) and c-myc were detected.Results PCNA and c-myc ASODN could inhibit the proliferation of VSMC significantly,compared with control group(P＜0.05);Transferring PCNA and c-myc ASODN into VSMC obtained successfully;the corresponding gene was inhibited obviously; compared with control group(P＜0.05);Chuanxiongzine could inhibit the proliferation of VSMC significantly in a dose- and time-dependent manner,compared with control group (P＜0.05).The expression of PCNA and c-myc were inhibited obviously by chuanxiongzine and was correlated with the concentration of chuanxiongzine(P＜0.05).Conclusion PCNA and c-myc might play a considerable role in the VSMC proliferation process.The corresponding gene could be depressed successfully after transferring PCNA and c-myc ASODN into VSMC,and then the proliferation of VSMC was retarded;Chuanxiongzine may play a considerable role in the VSMC proliferation process.The inhibitory effect of chuanxiongzine in a dose- and time-dependent manner could be realized via down regulating the expression of PCNA and c-myc.In this study, great theoretical fundament about Chinese medicine,which is used to treat atherosclerosis (AS),has been obtained.更多还原