摘要
研究背景和目的：
冠心病(CHD)在全球范围内已成为威胁人类生命的首要疾病,而急性心肌梗死(AMI)是冠心病不同发展阶段中最为严重的一种,其主要的病理基础是动脉粥样硬化(AS)的形成。研究表明：AMI的发生、发展是极其复杂的过程,其中涉及到内皮细胞损伤、炎症细胞浸润、斑块崩裂和血栓形成等。
目前,经皮冠状动脉成形术(percu-taneous coronary intervention, PCI)已成为治疗CHD最有效手段之一。虽然血管再通后,辅以一些溶栓药物防止血栓形成,预防梗塞取得了一定的效果,但由于手术对血管内皮的损伤和对炎症反应的激发,术后仍有部分患者出现了不同程度的血管内皮损伤以及血栓的形成,影响PTCA的后期效果。血管内皮细胞的损伤是PCI术后首发及核心的损伤,是再狭窄的重要成因。尽早使受损血管重新内皮化,恢复内皮功能维持血管内环境稳态,进而抑制炎症反应与血管平滑肌的异常增生,是防止PCI术后再狭窄的有效手段。血管内皮损伤及功能障碍是许多疾病的始动环节,贯穿于疾病的全过程。其引起血管内皮功能障碍,与高血压、动脉粥样硬化、心力衰竭、糖尿病等心脑血管疾病和代谢疾病的发生发展密切相关。目前关于EPCs和心脑血管疾病的相关性研究比较多见,但是就EPCs和冠心病特别是急性心肌梗死的相关的详细研究并不多见。
有研究发现,炎症反应是AS斑块的重要特征之一,它与巨噬细胞、内皮细胞、平滑肌细胞(SMC)的激活和增生、细胞因子和生长因子的产生、补体的释放和激活以及其它炎症介质的分泌均有一定的关联。炎症反应在AS的发生发展中具有重要的意义。AS的形成对损伤的血管内膜而言,既是免疫反应的过程,又是炎症反应的过程。在众多有关炎症因子的研究中,白介素-18是最近研究的热点。白介素18(IL-18)是一种主要介导细胞免疫的多效细胞因子,其结构类似IL-1受体,功能类似IL-12,具有多种生物学活性。它主要诱导产生干扰素-γ(IFN-γ)、协同IL-12发挥作用,同时又能增强自然杀伤细胞、肿瘤特异性杀伤(T淋巴)细胞的溶解活性,在AMI局部炎症反应中起着十分重要的作用。研究发现,心肌对IL-18具有高度敏感性,IL-18可减弱心肌功能、诱导心肌损伤,但IL-18在AMI中的变化规律目前尚未完全明了。AMI时发生的化学性炎症反应,其详细变化的机制目前国内外研究不多,而IL-18在AMI病人中的动态变化及与病情的相关性研究报道罕见。
最近研究发现内皮祖细胞(EPCs)具有特异性归巢于缺血缺氧,血管损伤组织,促进新血管形成的特性。EPCs移植促进血管新生的机制可能有两方面：一是迁移到局部分化成成熟的血管内皮细胞；二是通过自分泌和旁分泌机制分泌促血管生成的物质,如VEGF、HGF(肝细胞生长因子)、IGF-1(胰岛素样生长因子1)、G-CSF等；EPCs还可横向分化为心肌细胞,起到细胞补充作用。当机体本身EPCs因病理性消耗过多而不足时,外来EPCs移植治疗血管内皮损伤性疾病成为新的研究热点。近年来,EPCs移植在心血管疾病中的应用主要为缺血下肢、缺血心肌的促血管新生治疗研究,以及损伤血管、人工血管和带膜支架的内皮化研究等。实验证明,EPCs移植可显著改善缺血部位的血流量,增加毛细血管密度,促进功能的恢复并在损伤血管、人工血管和带膜支架的内皮化防止再狭窄中发挥积极的作用。
AMI时有ST段持续抬高的称为STEMI,冠状内血栓形成导致相关动脉完全闭塞是STEMI的最常见原因,约占所有病因的80-90%以上。临床上,静脉注射纤溶酶被应用于STEMI的治疗,取得了很好的疗效。近年来的研究发现,蛇毒纤溶酶具有生长因子活性,对细胞具有营养作用,将纤溶酶与EPCs合并注射于兔心肌梗死模型中取得了较好的疗效。但蛇毒溶栓治疗怎样促进损伤血管内皮恢复,如何抑制炎症因子对血管内皮的进一步损伤,其机制目前还不是很清楚,特别是蛇毒溶栓辅以自体内皮祖细胞移植的研究比较少见。
本研究着重探讨蛇毒溶栓治疗辅以自体内皮祖细胞移植对心血管疾病患者血流的再灌注和内皮损伤的恢复的影响,以及对炎症反应中炎症因子的控制。首先,分离冠心病患者外周血中内皮祖细胞,进行培养鉴定,观察其数量及功能的变化,同时检测血清中IL-18的含量；其次,监测内皮祖细胞数量及功能的影响和炎症因子—IL-18浓度的变化,以探讨蛇毒溶栓治疗的治疗效果；最后,利用动物实验,建立血管损伤模型,在蛇毒溶栓治疗的基础上,同时进行自体内皮祖细胞移植,进一步观察内皮损伤后其恢复程度和炎症反应中IL-18的变化,以期为细胞移植在临床心血管病的治疗提供新的思路和理论依据。
研究方法
1、实验资料及分组
(1)：临床病例资料：2006年10月—2007年10月年南方医科大学珠江医院住院冠心病患者200例,分为稳定心绞痛组(SA)45例,不稳定性心绞痛组(UA)40例,急性心肌梗死组(AMI)115例(单支病变组45例、双支病变组38例、多支病变组32例),其中选取100例AMI患者做蛇毒溶栓治疗,分为治疗前组、治疗后组,同时以20例健康体检人群作为对照组。
(2)：动物实验及分组：28只日本兔平均分成四组：A-(培养液对照组)：仅以培养液处理；,B(细胞移植组)：以自体内皮祖细胞处理；C(蛇毒纤溶组)：以蛇毒纤溶酶处理；D(蛇毒纤溶和细胞移植组)：以蛇毒纤溶酶和自体内皮祖细胞同时处理。
2、标本采集
(1)血标本采集：所有冠心病患者入院后从外周采血20ml,蛇毒溶栓治疗前后分别抽血,实验动物处理后28天抽血,对照组清晨空腹抽血,所有血标本肝素钠抗凝,4小时内处理样本。
(2)病理标本收集：实验动物处死后做病理切片。
3、实验方法
(1)分离所有冠心病病例组以及蛇毒溶栓治疗前、后组外周血EPCs,进行培养、鉴定、计数和功能检测。采用密度梯度离心法分离获得单个核细胞,在RMP-1640专用培养基中进行培养。进行DiL-acLDL和FITC-UEA-I双染色,荧光显微镜下观察。双染色阳性的细胞被认为是正在分化的EPCs。在激光共聚焦显微镜下对每孔细胞进行计数(计数15个随机选择的200倍视野的EPC)。专用纤维连接蛋白培养板计数贴壁细胞,检测其粘附能力；改良Boyden小室计数其迁移能力。
(2)IL-18浓度检测：分离少量血浆采用双抗夹心-酶联免疫吸附(ABC-ELISA)检测IL-18浓度。
(3)动物处理：所有动物在麻醉状态下行冠状动脉左前降支接扎手术,1小时候再灌注,在接扎完和再灌注后分别进行心电图检查,并分别于A、B、C、D四组动物主动脉根部注入DMEM培养液、含内皮祖细胞的DMEM混合液、含有蛇毒纤溶酶的DMEM混合液、含有内皮祖细胞及蛇毒纤溶酶的DMEM混合液。所有动物移植手术4周后,先行心脏超声检查,然后抽取外周血,进行内皮祖细胞计数和IL-18浓度检测,最后处死所有动物,取其心脏,做病理切片,HE染色,做病理学检查,同时做Ⅷ因子免疫组化染色,任选10个视野计算染色阳性的毛细血管平均数作为新生血管数。
结果
1、细胞培养形态、数量变化及荧光双染色鉴定
通过密度梯度离心法,30mL外周血中可获得约3×107个MNC。培养5-6天,可见贴壁细胞汇聚形成大小不等的细胞簇。培养第7天,细胞形态不一,由梭形、圆形和不规则形细胞组成；培养7-9天,可见少量梭形的晚期EPC三五成群出现在早期EPC之间。并在培养的第9-12天,迅速长成集落,细胞呈铺路石样排列。
光镜下观察细胞形态发现,对照组细胞较大较圆,出现梭形细胞并连接成条索状；而AMI组与对照组细胞比较有明显差别,细胞数目较对照组明显减少。分离获得的单个核细胞培养7d后形成了梭形的内皮样细胞。用acLDL-DiL和UEA-Ⅰ双染色后,AMI患者EPCs与对照组比较,双荧光染色阳性细胞明显减少。
2、冠心病不同阶段组EPCs数量及功能的比较
比较冠心病不同发病阶段EPCs的数量,发现稳定性心绞痛组、不稳定性心绞痛组和心肌梗死组均明显低于对照组(13.26±4.03,11.88±3.88,8.88±2.69,41.620±6.89),有极显著性差异(P<0.01)；EPCs的粘附(贴壁细胞)及增殖能力,不稳定性心绞痛组较稳定性心绞痛组(17.32±5.41,0.28±0.06；23.85±6.74,0.41±0.12)下降更明显,有统计学意义(P<0.05),而心肌梗死组与稳定心绞痛、不稳定心绞痛组(11.11±4.77,0.22±0.05；23.85±6.74,0.41±0.12；17.32±5.41,0.28±0.06)比较,其EPCs粘附及增值能力也明显下降,相差显著(P<0.05)。
3、AMI不同病变程度EPCs数量、集落形成单位及IL-18浓度比较
AMI患者不同病变组比较后发现：AMI不同病变程度(单支、双支、多支病变)EPCs数量、集落形成单位(48.5±7.3,15.60±4.79；36.5.9±5.6,12.14±7.31,18.9±3.9,8.0±4.8；)均显著低于对照组(57.1±2.1,31.1±1.9)(P<0.05)；而IL-18浓度却有逐步升高趋势(135.6±86.9 pg／ml,151.2±90.8 pg／ml,266.8±122.4 pg／ml),与对照组(20.5±9.7 pg／ml)比较,相差显著(P<0.05)；而且多支病变组与单支、双支病变组比较,EPCs数量、集落形成单位逐步下降,IL-18浓度不断升高,也具有统计学意义(P<0.05)。
4、溶栓治疗前、后EPCs数量及IL-18水平比较
比较各组EPCs数量,发现AMI患者治疗前的EPCs数量与对照组比较,减少明显,差异具有统计学意义(P<0.01)；在治疗3d后,细胞数量明显增加,较治疗前相比差异有统计学意义(P<0.05)；与对照组比较数量差异不明显(P>0.05)；治疗7d细胞数量有所下降,但仍多于治疗前水平(P<0.05)。集落形成单位数(CFU)计数结果显示,AMI患者治疗前CFU数目少,而且细胞集落较小而稀疏,和对照组比较差异具有统计学意义(P<0.05)；治疗3d后CFU有所增加,但与治疗前相比差异无统计学意义(P>0.05),治疗后7d细胞CFU与治疗前相比无差异性,仍少于对照组(P<0.05)；治疗3天、七天后IL-18水平(148.900±86.55 pg／ml,77.5±46.8 pg／ml)虽然均高于对照组(20.5±9.7pg／ml),但与治疗前(221.8±100.4pg／ml)比较,显著下降(P<0.05)。
5、心功能检测
结扎兔冠状动脉左前降支后,心电图表现ST段抬高；其中手术中A组死亡一只。再灌注后,ST段缓慢下降,其中5例出现心动过速,2例出现过缓,D组出现室颤死亡1只。比较各组实验动物心脏超声结果,发现B、C、D组左心室射血分数(64.9±4.51,66.1±5.46,68.9±5.07)、左心室壁运动幅度(0.26±0.28,0.26±0.18,0.28±0.45)均高于A组(59.8±2.96,0.22±0.39),有统计学意义(P<0.05)；B、D组左室短轴缩短率(38.1±2.95,39.6±2.31)也高于A组(34.9±1.61),相差显著(P<0.05),而C组(35.8±2.64)与A组比较相差不大(P=0.16)。
6、病理学检查
所有实验动物处死后心肌病理切片结果显发现,C、A心肌梗死区心室壁厚度(1.99±0.47 mm,2.03±0.72mm)与正常心肌(1.95±0.89mm)相比没有明显变化,B、D组则心肌表面颜色变化不大,心室壁的厚度(2.29±0.29mm,2.34±0.17mm)接近正常的心肌的心室壁。D组(2.34±0.17mm)与A组(2.03±0.72mm)比较,差异有统计学意义(P<0.05)。其余各组比较,相差不明显。各组HE染色没有见到梗死区及周围有多核肌细胞生长,B、C、D组心肌状态明显好于对照组；A组HE染色可见坏死的心肌细胞排列紊乱,胶原纤维形成,中间可见残存的心肌,B、C、D组有部分残存的细胞多于A组。未见多核细胞排列与心肌走形一致。Ⅷ因子染色后,比较毛细血管密度发现,D组(10.3±3.5)均显著高于B、C、A三组(7.2±2.5,6.9±2.2,3.4±1.7),B、C两组相差不大,但与A组比较也有明显差别。
7、实验动物各组EPCs计数及IL-18浓度比较
比较各处理组EPCs计数及集落数量,发现B、C、D三组EPCs数量(39.9±3.9,26.9±5.5,38.9±5.6；24.3±4.9,57.1±12.6,33.8±8.7)与A组(24.0±1.6,13.8±3.3)比较,均有较明显增加,差异具有统计学意义(P<0.05),而且D组(57.1±12.6)与B、C组(39.9±3.9,38.9±5.6)比较,EPCs数量也增加明显,具有显著性差别；IL-18浓度检测结果显示,B、C组(122.5±36.9 pg／ml,119.8±36.5 pg／ml)与A组(142.1±88.5 pg／ml)比较,IL-18浓度略有下降,相差不明显；而D组(40.2±22.1 pg／ml)与A组比较,IL-18浓度下降明显,相差显著(P<0.05)。
结论
1、冠心病不同发病阶段(稳定心绞痛—不稳定心绞痛—急性心肌梗死),急性心肌梗死不同病变程度(单支病变—双支病变—多支病变),血管损伤不断加重,内皮细胞不断下降,外周血EPCs逐步消耗,炎症反应进一步增强,外周血IL-18浓度逐步升高。
2、蛇毒溶栓治疗可提高AMI患者外周血循环EPCs数量,并抑制炎症因子IL-18表达,从而促进内皮损伤的恢复,并降低炎症反应对血管内皮的进一步损伤。
3、蛇毒溶栓治疗辅以自体EPCs移植,可显著提高内皮祖细胞的数量及集落,降低IL-18浓度,从而有效促进内皮损伤的恢复,抑制炎症反应对血管内皮的损伤。
4、蛇毒溶栓治疗辅以自体EPCs移植,可改善坏死的心脏功能,对梗死后心肌有一定的修复作用,效果明显好于单独EPCs移植或蛇毒溶栓治疗。
主要创新点：
1、首次联合监测冠心病患者外周血中内皮祖细胞和白介素-18的表达,由于其与冠心病有很强的相关性,因此其在一定程度上可以协助临床诊断冠心病的分型及分级,同时对其治疗和预后判断也有比较好的临床意义。
2、首次通过动物实验表明：在药物溶栓治疗的基础上,辅以自体内皮祖细胞移植,可以明显改善心功能、促进损伤内皮恢复、有效抑制炎症反应,为临床对冠心病特别是急性心肌梗死提供了一种新的可能的治疗手段。
Background&Objective:
Coronary heart disease (CHD) has become the primary threat to human life diseases, and acute myocardial infarction (AMI) are the most serious one in different stages of development of coronary heart disease, its main pathology foundation is atherosclerosis (AS) formation. Study shows that:occurrence and development of AMI are extremely complex process, involving endothelial cell injury, inflammatory cell infiltration, plaque and thrombosis, crack, etc.
At present, percutaneous transluminal coronary angioplasty (percu-taneous coronary intervention, PCI) has become one of the most effective the treatment of CHD. Although it have made some effect to thrombolytic therapy with some drugs in prevention of rethrombosis and the prevention of reinfarction after the vascular recanalization, but because of the vascular endothelial damagment and stimulating inflammatory response on surgery, and there will be vascular endothelial damagment and thrombus formation in some postoperative patients, so the late impact of PTCA is seriously effected. The injury of vascular endothelial cell is the first and core injury in PCI and the important causes of restenosis. It is necessary of re-endothelialization of damaged blood vessels and restore endothelial function in the maintenance of vascular homeostasis within the environment, inhibiting inflammatory reaction and abnormal hyperplasia of vascular smooth muscle as soon as possible, that is an effective means to to prevent restenosis after PCI. Vascular endothelial injury and dysfunction in many diseases are initiated, through the whole process of disease. Its cause vascular endothelial dysfunction, and hypertension, atherosclerosis, heart failure, diabetes and other cardiovascular and cerebrovascular diseases and metabolic diseases are closely related to the occurrence and development. Recent studies have found that endothelial progenitor cells (EPCs) with specific homing in hypoxia-ischemia, vascular injury, and promote the characteristics of the formation of new blood vessels, so when the body itself, excessive consumption of EPCs due to insufficient, external EPCs transplantation in the treatment of vascular endothelial injury disease has become a new research focus.
Research has found that inflammatory response are important characteristics of AS plaques and have a certain degree of associated on macrophages, endothelial cells, smooth muscle cells (SMC) activation and proliferation, cytokine and growth factor production, complement activation and unperturbed, as well as the other emergence of inflammatory mediators.The inflammatory response is of great significance in the occurrence and development of AS. To intimal injury, the formation of AS is not only a process of immune response, but also a process of inflammatory response. Among the various studies about inflammatory factors, the IL-18 is a recent hotspot. IL-18 is a major cell-mediated immune pleiotropic cytokine, and its structure similar to IL-1 receptor, functions similar to IL-12, with a wide range of biological activity. It mainly induces to product interferon-γ(IFN-γ),and plays a synergistic role with cooperation of IL-12, and enhances lysis activity of the natural killer cells and tumor-specific anti-(T lymphoid) cells, and plays an important role at AMI and I/R inflammatory response. The study found that there is a high sensitivity to IL-18 in cardiomyocytes. IL-18 may diminish cardiac function and induce myocardial injury, but the changes of IL-18 is not yet fully understand at the patients of AMI. There is little study about the chemical inflammatory response and its detailed change at home and abroad when AMI occurs, and it is rarely reported to the condition of dynamic change and the relevance of studies of IL-18 in AMI patients.
Recent studies have found that endothelial progenitor cells (EPCs)have the characteristics with specific homing to ischemia and hypoxia, vascular damage tissue, and promoting the formation of new blood vessels.Transplanted EPCs promote angiogenesis mechanism may have two aspects:One is migrated to the location into mature vascular endothelial cells; Second, secretion of angiopoietin substances through autocrine and paracrine mechanism, such as VEGF, HGF (hepatocyte growth factor), IGF-1 (insulin-like growth factor 1), G-CSF, etc; EPCs can differentiate into myocardial cells horizontally. In recent years, the applications of EPCs transplantation in cardiovascular disease were mainly to lower extremity ischemia, ischemic myocardium and promote angiogenesis therapy research, as well as vascular injury, artificial blood vessels and stent endothelialization. Experiments show that EPCs transplantation can significantly improve blood flow in ischemic area, increased capillary density, and promote functional recovery and play an active role in damaged blood vessels, artificial blood vessels and endothelial stent to prevent restenosis.
The coronary thrombosis is the most common cause in AMI with ST egment (AMI) accounting for more than 80-90% of all cause of. Clinically, It has been proved effective by intravenous fibrinolytic therapy. In recent years, it shows that snake venom fibrinolytic enzyme has growth factor activity and nutrition effects on the cells. It was good to inject EPCs and fibrinolytic enzyme into the rabbit model of myocardial infarction. However, it was not yet clear that how to promote vascular endothelial recovery in thrombolytic therapy for snake venom and how to suppress the inflammatory cytokines on vascular endothelial further injury, and the research is relatively rare in particular venom thrombolysis complemented by autologous progenitor cell transplantation.
This study focused on the effection of reperfusion of blood flow in patients with cardiovascular disease and recovery of injuried endothelial, as well as the control of inflammatory cytokines in the inflammatory response by the therapy with venom thrombolytic and transplantation of endothelial progenitor cells derived from the vivo. First of all, the peripheral blood endothelial progenitor cells were separated in the patients with coronary artery disease, and cultured identified. We observe the changes in quantity and function of endothelial progenitor cells,measure the concentration of serum IL-18. Secondly, to explore the therapeutic effect of thrombolytic therapy in snake venom, the number and function of endothelial progenitor cells and concentration of IL-18 were checked. ..Finally we establish animal model of vascular injury, then transplante endothelial progenitor cells derived from the vivo on the basis of the venom thrombolytic therapy. and finally observe the recovery after endothelial injury and the concentration changes of serum IL-18 in inflammatory response, aim to provide new ideas and theoretical basis to cell transplantation in the clinical treatment of cardiovascular disease
Method
1.Experimental group
(1) Case Information:There were 200 cases of hospitalized patients with coronary artery disease at Zhujiang Hospital, Southern Medical University in 2006 October to 2007 October,45 cases with stable angina,40 cases of unstable angina,115 cases with acute myocardial infarction (45 cases of single lesions,38 cases of double vessel disease,32 cases of multivessel disease),100 cases of AMI with venom thrombolytic therapy were divided into before treatment group and after treatment group.,while 20 cases of healthy people as controls.
(2) Animal grouping:An average of 28 Japanese rabbits were divided into four groups:A-culture medium control group, B-cell transplantation group, C-venom plasminogen Group, D-venom plasminogen and cell transplantation group
2. Specimen collection:
(1) Blood:All patients hospitalized were blooded from 20ml with heparin anticoagulation, the same to control group in the morning, Blood was collected for the patients before and after thrombolytic therapy, and for the animals dealed after 28 days,dealing with samples 4 hours later.
(2) Pathology specimen collection:experimental animals were killed for pathological slices.
3. Experimental method
(1) The blood EPCs were separated from all coronary heart disease patient group and the venom thrombolytic therapy for culture, identification, counting and functional testing. By density gradient centrifugation and mononuclear cells were isolated, and cultured in the RMP-1640 By DiL-acLDL and FITC-UEA-I double staining, Double-stained cells are considered to be differentiating EPCs.in fluorescence microscope.The cells were counted in the confocal microscope (Count 15 randomly selected field of vision of the EPC) in 200 times. The adherent cells were counted in special fibronectin plate,and their migration cells were counted in Boyden chamber.
(2) The concentration of IL-18:Measured the concentration of IL-18, using double-antibody sandwich-enzyme-linked immunosorbent assay (ABC-ELISA)
(3) Left anterior descending coronary artery surgery was bar after anesthesia, reperfused an hour later, ECG were carried out in the next bar-end and after reperfusion, and separately injected DMEM culture medium, DMEM culture medium containing endothelial progenitor cells, DMEM culture medium containing snake venom plasminogen, DMEM culture medium containing endothelial progenitor cells and snake venom fibrinolytic enzyme into the artery root of A, B, C, D groups of animals.
All animals were first checked by heart ultrasound after transplant surgery 4 weeks,later, and then collected blood, for endothelial progenitor cell count and IL-18 concentration detection.Finally all the animals were sacrificed for biopsy.HE stain and factor VIII immunohistochemistry.10 optional vision calculated as the average number of capillaries stained for the number of new vessels.
Results
1. Changes of morphology and the number, identification of fluorescent double staining
Through density gradient centrifugation, about 3×107 MNC will be collected in 30mL peripheral blood. Cultured 5-6 days later, adherent cells can be seen together to form cell clusters of varying sizes. After cultured 7 days, cell morphology was different from the spindle, round and irregular-shaped.After cultured 7-9 days, a small number of spindle can be seen in small groups late EPC arise at an early stage between the EPC. And in the development of the first 9-12 days, colonies cells as cobblestone were rapidly grown up.
It revealed that cell morphology observed under light microscope appeared to be spindle cell and connected into a cord-like, larger and more round in the control group than the treatment group.The treatment group compared with the control group cells had significant differences; the number of cells of AMI group before treatment, was significantly reduction than that of the control group,
Mononuclear cells obtained after separated and cultured 7d formed a spindle-like cells of the endothelium. With acLDL-DiL and UEA-I double staining, cells were identificated through confocal microscope, EPCs were charcterized as adherent cells of double positive for acLDL-DiL and UEA-I staining. The number of cells of double-fluorescence staining positive decreased significantly in the patients with AMI of no treatment compared with the control group;
2. The number and function of EPCs in coronary heart disease at different stages
It showed that the numbers of EPCs in stable angina group, unstable angina and myocardial infarction group were significantly lower than that in the control group(P <0.01). The capacity of adhesion (adherent cells) and proliferative of EPCs in unstable angina group decreased significantly than that in stable angina group (P<0. 05); the capacity of adhesion (adherent cells) and proliferative of EPCs in myocardial infarction group also decreased significantly than that in stable angina and unstable angina group (P<0.05).
3. The number of EPCs,colony-forming units and concentration of IL-18 in different lesions of AMI before thrombolytic therapy
The number of EPCs,colony-forming units in different lesion group of AMI (single, double, multi-vessel disease) were significantly lower than those in the control group (P<0.05); and concentrations of IL-18 were gradually increasing trend, significantly higher than that in the control group (P<0.05); compared many-vessel lesion group with single vessel, double vessel lesion group, the number of EPCs,colony-forming units significantly decreased, IL-18 concentration significantly increased (P<0.05).
4. The number of EPCs and concentration of IL-18 before and after thrombolytic therapy
The number of EPCs reduced significantly in AMI group than that in the control group (P<0.01); it increased significantly in the treatment group of treatment 3d than that in no treament group(P<0.05); but compared with the control group there was no obvious difference (P> 0.05); the number of cells after treatment 7d decreased, but still more than that before treatment (P<0.05). The number of colony-forming units (CFU)was less and CFU was smaller and sparser in the AMI patients than those in the control group (P<0.05); The number of CFU increased after treatment 3d,but compared with no treatment there was no significant difference (P>0.05); after cells, there was no difference compared treatment 7d with no treatment, and the number of CFU was less than that in the control group (P<0.05). The concentration of IL-18 in the treatment of three days, seven days was higher than that in the control group(P<0.05), but decreased significantly compared to no treatment (P<0.05).
5. Heart function
Rabbit coronary artery ligation of left anterior descending coronary artery, the ECG demonstrated ST-segment elevation; one of A group died during surgery. After reperfusion, ST segment declined slowly, five cases appeared tachycardia, two cases appeared bradygastria, D group appeared ventricular fibrillation, one died.
Comparison of echocardiography in each group animals, left ventricular ejection fraction (64.9±4.51,66.1±5.46,68.9±5.07), Rate of left ventricular wall motion of B, C, D group (0.26±0.28,0.26±0.18,0.28±0.45)were significantly higher than group A(59.8±2.96,0.22±0.39) (P<0.05); Left ventricular fraction shortening in group B and D (38.1±2.95,39.6±2.31)were significantly higher than group A(34.9±1.61) (P<0.05),while those in group C(35.8±2.64) was near to group A(34.9±1.61) (P=0.16).
6.Pathological examination
When all experimental animals were killed for myocardial pathology, it showed that myocardial infarction zone in C, A group(1.99±0.47 mm,2.03±0.72mm) was no significant different compared to normal myocardium (1.95±0.89mm); surface color in B, D group myocardial only little change; thickness of B,D ventricular wall myocardium (2.29±0.29mm,2.34±0.17mm) was near to that of normal ventricular wall of the myocardium; thickness of D group (2.34±0.17mm) was more than that of A group (2.03±0.72mm) (P<0.05).For the remaining group, the difference was not obvious. FactorⅧstaining, compared capillary density was found, D group (10.3±3.5) were significantly higher than B, C, A three-group (7.2±2.5,6.9±2.2,3.4±1.7), B, C groups did not differ large, but compared with the A group are also significant differences.
7.The number of EPCs and concentration of each group animals
The number of EPCs and CFU in B, C, D group((39.9±3.9,26.9±5.5; 38.9±5.6, 24.3±4.9; 57.1±12.6,33.8±8.7) increased significantly than that in A group (24.0±1.6,13.8±3.3)(P<0.05);there was a more marked increase compared D group to the B, C group (P<0.05); the concentrations of IL-18 in B, C group (122.5±36.9 pg/ml,119.8±36.5 pg/ml) decreased slightly compared with the A group (142.1±88.5 pg/ml)(P=0.09); but concentrations of IL-18 in D group (40.2±22.1 pg/ml) decreased significantly compared with A group (P<0.05).
Conclusions
1.Along as different stages of coronary heart disease incidence (stable angina pectoris-unstable angina-acute myocardial infarction) and acute myocardial infarction in different lesions (single-vessel disease-double-vessel disease-multi-vessel disease), vascular injury was increasing and inflammatory response enhanced further, the number and the function of endothelial progenitor cells were decreased.
2.The therapy of venom thrombolytic can improve the number of circulating EPCs and inhibited IL-18 expression, thus contributing to the restoration of endothelial injury and reduce the inflammatory response of vascular endothelial further injury.
3. The therapy of venom thrombolytic with autologous EPCs transplantation could improve cardiac function necrosis, and it was good to restoration for myocardial infarction.
4. The therapy of venom thrombolytic with autologous EPCs transplantation could significantly improve the number of endothelial progenitor cells and colony-and lower the of concentration IL-18, so as to effectively promote the restoration of endothelial injury, control of vascular endothelial damage in inflammatory response.
Main innovation:
1, It was the first to monitor of peripheral blood progenitor cells in patients with coronary heart disease and the expression of IL-18, and coronary heart disease due to its strong correlation to some extent, so the clinical diagnosis of coronary heart disease can help the sub-type and grade, and its treatment and prognosis is also good clinical significance.
2, It was the first animal experiment to show that:the drugs on the basis of thrombolytic therapy combined with autologous progenitor cell transplantation can improve cardiac function, and promote endothelial injury recovery, effectively inhibit the inflammatory response, clinical coronary heart disease, especially acute myocardial infarction provides a new possible treatment.
引文
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