【作者】 王绚卉；

【导师】 睢大筼；

【作者基本信息】 吉林大学 ， 药理学， 2010， 博士

【摘要】 急性心肌梗死是严重危害人类健康的常见多发病,溶栓及PCI是最有效的治疗方法,可使缺血心肌得到再灌注,改善心肌梗死的预后。然而,越来越多的证据显示,缺血心肌再灌注后一段时间内心肌常出现再灌注损伤,表现为心律失常、无复流现象、心肌顿抑及细胞坏死等。开发安全有效的抗心肌缺血药物是一项重要的课题。五加科人参属植物西洋参(Panax Quinquefolium L.)历来以根入药,茎叶则多弃之山岭不作药用。化学成分分析表明:西洋参皂苷在叶部的含量(9.05～10.45%)明显高于根部(3.89～6.49%)。西洋参叶总皂苷含有20s-原人参二醇组皂苷(PQDS)及20s-原人参三醇皂苷(PQTS),我们以PQDS为原料研制出洋参二醇皂苷注射液(IPQDS)按治疗急性心肌缺血的中药五类新药进行开发。本文建立大鼠心肌缺血再灌注损伤模型及犬急性心肌梗死模型,从形态学、心肌酶学、心外膜心电图、血液生化学、心功能血流动力学、冠脉循环及心肌氧代谢等方面观察了IPQDS对实验性心肌缺血的保护作用及其机制。结果表明,IPQDS可明显缩小心肌梗死面积,降低心肌缺血程度及缺血范围,抑制心肌三酶活性,并纠正急性心肌梗死时的泵衰竭。可能通过减轻氧自由基的堆积,提高内源性抗氧化酶活性,降低血小板粘附及聚集功能,纠正心梗死血时FFA代谢紊乱及血液高粘滞状态,保持血管活性因子(NO/ET及PGI2/TXA2)及心肌供氧与需氧平衡,改善心肌舒缩功能等多种途径发挥抗心肌缺血作用。此外,IPQDS能抑制心肌缺血再灌注损伤时原癌基因c-myc、c-fos及c-jun mRNA的表达,并明显降低血浆ET及AngⅡ水平,提示其可能具有防治急性心肌缺血后心室重构的作用。心肌肥大(Myocardial Hypertrophy,MH)是对心肌工作负荷增加的一种代偿,已成为导致心衰、心肌梗死及心源性猝死的独立危险因素。预防或逆转潜在的MH是治疗慢性高血压和心衰患者的主要目标。寻找有效的治疗方法是医学界面临的主要任务之一。许多证据显示,心肌局部产生的肾上腺髓质素(AM)具有减弱心脏肥大/重构以及对抗心肌缺血再灌注损伤等心脏保护作用。通过腺病毒靶向性递送AM基因至心脏,补充外源性AM是否能预防或逆转肥大心肌的结构及功能改变是一项有意义的研究工作。为探讨补充外源性AM的应用潜能,本研究采用给大鼠饮用NOS抑制剂L-NAME 8周建立心肌肥大模型,在巨细胞病毒(CMV)启动子的调控下,用腺病毒(Ad)递送外源性AM基因—Ad.CMV-AM对模型大鼠进行干预,检测相关指标,评价其作用及可能的机制。实验①进行重组基因腺病毒扩增、纯化并测定滴度;②尾静脉注射Ad.CMV-GFP,以荧光倒置显微镜检测心肌细胞GFP的存在,对Ad.CMV-AM在体研究进行靶向性预测;③大鼠饮用L-NAME(35mg/kg/d)8周,饮用L-NAME当天及第5周分别尾静脉注射Ad.CMV-AM 2×1010 pfu,观察外源性AM基因靶向性递送对大鼠心肌肥大的预防作用。结果显示:①重组腺病毒经过扩增、纯化,滴度达到1×1012 pfu/mL,可用于在体动物实验;②通过Ad.CMV-GFP感染大鼠,在心肌细胞检测到GFP,预测Ad.CMV-AM能靶向定位到心肌细胞;③大鼠心肌肥大预防作用的研究表明,与对照组比较,L-NAME可引起收缩压(SBP)明显升高;心肌细胞宽度、心重与体重比(HW/BW)、心钠素(ANP)及骨骼型α-actin(sk-α-actin)表达均明显增加;NADPH氧化酶、抗氧化酶SOD3和GPx基因表达及心肌细胞膜蛋白氧化均明显增加;内源性AM基因表达亦明显增加。与模型组比较,Ad.CMV-AM除了可明显对抗心肌细胞宽度增加,对上述各项指标均无明显对抗作用。没有检测到外源性AM基因的表达。实验未观察到AM基因对大鼠心肌肥大的预防作用,考虑可能与携带外源目的基因的腺病毒载体在动物体内递送过程及靶向定位不稳定,CMV启动子调控不稳定以及腺病毒递送的外源目的基因在体内表达不稳定等因素有关。对此实验模型体系中递送外源性AM基因的可行性仍需进一步探讨及评估。更多还原

【Abstract】 Part 1 Effects of IPQDS on experimentaL myocardial ischemia Background:Myocardial ischemia, which is caused by coronary atherosclerosis in most casses, is a clinical state with coronary blood flow decrease, myocardial oxygen supply deficiency and less removal of metabolic products.Myocardial ischemia is a higher incidence cardiovascular disease, its mortality is high when acute attack. Present therapeutic measure, such as thrombolylic drug treatment, interventional therapy and surgical treatment, have get good clinical efficacy in usual cases. But after applying them, the myocardial ischemia reperfusion injury which easily cause arrhythmia or heart failure have become new disadvantages. Therefore, looking for safe and effective drug for the prevention and treatment of ischemic heart disease is one of the important issues.Panax quinquefolius L. is one of the special local products of the east region of Changbai Mountain. Its main root and fibrous root are recorded in China Pharmacopeia, while its stem and leaves are not sufficiently used. Therefore, exploitation the overground part of Panax quinquefolius L. is meaningful to systematically utilize the medical resource of Panax quinquefolius L. We have been studying the overground part of Panax quinquefolius L. and confirmed with systemic reseach in chemistry, pharmacology and toxicology that panaxsaponins derived from stem and leaves of Panax quinquefolius L. (PQS) are main effective parts. Panaxsaponin-Rb2,-Rb3,-Rd,-Re,-Rg1,-F2 and para-panaxsaponin-F11,-RT5 were separated from PQS. Panax quinquefolium 20-sprotopanaxdiolsaponins (PQDS) were separated by us from PQS and confirmed that PQDS mainly contain panaxsaponin-Rb2,-Rb3, -Rd and -Rc and they have not haemolysis. Therefore, by means of raw material of PQDS derived from PQS, we reseached and exploited IPQDS which a new drug of traditional Chinese drug about treatment of acute myocardial ischemia diseases with patent technique and got the supporting of country 863 plan.In this paper, after the establishment of rat and dog myocardial ischima model, we analyse protective effects of PQDS on experimental myocardial ischemia and its mechanism on morphology, enzymes, blood biochemistry, epicar dial ECG, cardiac function, hemodynamics, myocardial oxygen metabolism and molecular levels to provide pharmaco dynamic basis for the development of five new Chinese medicine to the treatment of acute myocardial ischima.Methods:1. Establish a rat model of myocardial ischemia reperfusion injury and measure the following indicators:(1)Determinate serum the activities of aspartate aminotransferase enzyme (AST), creatine phosphate kinase (CK) and lactate dehydro genase (LDH) By COBAS-FARA automatic analyzer ;(2)Draw blood from abdominal aorta, detect serum the content of nitric oxide (NO) and malondial dehyde (MDA), activities of superoxide dismutase (SOD) and glutath ione peroxidase (GSH-Px) by kit;(3)Draw 1ml Blood from the abdominal aorta, anticoagulate with 3.8% sodium citrate by ratio of 1:9, add the blood into platelet adhesion LBY-F5 Miriam ball, count the number of platelets after adhesion in the light microscope, calculate platelet adhesion rate (PAR) ;(4)Draw 3mL blood from the abdominal aorta, anticoagulate with 3.8% sodium citrate by ratio of 1:9, utilize LBY-NJ2 to measure platelet aggregation rate(PAG)and maximum platelet aggregation rate (MPAG) ;(5)Stain organizations with chlorination NBT (NB-T) to calculate myocardial infarct size (MIS) with the ratio of myocardial ischemia and ventricular wet weight and myocardial histopathology was observed under light and electron microscope;(6)The levels of plasma endothelin (ET), angiotensinⅡ(AngⅡ) and PGI2 and TXA2 were detected by Radioimmunoassay;(7)mRNA expression of cardiac proto-oncogene c-fos, c-myc and c-jun were detected by RT-PCR.2. Establish canine myocardial infarction model, determinate the following indicators:(1)Separate the right common carotid artery, connecte pressure transducer after intubation, measure systolic blood pressure (SBP) and diastolic blood pressure (DBP) by carrier amplifiers (AP-601G); (2)Separate the left femoral artery, insert the catheter to the left ventricle, connecte carrier amplifier (AP-601G) to detect left ventricular pressure (LVP), left ventricular end diastolic pressure (LVEDP) and the maximum change rate of left ventricular pressure (±dp/dtmax);(3)Separate the aortic root, place the suitable electromagne tic flowmeter probe, measure cardiac output (CO) and compute cardiac index(CI) ;(4)Separate LCX, place the suitable diameter electromagnetic flowmeter probe to measure coronary flow (CBF) and compute myocardial blood flow (MBF) and coronary vascular resistance (CVR) ;(5)Connect ECG electrodes, recordⅡ-lead ECG and heart rate (HR) by AB-601 bioel- ectrical amplifier ;(6)Map the epicardial electrocardiogram in normal, the edge of infarction zone and the central area of infarction by wet-type multi-point adsorption ;(7)Draw blood from the left femoral artery at the different time, measure the content of serum free fatty acid (FFA) with one abstraction colorimetry ;(8)Draw blood from the coronary sinus and the left femoral artery at the same time, measure arterial and venous blood oxygen by the CORNIHG 178 blood gas analyzer, calculate myocardial oxygen consumption (COC), myocardial oxygen utilization (MOUR) and myocardial oxygen consumption index (MOCI) ;(9)Draw Blood from the femoral artery, determinate activities of serum aspartate aminotransferase enzyme (AST), creatine phosphor kinase (CK) and lactate dehydrogenase (LDH) by COBAS-FAARA automatic biochemical analyzer ;(10)Blood from the femoral artery, anticoagulate with heparin, utilize LBY-N6A to measure viscosity of blood and plasma ;(11)Stain tissues with chlorination NBT (NB-T) to calculate myocardial infarct size (MIS) with the ratio of myocardial ischemia and ventricular wet weight.Results(1)IPQDS have significant protective effects on myocardial ischemia reperfusion injury in rats and acute myocardial infarction in dogs, which relate to l as the extent of myocardial ischemia in dogs and ischemic area,and can alleviate the level of the acute myocardial ischemic electrocardiogram and decrease activities of serum CK, LDH and AST ; (2)IPQDS can significantly reduce the content of serum MDA, increase activities of serum SOD and GSH-Px in myocardial ischemia reperfusion injury rats and acute myocardial infarction dogs ;(3)IPQDS can significantly reduce PAR, PAG and MPAG in myocardial ischemia reperfusion injury rats,as well as decrease the viscosity of blood and plasma in acute myocardial infarction dogs ;(4)IPQDS can significantly reduce the level of FFA in myocardial infarction dogs ;(5)IPQDS can significantly reduce levels of serum NO, plasma ET, AngⅡand TXA2, increase plasma levels of PGI2 and PGI2/TXA2 ratio in rats with myocardial ischemia reperfusion injury ;(6)IPQDS could inhibit the expression of proto-nocogene c-myc,c-fos and c-jun after myocardial ischemia reperfusion injury ;(7)IPQDS can significantly reduce oxygen utilization, reduce oxygen consumption in dogs with acute myocardial infarction ;(8)IPQDS can significantly prevent blood pressure (SBP, DBP and MAP) from decreasing, markly increase MBF, LVP,±dp/dtmax, CO and CI, and decrease LVEDP ;ConclusionIPQDS have protective effects on myocardial ischemia reperfusion injury in rats and acute myocardial infaction in dogs, which maybe relate to elevating the activities of endogenous antioxidase,decreasing function of platelet adhesion and aggregation,improving metabolic disorder of myocardial FFA, keeping physiologic equilibrium of vasoactive substance (NO/ET and PGI2/TXA2), reducing cardiac work,decreasing myocardial oxygen consumption and improving heart diastolic and systolic funtion, as well as apposing pump failure under myocardial ischemia,et al, which indicate protective effects of IPQDS on myocardial ischemia should be the result from many synthetic path action. In addition, IPQDS could inhibit the expression of proto-nocogene c-myc,c-fos and c-jun after myocardial ischemia reperfusion injury and significantly decrease levels of plasma ET and AngⅡ,which indicate it maybe prevent and treat ventricular remodelling after acute myocardial ischima. Part 2 Prevention of Ad.CMV-AM on myocardial hypertrophic ratsBackground:AS one major type of cardiac hypertrophy, Left ventricular hypertrophy (LVH) is a compen-satory response to increased cardiac workload and is an independent risk factor for heart failure, myocardial infarction and sudden cardiac death. Prevention of underlying LVH is a major goal in the management of patients with chronic hypertension and heart failure and understanding its mechanistic basis is one of the most important tasks facing medicine and science.Chronic administration of the pharmacological inhibitor of NOS, (L-NAME) into normotensive rats, results in hypertension accompanied by myocardial remodelling, cardiac ischemia and necrosis, and mechanical dysfunction. Reports of prevention or regression of increased LV mass by ACE inhibitors and Ca2+ channel antagonists but not by blood pressure reduction per se indicate that NOS inhibition may have direct consequences for the myocardium.Many evidences support a direct role of Adrenomedullin (AM) produced locally within the heart, in the attenuation of cardiac growth/remodelling and protection against ischemia-reperfusion injury. However, the necessity for intravenous infusion may limit the therapeutic potential of this peptide. Such limitations could conceivably be overcome by delivery of the AM gene. Adenovirus Ad.CMV-AM, in which human AM cDNA is under the control of cytomegalovirus (CMV) promoter, will be administered to Sprague-Dawley rats treated with the inhibitor of nitric oxide synthase, L-NAME (35mg/kg/day in drinking water), which normally exhibit increased oxidative stress, hypertension, left ventricular hypertrophy and myocardial ischemia. Ad.Null injected rats age-matched SD rats given L-NAME (35mg/kg/day in drinking water) and untreated SD rats will serve as controls for treatment, disease and normal ageing, respectively. We measured SBP、parameters of hypertrophy、indicators of oxidative stress in cardiomyocytes and mRNA expression of endogenous AM and the foreign gene. These findings should provide a basis for ethical approval for small pilot studies to be undertaken to assess the feasibility of administration of such a treatment to patients with clinically significant abnormal thickening of heart muscle, in order to reverse this structural change and thereby reduce these patients’cardiovascular risk. Hope to attain the objectives: obtain a supply of Ad.CMV-AM and knowledge of its use for in vivo studies; ascertain the feasibility of delivery of the human AM gene into a novel experimental model system; investigate if increased levels of AM attenuate development or regress established ventricular of AM attenuate development or regress established ventricular remodeling, oxidative stress and ischemia; provide the rationale for pilot clinical studies.1. Large scale production,purification and amplication, as well as titer detection for recombinant Adenovirus.(1)Culture, passage HKE293T cells, furthermore culture the cells in large scale.(2)Amplication of recombinant Adenovirus in cultured HEK293T via infecting the cells with virus at a MOI of 10.(3)Generate cesium chloride ( CsCl ) gradient, spin the virus solution by Optima L-90K ultra low temperature high speed centrifugation to purify and recombinant adenovirus at two steps. Determinate the titer of purified virus by VP and TCID50.2. Ad.CMV-GFP transfect cardiomyocyte in vivo.(1)Ad.CMV-GFP was administered to SD rats by tail intravenous injection.(2) Isolation of ventricular cardiomyocytes was taken by Perfusion of excised hearts with a solution of collagenase in calcium-free Krebs Ringer solution using a Langendorff perfusion apparatus.(3)Observe the infected cardiomyocyte using Axiovert IM35 phase-contrast invert microscope and take pictures using WV BL600 camera.3. Establish cardiomyocyte hypertrophy Model, determinate the following parameters:(1)SD rats at 8 weeks will receive a single intravenous injection of Ad.CMV-AM at a dose of 2x1010 plaque-forming units/rat via the tail vain at 4-weekly intervals concurrently with L-NAME (35mg/kg/day in drinking water) for 8 weeks prior to isolation of left, and for comparison right, ventricular cardiomyocytes at 16 weeks.(2)Isolate ventricular cardiomyocytes by the same way as the second experiment.(3)Measure the systolic blood pressure (SBP) at weekly intervals in each animal during its lifetime by tail cuff sphygmomanometry .(4)Weigh the heart mass before perfusion and calculate the HW/BW ratio.(5)Cell was visualised using an inverted phase contrast microscope and width(μm) was detected.(6)Oxidative status of membrane proteins was assess via OxyblotProtein Oxidation Detection Kit and ECL Advance Western Blotting Detection Kit, and quantified by densitometry, compared to the signal intensity of the molecular weight protein standards provided.(7)Total RNA will be extracted from cardiomyocytes by standard acid guanidinium thiocyanate-phenol-chloroform extraction. Genes expression of c-fos ANP, BNP, sk-α-actin, NADPH oxidase, SOD3, GPx, human-AM and rat-AM were all performed by real-time RT-PCR: reported sequences for each gene will be used to design on Primer Express software, rat and human specific primers adapted to RT-PCR conditions, synthesised by Invitrogen. RT-PCR will be performed in duplicate using Roche FastStart Universal SYBR Green Master (Rox) in a 2:1 reaction, and an ABI Prism Sequence Detector (PE Applied Biosystems). Analysis will be performed using ABI 7000 Prism software and normalised to GAPDH.(8)Detect the expression of exogenous AM by imunofluorescence technique.Results(1)Adequate level of titer of purified recombinant Adenovirus were obtained.(2)Greenfluorescent was visible in infected cardiomyocyte using Ad.CMV-GFP weekly after treatment.(3)SBP was Elevated, Ad.CMV-AM can not prevent this effect.(4)Ad.CMV-AM did not prevent the increase of heart weight and HW/BW ratio under current condition, indicate not to prevent cardiac hypertrophy at organ level.(5)Width of cardiomyocyte was not widened too much with the effect of Ad.CMV-AM, indicate not to prevent cardiac hypertrophy at cell level.(6)Ad.CMV-AM did not appear preventative effect mRNA expression of hypertrophic genes, sk-α-actin and promote increase of ANP level.(7)Ad.CMV-AM did not affect elevated mRNA expression of NADPH oxidase and increased expression of SOD3 and GPx mRNA.(8)Ad.CMV-AM seems not to affect increased oxidation status of cardiomyocyte membrane protein.(9)Exogenous AM seems no expressed and Ad.CMV-AM seem not to promote mRNA expression of endogenous AM.(10)Peptide of exogenous AM was not expressed in cardiomyocyte cells.ConclusionUnder the current experiment conditions, AM gene delivery did not obviously attenuate manifestation of parameters of ventricular cell hypertrophy and oxidative stress in these parameters besides the width of cardiomyocyte in a model of pressure overload and cardiac ischemia induced by chronic nitric oxide deficiency.Expected result that an exogenous supply of AM, acting with elevated levels of the endogenous peptide could potentially prevent or reverse the phenotypic changes occurring in hypertrophying cardiomyocytes seems not to be attained. The close reasons are considered perhaps relevant to titer and activity stability of and necessary targeting confirmation and stability of exogenous genes delivered by Adenovirus.更多还原