【作者】 曹广庆；

【导师】 吴树明；

【作者基本信息】 山东大学 ， 心血管外科学， 2008， 博士

【摘要】 目的自体静脉移植为冠心病或周围血管疾病患者提供了一个有效的治疗方法,但是,自体静脉移植后再狭窄的发生率较高。移植静脉再狭窄的机制涉及到一系列复杂的病理生理学过程,传统的药物治疗效果不明显。雷帕霉素被证实可抑制血管内膜增生,雷帕霉素药物涂层支架在动物实验和临床研究中也被证实可预防血管再狭窄,但将雷帕霉素制成纳米粒剂型,血管腔内局部灌注治疗移植血管再狭窄的研究未见有报道。因而,本研究欲通过动物实验和临床试验,达到如下目的:1.建立可靠的兔移植静脉再狭窄模型,应用彩色多普勒超声和组织病理学检查判断移植静脉狭窄的程度并评价两者的相关性,寻求一种操作简单、结果精确可用于在动物实验中判定移植静脉再狭窄的检查方法;2.制备出生物学相容性好的雷帕霉素纳米粒,通过对其理化性质和生物学特性进行评价,确定该纳米粒具有良好的缓释效能和局部组织滞留能力;3.在移植静脉模型中,了解雷帕霉素纳米粒移植静脉血管腔内局部灌注处理后局部静脉组织和血浆中雷帕霉素的浓度、毒副作用以及对移植静脉内膜和血浆肾上腺髓质素(ADM)及内皮素-1(ET-1)的影响;4.探讨血浆ADM和ET-1与兔移植静脉再狭窄的关系;5.在临床试验中,探讨体外循环和非体外循环冠状动脉旁路移植术(CABG)对血浆ADM和ET-1的影响。方法将研究分成两部分进行,第一部分为动物实验,第二部分为临床试验。在动物实验部分,首先对建立的兔移植静脉模型进行评价。采用游离的颈外静脉间位移植至颈总动脉之间的方法建立兔移植静脉模型,共18只,分别于术后7天、14天及28天各处理6只兔,在处死前,彩色多普勒超声检测移植静脉内膜厚度、狭窄处静脉内径、最大血流速度、最小血流速度、搏动指数(PI)和阻力指数(RI);处死后,组织学病理定量分析移植静脉内膜厚度和内径,相应的对侧正常颈外静脉作为对照;Spearman相关分析法分析彩色多普勒超声和组织病理学检查结果的相关性。其次,雷帕霉素纳米粒的制备和理化特性评价。采用溶剂扩散法,以不同浓度的卡波姆为乳化剂制备具有生物粘附性的雷帕霉素纳米粒,并用相应的方法对纳米粒的理化性质表征,包括形态学、平均粒径和粒径分布、Zeta电位、包封率、载药量以及体外释放等进行评价;在兔移植静脉模型中,对纳米粒的生物学性质进行评价,分为两组,每组各5只,一组用自制的药物灌注装置对移植静脉血管腔内局部灌注不含纳米粒子的生理盐水,另一组用同样的方法灌注所制备的雷帕霉素纳米粒,术后7天切取两组的移植静脉,对其行形态学分析,显微镜下观察血管周围炎性反应程度,评价纳米粒的生物学相容性。再次,探讨不同浓度雷帕霉素纳米粒移植静脉血管腔内局部灌注处理后的药理学行为。根据所处理的雷帕霉素纳米粒浓度的不同,将模型兔分为两组,每组各20只,低浓度组为100μg/ml,高浓度组为500μg/ml,分别于术后1天、7天、14天及28天各处理5只兔,心脏穿刺留取血标本后处死,采用高效能液相层析法(High performance liquid chromatography,HPLC)检测局部静脉组织中雷帕霉素的药物浓度,血标本用于肝肾功能检查和血浆雷帕霉素药物浓度的测定,组织病理学检查术后28天的移植静脉内膜厚度。最后,分析雷帕霉素纳米粒移植静脉血管腔内局部灌注对兔移植静脉内膜和血浆ADM及ET-1的影响。将模型兔分为三组,每组各10只,实验组用雷帕霉素纳米粒(500μg/ml)对移植静脉行血管腔内局部灌注处理,对照组用相同浓度的雷帕霉素纳米粒周围静脉给药,空白组不做任何处理,分别于术前和术后即刻、1天和7天抽取静脉血,采用放射免疫法测定三组血浆ADM及ET-1的浓度;术后28天时切取移植静脉,行苏木精—伊红染色(HE染色)和Van Gieson胶原染色,采用Image proplus 6.0图像分析系统检测各组移植静脉内膜厚度、内径、内膜/中膜厚度比以及胶原容积指数;Pearson相关分析法分析移植静脉内膜厚度、内径、内膜/中膜厚度比以及胶原容积指数和血浆ADM及ET-1浓度的相关性。在临床试验部分,探讨体外循环和非体外循环CABG对患者血浆ADM和ET-1的影响。选取体外组、非体外组以及对照(普通开胸手术)组患者各40例,分别于术前和术后即刻、1天、7天及14天抽取静脉血,采用放射免疫法测定三组血浆ADM及ET-1的浓度。结果1.彩色多普勒超声检测结果显示,静脉移植术后,移植静脉的内膜增厚,内径缩小,血流速度减慢,PI和RI升高,与正常对侧颈外静脉比较,差异有统计学意义(P＜0.05);且随着静脉移植术后时间的延长,这些指标变化更明显,即术后7天、术后14天和术后28天之间比较,差异也有统计学意义(P＜0.05)。组织病理学检查结果也显示,术后7天、术后14天和术后28天各时间点移植静脉的内膜均增厚,内径均缩小,与正常对侧颈外静脉比较,差异均有统计学意义(P＜0.05);且术后7天、术后14天和术后28天各时间点之间比较,差异也有统计学意义(P＜0.05)。Spearman相关分析显示,术后7天、术后14天和术后28天移植静脉内膜厚度和内径的彩色多普勒超声测量结果与组织病理学定量分析结果呈正相关关系(r=0.905,r=0.971,r=0.928;r=0.924,r=0.829,r=0.940)。2.采用溶剂扩散法,以卡波姆为乳化剂制备的载雷帕霉素纳米粒,其粒径200nm左右,包封率70%以上且表面带负电荷,体外药物释放符合Higuchi释放模型,呈缓释相释放;术后7天时,移植静脉血管腔内局部灌注雷帕霉素纳米粒组和移植静脉血管腔内局部灌注不含纳米粒的生理盐水组,组织病理学检查发现,两组移植静脉周围均未出现中性粒细胞浸润。3.低浓度组和高浓度组雷帕霉素纳米粒移植静脉血管腔内局部灌注处理术后1天时,局部静脉组织和血浆中均可检测出一定浓度的雷帕霉素,但局部静脉组织中的药物浓度高,与血浆比较,差异有统计学意义(P＜0.05),高浓度组局部静脉组织和血浆药物浓度分别为(8.88±0.26)μg/10mg和(1.00±0.03)μg/10mg,低浓度组为(6.24±0.24)μg/10mg和(0.51±0.02)μg/10mg;术后7天,两组局部静脉组织和血浆中的雷帕霉素药物浓度均达到高峰;术后14天,两组局部静脉组织中的药物浓度仍稳定在高峰期水平,而血浆药物浓度己明显下降(P＜0.05);术后28天,两组局部静脉组织中的药物浓度均开始下降,而血浆药物浓度为零;术后1天、7天和14天,高浓度组的血浆和局部静脉组织中的药物浓度均始终高于低浓度组(P＜0.05)。术后1天、7天、14天和28天各时间点高浓度组和低浓度组的肝、肾功能在不断变化,但均在正常范围内。高浓度组和低浓度组雷帕霉素纳米粒对移植静脉血管腔内局部灌注处理术后28天时的移植静脉内膜厚度比较,差异有统计学意义(P＜0.05),高浓度组雷帕霉素纳米粒对移植静脉内膜增生的抑制作用显著。4.静脉移植术后28天时,实验组(雷帕霉素纳米粒移植静脉腔内局部给药)、对照组(相同的雷帕霉素纳米粒周围静脉给药)和空白组(未做任何处理)移植静脉的HE染色和Van Gieson染色组织病理学结果显示,与正常对侧颈外静脉比较,空白组移植静脉内膜明显增厚,内径明显缩小,内膜/中膜厚度比升高,胶原容积指数明显增加,差异有统计学意义(P＜0.05);对照组也呈现类似变化,但与空白组比较差异不显著(P＞0.05);而实验组的这些指标变化不明显,与正常对侧颈外静脉比较,差异无统计学意义(P＞0.05),实验组与对照组和空白组比较,差异均有统计学意义(P＜0.05)。血浆ET-1及ADM检测结果显示,三组术前血浆ET-1及ADM浓度比较,差异无统计学意义(P＞0.05);术后即刻三组血浆ET-1及ADM浓度较术前均明显升高,与术前比较,差异均有统计学意义(P＜0.05),但实验组升高较少,与空白组和对照组比较,差异均有统计学意义(P＜0.05),而空白组和对照组比较,差异无统计学意义(P＞0.05);术后1天,三组ET-1及ADM浓度均下降;术后7天,实验组ET-1及ADM浓度基本恢复至术前水平,而对照组和空白组尚处于较高水平,实验组与对照组和实验组与空白组比较,差异均有统计学意义(P＜0.05),但对照组和空白组之间比较,差异无统计学意义(P＞0.05)。Pearson相关分析法显示三组术后28天的移植静脉内膜厚度、内径、内膜/中膜厚度比以及胶原容积指数和术后即刻、1天和7天血浆ADM及ET-1的浓度均有强相关性。5.术前和术后不同时间点体外和非体外循环CABG患者以及普通开胸手术患者血浆ADM及ET-1的浓度测定结果显示,三组术前血浆ADM及ET-1的浓度无差异(P＞0.05);体外组和非体外组患者术后即刻ET-1及ADM浓度均明显升高,与术前比较,差异均有统计学意义(P＜0.05),但体外组升高更明显,与非体外组比较,差异有统计学意义(P＜0.05);术后1天ET-1及ADM浓度均开始下降;术后14天,非体外组患者ET-1及ADM浓度已降到正常水平,而体外组患者尚未恢复至术前水平,仍维持较高浓度;而普通开胸手术组患者术后即刻ET-1及ADM浓度仅轻度升高,与术前比较变化不明显(P＞0.05);且术后1天时,基本恢复至术前水平。结论1.采用兔自体游离颈外静脉间位移植至颈总动脉之间的方法建立的静脉移植模型能引起术后移植静脉再狭窄;彩色多普勒超声能精确检测实验动物的移植静脉再狭窄的程度,与组织病理学检查有良好的相关性;彩色多普勒超声检查操作方便、无创伤、可以动态观察移植静脉再狭窄的程度,为今后观察移植静脉再狭窄提供了较好的实验技术方法。2.本实验所制备的雷帕霉素纳米粒具有一定的生物粘附性,较高的包封率和缓释效能,与兔血管有良好的生物学相容性。3.移植静脉血管腔内局部灌注雷帕霉素纳米粒,可在局部静脉组织中维持较高的药物浓度,而血浆中的药物浓度较低,增加了药物对靶器官或组织的作用,避免了全身给药对其他器官或组织所造成的影响;雷帕霉素纳米粒浓度的不同,其在局部静脉组织和血浆中的浓度也不同,但对肝肾功能的影响不明显;雷帕霉素纳米粒浓度不同,对移植静脉内膜增生的抑制作用也不同;雷帕霉素纳米粒移植静脉血管腔内局部灌注处理,可在局部静脉组织中表现出良好的药物缓释能力,可维持较长时间的药物作用。4.雷帕霉素纳米粒移植静脉血管腔内局部给药可抑制移植静脉内膜增生,抑制血浆ET-1及ADM浓度升高,保护血管内皮功能,预防移植静脉再狭窄;而雷帕霉素纳米粒周围静脉给药则无此作用;血浆ET-1及ADM与移植静脉再狭窄有较强的相关性。5.体外循环和非体外循环CABG均可引起血管内皮功能紊乱,导致术后血浆ET-1及ADM水平升高,但非体外循环CABG对其影响相对较小;普通开胸手术对血浆ET-1及ADM基本无影响。更多还原

【Abstract】 Objective Surgical bypass using autogenous vein provided an effective treatment for many patients with coronary or peripheral therosclerosis. However, the vein graft restenosis occured in a significant percentage. The pathophysiology of vein graft failure involved a complex mechanism. Conventional pharmacotherapy had limited impact on graft failure. Rapamycin was an immunosuppressive agent which also exhibited antiproliferative properties. Rapamycin coated stents had been demonstrated to suppress restenosis in experimental and clinical studies of percutaneous coronary catheter intervention. PLGA (hydroxy acid and lactic acid copolymer) as a good carrier of controlled release drug could increase the drug retention time in the body, release drug at a constant velocity. However, the study of local infusion intravascular with rapamycin nanoparticles inhibiting neointimal hyperplasia in vein graft had not been reported. Therefore, the aim of this study was to observe the effect of vein graft after local infusion intravascular with rapamycin PLGA nanoparticles on animal experiment and to investigate the effect of plasma adrenomedullin and endothelin-1 in patients with on-pump and off-pump coronary artery bypass grafting on clinical trial. The specific purpose on animal experiment of the present study was described as follows: the established vein bypass graft models in rabbits were verified if vein graft restenosis could be caused. The correlation of pathological examination and color Doppler echocardiography was analyzed to evaluate the reliability of color Doppler echocardiography in detecting the degree of vein graft stenosis in the rabbit model. A good bioadhesive rapamycin nanoparticle was prepared, and its physicochemical and biological characterizations were evaluated. The perfect biological compatibility and drug-release capability of rapamycin nanoparticle were verified. After rapamycin nanoparticle was applied locally through intravascular infusion in vein graft, its concentrations in vein tissue and plasma were tested, the side effects of liver and renal function monitored, the vein graft intimal and plasma adrenomedullin (ADM) and endothelin-1 (ET-1) levels detected. The relationship between vein graft restenosis and plasma adrenomedullin, between vein graft restenosis and plasma endothelin-1 were analyzed.Methods The study was divided into two parts. One part was animal experiment. The other was clinical trial.Firstly, the vein bypass graft model, which external jugular vein was end-side anastomosed at the proximal and distal side of the ligated carotid artery was established in a rabbit. Eighteen rabbits were conducted. Six rabbits were euthanized at the seventh, fourteenth, and twenty-eighth postoperative day respectively and pathological examinations of the vein graft were made. Before enthanizing, Color Doppler echocardiography was done to detect the thickness of endothelium, internal diameter, the maximum flow velocity, the minimum flow velocity, pulsatility index (PI) and resistance index (RI) of the graft vein. The contralateral external jugular vein was regarded as control. The correlation of color Doppler echocardiography and pathological examination was analyzed.Secondly, rapamycin nanoparticle was prepared and its physicochemical characterization was evaluated. Solvent diffusion was applied to prepare for bioadhesive rapamycin nanoparticle, using different concentrations carbopol 940 or PVA as stabilizers. The physicochemical characterizations were evaluated in terms of morphology, average particle size and particle size distribution, Zeta potential, encapsulation efficiency, the drug loading and in vitro release. The biological characterization of the nanoparticle was evaluated in a rabbit model. The experiment model was divided into two groups. Each group had five rabbits. One group was treated with free nanoparticles saline infused into graft vein through a self-made drug infusion device. The other group was treated similarily with rapamycin nanoparticle At the seventh day after the treatment, the morphology of vein graft was analyzed under microscope to observe perivascular inflammatory reaction.Thirdly, the pharmacological behavior of different concentrations rapamycin nanoparticles treatment locally on vein graft was investigated. The rabbit vein graft model was divided into the low concentration group and the high concentration group according to treated rapamycin nanoparticles concentrations (100μg/ml and 500μg/ml). Each group had twenty rabbits. At the first, seventh, fourteenth and twenty-eighth postoperative day, five rabbits were euthanized respectively for detection of rapamycin concentrations in plasma and vein tissue by high performance liquid chromatography (HPLC). Their liver and kidney function were tested at the same time. At the twenty-eighth postoperative day, pathological examinations were made for the vein graft intimal thickness.Lastly, the variation of plasma endothelin-1 (ET-1), adrenomedullin (ADM) levels and vein graft intimal after local application of rapamycin nanoparticles were observed in rabbit vein graft models. Thirty rabbits were conducted and divided into the experimental group which high concentrations of rapamycin nanoparticles (500μg/ml) was used to deal with vein graft in the same way, the control group (peripheral venous injection with the same rapamycin nanoparticles) and the blank group (without treatment). At preoperation, postoperation immediately, the 1st and 7th postoperative day, the blood samples were extracted to measure the concentrations of plasma ET-1 and ADM by radioimmunoassay in three groups. At the twenty -eighth postoperative day, the graft external jugular vein in rabbits were removed for Hematoxylin and Eosin(HE) and Van Gieson(VG) eosin to analyze the intimal thickness, internal diameter, intimal/media thickness ratio and collagen volume index. The correlation of plasma ET-1 and ADM concentrations and the intimal thickness, internal diameter, intimal/media thickness ratio and collagen volume index of vein graft were analyzed by pearson correlation.On clinical trial, patients with on-pump and off-pump coronary artery bypass grafting and general thoracotomy were conducted. At preoperation, postoperation immediately, the 1st, 7th and 14th postoperative day, the concentrations of plasma ET-1 and ADM were measured by radioimmunoassay. Results 1. Compared with the contralateral external jugular vein, the intimal of vein graft was thickened, the internal diameter narrowed, the maximun and minimun flow velocity slowed, PI and RI increased on color Doppler echocardiography after operation. There were significant statistical difference (P<0.05). And with the extension of time, the above mentioned criteria had changed more obviously. Which implied that there was significant statistical difference (the seventh postoperative day vs. the fourteenth postoperative day, the seventh postoperative day vs.the twenty-eighth postoperative day, and the fourteenth postoperative day vs. the twenty-eighth postoperative day). On pathological examination, the intimal of vein graft after operation was thickened, the internal diameter narrowed, too. Compared with the contralateral normal external jugular vein, the difference had statistical significance (P<0.05). And there were significant statistical difference between the different time point (the seventh postoperative day vs.the fourteenth postoperative day, the seventh postoperative day vs.the twenty-eighth postoperative day, and the fourteenth postoperative day vs. the twenty-eighth postoperative day). The results of color Doppler echocardiography and pathological examination showed positive correlation in vein graft intimal thickness and internal diameter (r = 0.905, r = 0.971, r = 0.928; r = 0.924, r = 0.829, r = 0.940).2. The rapamycin nanoparticle was successfully prepared with solvent diffusion. Its average diameter was about 200nm and encapsulation efficiency was about more than 70 percent. The surface of nanoparticle had negative charge. The drug release in vitro was fit to Higuchi release model and was performed with sustained-release phase. No neutrophil infiltration was found in perivascular tissue at the seventh postoperative day both in rapamycin nanoparticles treatment group and saline control group. The biological compatibility of rapamycin nanoparticle was proved perfect.3. At the first day after treatment with low and high concentration rapamycin nanoparticles, a certain concentration of rapamycin in the vein tissue could be detected. The high concentration rapamycin nanoparticle was about (8.88±0.26)μg/10 mg and the low concentration was (6.24±0.24)μg/10 mg. At the seventh day after operation, the drug concentration arrived at its peak. At the fourteenth day after operation, the drug concentration stood at the peak level steadily. At the twenty-eighth day after operation, the drug concentration began to decline. The rapamycin in plasma could be detected at the first postoperative day, but its concentration was very low. The high concentration rapamycin nanoparticle was about (1.00±0.03)μg/10mg, the low concentration was (0.51±0.02)μg/10mg. Compared with the vein tissue, there was significant difference (P<0.05). At the seventh postoperative day, the drug concentration in plasma reached the peak point, however it was still at a low level. It declined significantly at the fourteenth postoperative day, and declined to zero at the twenty-eighth postoperative day. The drug concentrations of plasma and vein tissue in the high concentration group were always higher than those in the low concentration group at the same postoperative day (P<0.05). The drug concentrations of vein tissue were always higher than those of plasma, too (P<0.05). The liver and kidney function were normal at each postoperative day both in low and high concentration rapamycin nanoparticles group. At the twenty-eighth postoperative day, the graft vein intimal thickness got less significant in high concentration rapamycin nanoparticles than that in low concentration rapamycin nanoparticles (P<0.05).4. At the twenty-eighth postoperative day, compared with the normal external jugular vein, the vein graft intimal was thickened obviously, the internal diameter was significantly narrowed, the intima/media thickness ratio increased and the collagen volume index increased significantly in the blank group and the control group in vein graft HE and Van Gieson eison stain. The above mentioned indicators in the experimental group had no apparent change compared with the normal external jugular vein (P>0.05). There were significant difference between experimental group, the control group and the blank group (experimental group vs. control group, and experimental group vs. blank group). The plasma ET-1 and ADM concentrations had no significant statistical difference in three groups before operation (P>0.05). Immediately after operation, the plasma ET-1 and ADM concentrations were increased in three groups, but the experimental group increased slightly. Compared with the preoperative level, the difference had statistical significance (P<0.05). But between the control group and the blank group, there was no significant difference (P>0.05) in plasma ET-1 and ADM. There were significant difference between the experimental group and the control group, and between the experimental group and the blank group. At the first postoperative day, the plasma ET-1 and ADM concentrations started to decline. At the seventh postoperative day, the plasma ET-1 and ADM concentrations returned to the preoperative level in the experimental group, but in the blank group and the control group, they stood at a high level. There were strong correlations between plasma ET-1, ADM concentrations and the intimal thickness, internal diameter, intimal / media thickness ratio and collagen volume index of vein graft.5. In comparison of preoperative plasma ADM and ET-1 concentration, there was no significant difference in three groups. But they increased immediately after operation in the off-pump and on-pump groups, and increased more significantly in the on-pump group. Compared between the off-pump group and the on-pump group, there were significant difference (P<0.05).The plasma ADM and ET-1 levels started to decline at the first postoperative day, and continued to decline at the fourteenth postoperative day. But their concentrations in the off-pump group and the on-pump group still stood at a higher level than normal. The concentrations of plasma ADM and ET-1 in the on-pump group were higher than those in the off-pump group at each postoperative day. However, in the thoracotomy group, the concentrations of plasma ADM and ET-1 had almost not increased after operation. Compared with preoperative level, there was no significant difference (P>0.05). Their concentrations declined quickly and closed to normal level at the first postoperative day.Conclusion 1. The rabbit autologous graft vein model in which external jugular vein was end-side anastomosed at the proximal and the distal side of the same carotid artery could lead to vein graft restenosis. Color Doppler echocardiography was a new experimental technique which might be used to observe the vein graft stenosis continuously and noninvasively. Color Doppler echocardiography and pathological examination had good correlation in evaluation of vein graft restenosis. 2. The rapamycin nanoparticle which was prepared with solvent diffusion in the experiment had a certain biological adhesion, high encapsulation efficiency and slow-release effectiveness. It showed good biocompatibility with rabbit vessels without perivascular inflammatory reaction.3. Rapamycin nanoparticles could maintain high concentrations in the vein tissue after local application, but a few rapamycin nanoparticles in plasma could be detected. The different concentration of rapamycin nanoparticle could release different concentrations in the local tissues and plasma. The effective time of rapamycin nanoparticles could maintain for several months, and had no damage and no significant toxicity to other organization. The different concentration of rapamycin nanoparticle could inhibit graft vein intimal hyperplasia differently.4. Rapamycin nanoparticles were applied locally for the vein graft which could reduce the release of vasoactive peptide after the injury of endothelial cell, decrease the plasma concentration of ET-1 and ADM, and protect the vascular endothelia function. It could inhibit vein graft intimal hyperplasia, and prevent the restenosis of vein graft. The function of rapamycin nanoparticles applied through peripheral venous had no less. There were strong correlations between plasma ET-1, ADM concentration, and the intimal thickness, internal diameter, intimal/media thickness ratio and collagen volume index of vein graft.5. Both on-pump and off-pump coronary artery bypass grafting could cause endothelial dysfunction, increase plasma levels of ET-1 and ADM. But compared with the on-pump coronary artery bypass grafting patient, there was less affect in the off-pump coronary artery bypass graft patient. The effect of ET-1 and ADM in general thoracotomy had no less.更多还原