【作者】 林清飞；

【导师】 陈良龙；

【作者基本信息】 福建医科大学 ， 内科学， 2011， 博士

【摘要】 目的1.在新西兰大白兔的腹主动脉构建动脉粥样硬化易损斑块的动物模型。2.通过血管内超声技术VH-IVUS与病理检测结果进行比较,评价血管内超声技术在识别易损斑块中价值。3.通过观察血脂及主要致炎因子hs-CRP、MMP-3、IL-1、IL-10、TNF-α及ox-LDL等的水平变化,探讨血脂及炎症因子与斑块稳定性的关系。方法4月龄新西兰大白兔16只,随机分成单纯高脂组(6只)和高脂+球囊损伤组(10只)。2周后腹主动脉内膜球囊损伤术,术后继续高脂饲料喂养10周,总共12周后。两组兔分别于实验开始、球囊损伤后及处死动物前作血液生化检查。禁食12h以上,抽取空腹血,留取自凝血检测血脂水平。同时采用ELISA法测定血清高敏C反应蛋白(hs-CRP),肿瘤坏死因子(TNF-α),氧化型低密度脂蛋白(ox-LDL),白介素1(IL-1),白介素10(IL-10),基质金属蛋白酶3(MMP-3)的浓度。12周处死前高脂+球囊损伤组(8只)进行血管内超声检查。两组实验兔处死后,切取左肾动脉向远端的5cm长动脉,切成3mm厚,石碏包埋,5um的组织切片固定在载玻片上,分别进行病理学及免疫组织化学染色检查。结果1.实验过程中,高脂+球囊损伤组有两只兔意外死亡。2.两组兔血生化指标和炎性因子水平检测结果与基础状态相比,建模前后两组兔血脂各项指标比较均有显著性差异,只有LDL-c和Fig在处死前进一步升高,与球囊损伤时相比有显著差异,但两组间比较差异无统计学意义。基础状态和球囊损伤时,两组间对比hs-CRP、MMP-3、IL-l、IL-l0、TNF-α、ox-LDL水平无明显差异,在十二周处死前高脂+球囊损伤组与单纯高脂组比较,hs-CRP、MMP-3、IL-l、IL-l0、TNF-α、ox-LDL水平明显升高(P=0.00)。而且通过动态观察血清炎性因子的情况,发现两周后两组的hs-CRP、IL-l、IL-l0、TNF-α、ox-LDL水平较基础均有升高,但是与单纯高脂组不同,12周处死前高脂+球囊损伤组与两周时对比都有进一步提高,除IL-l的(P<0.05),其余(P均<0.01),而MMP-3在单纯高脂组动态观察并没有明显变化,高脂+球囊损伤组MMP-3在两周时虽有升高但没有统计学意义,12周处死前有明显升高(P<0.01)3.虚拟组织学-血管内超声(VH-IVUS)检查及与病理组织学对比在球囊损伤+高脂饮食组,8只实验兔均进行了血管内超声(IVUS)检查,并与病理检查的结果进行比较,总共276个动脉粥样硬化性斑块被分析,对于不同斑块的特异性,敏感性和阳性预测值被总结, VH-IVUS对非钙化和钙化的薄帽纤维斑块(TCFA)的敏感性是83.3和92.9%,VH-IVUS能正确的识别83.3和81.3%的非钙化和钙化的纤维斑块(FA),对于致病性内膜增生(PIT),VH-IVUS识别PIT的敏感性和阳性预测值分别是81.7和75.4%。以上结果提示VH-IVUS对于识别TCFA具有高度的敏感性和特异性。病理组织学与VH-IVUS对不同的组织成份识别存在明显的相关,线性回归分析显示,在斑块成份分析方面,病理组织学与虚拟组织学(VH-IVUS)之间存在明显相关:纤维组织(R=0.845,P<0.001),纤维脂肪组织(R=0.791,P<0.001),坏死钙化组织(R=0.793,P<0.001),并且坏死核心(R=0.731,P<0.001)。在内弹力膜和中外弹力膜边界的测量方面,不同的观察者之间变化很小,线性回归分析显示测量值明显相关各为(R=0.993和0.996)。4.两组病理学测量结果之比高脂与球囊损伤结合组12周时与单纯高脂组对比,除内中膜厚度变化不明显外,斑块厚度,纤维帽厚度及斑块厚度与内中膜厚度比值经分析,差异有非常显著性意义(P<0.01)。5.两组免疫组化染色对比高脂+球囊损伤组的斑块内有大量染色阳性CD68(巨噬细胞)聚集,主要集中于内膜近管腔面的数层细胞内及外膜处,而单纯高脂组几乎不着色,示炎症较轻;α-actin肌动蛋白两组均有染色,与单纯高脂组比,高脂+球囊损伤组的染色稍浅,两组CD68巨噬细胞和α-actin肌动蛋白阳性面积比有明显区别(P<0.01)。结论1.通过高脂+球囊损伤兔腹主动脉内膜,成功建立了易损斑块的动物模型。2.通过动态观察血脂水平和炎性因子水平的变化,得出易损斑块形成与高血脂水平基础上血管内膜损伤有关。3. VH-IVUS与病理检查结果存在良好的相关性,其对易损斑块检测的敏感性,特异性和阳性预测值高,是一个重复性好,有价值的检查手段。目的1.通过分组横向对比病理学、免疫组织化学及VH-IVUS的观察结果,了解短期不同剂量匹伐他汀干预对斑块成份和稳定性的影响。2.通过纵向动态观察VH-IVUS以及炎症因子水平在不同剂量匹伐他汀治疗前后变化,以了解不同剂量他汀干预对斑块的影响,探讨短期大剂量他汀对斑块的影响及其作用机制。方法1.动物实验易损斑块模型的建立同论文第一部分,采用高脂+球囊损伤腹主动脉建模,经过VH-IVUS检查确定已产生易损斑块的新西兰大白兔24只入选,随机分成对照组,匹伐他汀普通剂量组(1㎎/kg/d)和大剂量组(10㎎/kg/d) ,各8只,对照组仅予高脂饲料,1周后均进行VH-IVUS检查。2.血清学检查实验后12周末和13周末抽取兔耳缘静脉血4ml,自凝后留取血清分装于EPPendorf管中,分别检测血清总胆固醇(TC)、甘油三酷(TG)、低密度脂蛋白(LDL-C)及高密度脂蛋白(HDL-C)。部分血清按试剂盒说明采用ELISA法测定血清高敏C反应蛋白(hs-CRP),基质金属蛋白酶3(MMP-3),基质金属蛋白酶9(MMP-9),肿瘤坏死因子-α(TNF-α),白介素1(IL-1),白介素10(IL-10),氧化型低密度脂蛋白(ox-LDL)。3.血管内超声检查实验12周末及13周末检测以下指标:腹主动脉管腔面积(lumen area,LA),血管外弹力膜面积(external elastic membrane area,EEMA),斑块面积(Plaque area,PA),斑块负荷(Plaque burden,PB)。VH-IVUS检测根据成像原理,动脉粥样斑块成份分为四种:纤维成份(fibrous tissue,FT;绿色标记)、纤维脂质成份(fibro-fatty,FF;黄色标记)、钙化成份(dense calcium,DC;白色标记)、坏死成份(necrotic core,NC;红色标记)。4.病理组织学及免疫组织化学检查苏木素-伊红(HE)染色、天狼猩红染色和Masson染色,应用图像分析软件(Image-ProPlus 6.0 software,Mediacybemeties)进行测量分析,平均纤维帽厚度和血管内-中膜厚度(IMT)是在每个切片血管内膜的十个等距点测量的均值,并计算斑块厚度/血管内-中膜厚度的比值。应用MMP-3、MMP-9、CD68及α-actin等抗体检测局部基质金属蛋白酶,巨噬细胞及平滑肌细胞的表达。5,实时定量反转录多聚酶链式反应RT-PCR检测LOX-1、MMP-3、MMP-9、TIMP1及TIMP2等mRNA的表达。结果1.治疗前后血清学检测结果匹伐他汀组TC、TG、LDL-c等治疗后均有明显的减少(P<0.01),而大剂量匹伐他汀组的HDL-c有明显升高(P<0.01);组间对比,治疗后(13周末),与对照组相比,匹伐他汀治疗的两组TC、TG、LDL-c等均有明显的减少,且与普通剂量匹伐他汀组对比,大剂量组LDL-c有进一步的减少但没有统计学意义(P=0.066),而HDL-c明显升高有意义(P<0.01)。匹伐他汀组hs-CRP、MMP-3、MMP-9、TNF-α、IL-1、IL-10、ox-LDL等治疗后均有明显的减少(普通剂量组的TNF-α,hs-CRP, MMP-9的P<0.05外,其余的指标以及大剂量组的所有指标均P<0.01),且大剂量组更明显。组间对比,治疗后(13周末时),与对照组相比,普通和大剂量匹伐他汀组hs-CRP、MMP-3、MMP-9、TNF-α、IL-1、IL-10、ox-LDL等水平均有明显减少(P<0.05和P<0.01),且与普通剂量组相比,除ox-LDL和MMP-9外,其他指标大剂量组有更明显的减少(P<0.01)。2.治疗前后VH-IVUS血管容积和斑块成份改变大剂量匹伐他汀组与治疗前相比,治疗后管腔面积显著增大(P=0.01),斑块面积和斑块负荷显著减少(P<0.01),VH-IVUS示纤维脂质成份面积和纤维脂质成份含量有明显减少(P<0.05)。组间对比,治疗后(13周末)大剂量匹伐他汀组纤维脂质成份面积较对照组和普通剂量匹伐他汀组明显减少(P<0.01和P<0.05)。3.治疗前后斑块病理组织学检查结果根据特殊染色的结果,治疗后大剂量匹伐他汀组斑块厚度与对照组和普通剂量匹伐他汀组相比有明显减少而纤维帽厚度明显增厚(P<0.05和P<0.01),内中膜厚度指标三组间没有明显不同。通过特殊染色和免疫组化检测斑块内胶原、巨噬细胞、平滑肌细胞含量。巨噬细胞(CD68)染色阳性百分比大剂量匹伐他汀组较其他两组明显减少而α-actin染色阳性百分比明显增加(P<0.01),天狼猩红染色胶原阳性百分比大剂量匹伐他汀组与对照组相比有增加趋势但没有统计意义(P=0.06)。4.治疗前后实时定量反转录多聚酶链式反应(RT-PCR)匹伐他汀两组斑块内MMP-3、MMP-9mRNA和LOX-1表达明显减少,TIMP-l和TIMP-2的mRNA表达量增多,且存在剂量依赖性。结论1.普通和大剂量匹伐他汀短期内均有调脂作用,且存在剂量依赖性。2.普通和大剂量匹伐他汀短期内均有抗炎作用,且存在剂量依赖性。3.大剂量匹伐他汀短期内可稳定易损斑块,并逆转斑块的进展。4.在易损斑块动物模型中,匹伐他汀通过调脂、抗炎和抗氧化而发挥稳定及逆转斑块的作用。更多还原

【Abstract】 Objectives1. To establish an animal model of vulnerable atherosclerotic plaque in New Zealand white rabbits abdominal aorta.2. To evaluate the value of intravascular ultrasound in recognition of vulnerable plaque by comparison with pathological results.3. To explore the relationship between plaque stability and the lipids and the major pro-inflammatory cytokines.Materials and methodsNew Zealand rabbits aged 4 months(n=16)were randomly divided into 2 groups: fat-rich diet group(group A, n=6) and fat-rich combined with arterial intimal injury group(group B, n=10). Rabbits of group B after 2 weeks underwent balloon-induced endothelial injury in the abdominal aorta and thereafter were fed a fat-rich diet (1%cholesterol) for 10 weeks. Blood was drawn from rabbits fasting overnight to measure lipid profile at the beginning of the study, after week 2, the end of week12 on termination of experiment. At the same time,ELISA was used to quantify the amount of different inflammation mediators such as hs-CRP,MMP-3, IL-1, IL-10, TNF-α, and ox-LDL. Rabbits of group B underwent intravascular ultrasound studies after anesthesia on termination of experiment. Rabbits of 2 groups were sacrificed, the abdominal aorta of 5cm was cut down distal to the takeoff of the left renal artery, and 3mm specimen was embedded in paraffin, and then cut into 5um-slices and fixed on glass slides for the purpose of pathological and immunohistochemical analysis.Results1. There were 2 rabbits of group B died in accident during experiment.2. Biochemical studiesThe serum lipids levels of all rabbits increased significantly after the fat-rich diet, with further increase of levels of LDL-c and Fig on termination of the experiment (P<0.01), but values did not significantly differ between two groups (P>0.05).Levels of hs-CRP,MMP-3, IL-1, IL-10, TNF-α, and ox-LDL were no significant differences between the 2 groups at baseline and week 2(all P>0.05). However,the levels of six inflammatory biomarkers were significantly higher in rabbits of group B than those in rabbits of group A on termination of the experiment (all P<0.01). The levels of hs-CRP,IL-1, IL-10, TNF-α, and ox-LDL of all rabbits were significantly higher at week 2 than at baseline, and the levels of hs-CRP,IL-1, IL-10, TNF-α, and ox-LDL of group B increased further on termination of the experiment, as compared to group A. The level of MMP-3 was significantly higher on termination of the experiment in group B.3. VH-IVUS imaging and histologyEight rabbits of group B underwent intravascular ultrasound, a total of 276 atherosclerotic plaques were analyzed. VH-IVUS had a sensitively of 83.3% and 92.9% for the detection of non-calcified and calcified TCFA, respectively. VH-IVUS correctly identified 83.3% of the non-calcified FA and 81.3% of the calcified FA. For the detection of PIT, VH-IVUS showed a sensitivity of 81.7% and a positive predictive value of 75.4%.Linear regression analysis showed a strong correlation between histology and VH-IVUS for the percent area of fibrous tissue (R=0.845, P<0.001), fibro-fatty tissue (R=0.791, P<0.001), necrotic calcified tissue (R=0.793, P<0.001) and confluent necrotic core (R=0.731, P<0.001).The inter-observer variability of the intimal boundaries and the medial- adventitial boundaries were low. Linear regression analysis shows a strong correlation between two measurements, which were performed by two observers (R=0.993 for intimal boundaries and 0.996 for medial-adventitial boundaries).4. Histological and immunohistochemical stainingIn addition to intra-media thickness, the plaque thickness, fibrous cap thickness and plaque thickness/ intra-medial thickness ratio were different significantly between the two groups (P<0.01). The results of immunostainingα-actin were less expression in group B than group A, the positive staining of CD68 were higher in group B than group A.ConclusionsWe have developed an animal model of vulnerable plaque in New Zealand white rabbit abdominal aorta. vulnerable plaque is successfully produced by fat-rich diet together with balloon-induced endothelial injury in the rabbit abdominal aorta. Linear regression analysis showed a strong correlation between histology and VH-IVUS, VH-IVUS is a feasible, reproducible and valuable means of the vulnerable plaque identification. Objectives1. To test the hypothesis that short-term high-dose pitavastatin intervention has anti-atherosclerotic progression and plaque-stabilizing effect in a rabbit model of vulnerable plaques.2. To elucidate the mechanisms of short-term high-dose pitavastatin treatment in stabilizing vulnerable plaques. Methods1. Anmal experiment protocolA rabbit model of vulnerable plaque was produced by the method in part I. The male New Zealand white rabbits underwent balloon-induced endothelial injury in the abdominal aorta and thereafter were fed with a high-cholesterol diet (l% cholesterol) for 10 weeks. A total of 24 male New Zealand white rabbits were identified vulnerable plaques in abdominal aorta by VH-IVUS. Then rabbits were randomly divided into 3 groups: group A was served as controls (without treatment, n=8), group B was given usual dose pitavastatin (1mg/kg/d, n=8), group C was given high-dose pitavastatin (10mg/kg/d, n=8). One week later, all rabbits underwent VH-IVUS examination.2. Biochemical studiesBlood was drawn from rabbits fasting overnightat the end of week12 and 13 to measure lipid profile and the amount of different inflammation mediators such as hs-CRP, MMP-3, MMP-9, TNF-α, IL-1, IL-10 and ox-LDL.3. Intravascular ultrasound studiesVH-IVUS was accomplished at the end of week 13 to measure the external elastic membrane area(EEMA), the lumen area(LA),plaque area(PA),the percentage of plaque burden(PB). VH-IVUS identified four different plaque components (fibrous, fibrofatty, necrotic and calcified tissue).4. Histological and immunohistochemical stainingSerial cross-sections underwent general histological staining with hematoxylin & eosin(HE), picrosirius red, Masson and specific immunohistochemical stainings. The primary antibodies included monoclonal antibodies against rabbit macrophages to identify macrophages,andα-smooth muscle cell actin to detect VSMCs. The fibrous cap thickness, the intra-media thickness, the plaque thickness, and the plaque thickness/ intra-medial thickness ratio were measured and values averaged. VSMCs and macrophages were expressed as a percentage of statining area divided by plaque area.5. Quantitative Real-time reverse transcriptase-polymerase chain reactionThe mRNA expression of various inflammation medicators LOX-l, MMP-3, MMP-9, TIMP-1 and TIMP-2 in the abdominal arterial atherosclerosis lesions was determined by RT-PCR. Results1. Biochemical changesThe levels of TC, TG and LDL-C were decreased significantly in pitavastatin groups (P<0.01), and HDL-C was increased in high-dose pitavastatin group(P<0.01). At the week 13, the levels of TC, TG and LDL-C in pitavastatin group were lower than those in control group, but only HDL-C in high-dose pitavastatin group was higher than that in usual dose pitavastatin group (P<0.01).The levels of MMP-3, hs-CRP, MMP-9, TNF-α, IL-1, IL-10 and ox-LDL were decreased significantly after treatment in pitavastatin groups (P<0.05 for TNF-α,hs-CRP, MMP-9 in usual dose group and P<0.01 for IL-1, IL-10, MMP-3, ox-LDL in usual dose group and all in high dose group). The levels of MMP-3, hs-CRP, MMP-9, TNF-α, IL-1, IL-10 and ox-LDL in pitavastatin groups was lower than those in control group, and the levels of MMP-3, hs-CRP, TNF-α, IL-1 and IL-10 in high-dose pitavastatin group was lower than those in usual dose group(P<0.01 for MMP-3, hs-CRP,TNF-α, IL-1, IL-10 ). 2. Intravascular ultrasound alterationsThe lumen area was increased significantly (P=0.01), and the plaque area and the plaque burden were decreased significantly after pitavastatin treatment in high-dose group (P<0.01), however, fibro-fatty volume and fibro-fatty (%) was decreased significantly after the treatment in high-dose group. Fibro-fatty volume in high-dose group was decreased significantly in control and usual dose group (P<0.01 and P<0.05).3. Histological changesPlaque thickness in high-dose pitavastatin group was decreased significantly than in control and usual dose groups (P<0.05), however, fibrous cap was increased significantly (P<0.01). Lower macrophage and higher SMCs were found in high-dose pitavastatin group than in control and usual dose groups (P<0.01).4. Quantitative Real-time RT-PCRThe relative mRNA expressions of LOX-l,MMP-3,MMP-9 were lower in treatment groups than in control group (all P<0.01), and high dose group was decreased significantly than usual dose group (P<0.01). On the contrary,TIMP-1 and TIMP-2 mRNA expressions were higher in treatment groups than in control group, and high dose group was increased significantly than usual dose group.Conclusions1. Short-term pitavastatin treatment is effective in lipid lowering with a dose-dependent way.2. Short-term pitavastatin treatment has anti-inflammation effect with a dose-dependent way.3. Short-term with high-dose pitavastatin intervention enhances the vulnerable plaque stability and inhibits the plaque progression.4. The mechanisms involved in stabalising vulnerable plaques by short-term high-dose pitavastatin may be mainly due to its lipid lowering, anti-inflammation and anti-oxidative effects.更多还原