阿托伐他汀对糖尿病下肢动脉硬化PTA术后再狭窄影响的实验研究

发布时间：2017-12-27 13:29

本文关键词：阿托伐他汀对糖尿病下肢动脉硬化PTA术后再狭窄影响的实验研究　出处：《山东大学》2017年博士论文　论文类型：学位论文

【摘要】：目的糖尿病患者动脉粥样硬化(atherosclerosis,AS)的患病率较高,而且容易发生在主动脉,冠状动脉,肢体外周动脉等部位。冠状动脉目前多采用血管内支架植入术,但是肢体外周AS常以下肢动脉病变为主,尤其是膝以下动脉及其分支。由于内径细、压力低,膝下动脉的治疗一直是个难题。新型的球囊导管由于剖面低、压力低、长段、顺应性较好的优点,大大增加了膝下动脉进行介入治疗的可行性。因此经皮腔内血管成形术(percutaneous transluminal angioplasty,PTA)成为目前治疗糖尿病下肢动脉特别是膝下AS的重要手段。但由于PTA术后再狭窄发生率高,平均约为30%,严重影响了 PTA的远期效果。如何防治PTA术后再狭窄成为影响糖尿病下肢AS治疗的关键问题。他汀类药物,是羟甲基戊二酰辅酶A(hydroxy-methylglutaryl-CoA,HMG-CoA)还原酶抑制剂。实验研究表明,除了降低胆固醇的作用外,它们还有非调脂的抗AS作用:抑制血管内皮的炎症反应,稳定粥样斑块,改善血管内皮功能,抑制动脉内膜增生等。基于上述原因,他汀类药物在临床上用于AS患者PTA术后再狭窄的预防。但是PTA术后再狭窄与AS的产生机制不尽相同,血管平滑肌细胞(vascular smooth muscle cells,VSMCs)的增殖与迁移在再狭窄的进程中起了非常关键的作用。尽管部分体外实验观察到他汀类药物能够抑制VSMCs的增殖、迁移、凋亡,但是有临床试验研究发现他汀类药物抑制再狭窄的临床疗效欠佳。他汀类药物是否可以抑制再狭窄?特别是糖尿病下肢AS的PTA术后再狭窄?这是尚未明确的问题。临床获得血管标本具有局限性,使得我们需要动物体内实验提供更直观和确切的证据。目前很多研究采用单次球囊拉伤术建立再狭窄动物模型。这种模型的缺点在于:直接在正常的血管上而不是发生AS的血管上建立再狭窄模型,与人体血管内再狭窄发生的过程有较大差异。为了明确上述问题,我们的研究目的总结如下:首先,我们的实验先通过一次球囊拉伤术建立糖尿病兔髂动脉AS模型,然后在此基础上进行PTA手术,更好的模拟了糖尿病患者下肢血管硬化PTA术后再狭窄的过程,应用双次球囊拉伤术/扩张术为再狭窄的研究建立更为准确的动物模型。其次,通过观察PTA术后不同时间点的狭窄率、内膜增生情况、VSMCs在血管内的分布情况等以明确PTA术后再狭窄的进程和特点。第三,通过应用他汀类药物,观察其对血管平滑肌细胞的迁移、增殖以及再狭窄程度的影响以明确其是否可以抑制PTA术后再狭窄的发生。方法一、建模与分组选取雄性新西兰白兔(体重约1.7kg),适应性喂养1周后,给予高脂饲料喂养至实验终止。1、双次球囊拉伤术/扩张术再狭窄模型建立(1)糖尿病模型建立:耳缘静脉注射四氧嘧啶(剂量为80mg/kg),1周后测量血糖确认糖尿病模型的建立。(2)AS模型建立:糖尿病模型建立后1周对髂动脉行球囊拉伤术,拉伤术后4周通过超声检查确认髂动脉AS模型的建立。(3)再狭窄模型建立:在AS形成的血管部位行PTA手术以建立再狭窄模型。2、实验分组与处理将造模成功的再狭窄模型动物随机分为四组:7天再狭窄组,14天再狭窄组,28天再狭窄组,28天阿托伐他汀组,另设对照组。各组处理方法(1)7天再狭窄组:给予相同体积生理盐水灌胃,PTA术后第7天处死。(2)14天再狭窄组:给予相同体积生理盐水灌胃,PTA术后第14天处死(3)28天再狭窄组:给予相同体积生理盐水灌胃,PTA术后第28天处死。(4)28天阿托伐他汀组:给予常规剂量阿托伐他汀(2.5mg/kg/d)灌胃28天后处死。(5)对照组:进行和再狭窄模型组动物相同的外科手术,但不进行球囊扩张,相同体积生理盐水灌胃。二、标本留取及指标检测1、根据实验分组,分别在不同时间点(PTA术后第7天,14天,28天)处死动物,留取血液样本及髂动脉标本。2、血脂指标的检测:(1)血清总胆固醇(total cholesterol,TC);(2)甘油三酯(Triglyceride,TG);(3)低密度脂蛋白(low density lipoprotein cholesterin,LDL-C);(4)高密度脂蛋白(high density lipoprotein cholesterol,HDL-C)。3、血管形态学检测:髂动脉段固定在4%多聚甲醛中,脱水后包埋于石蜡中切片,进行以下检测(1)通过HE染色观察血管的一般形态(2)通过Masson染色观察血管平滑肌的形态及胶原蛋白(3)通过EVG染色观察弹力纤维形态。(4)应用Image-Pro Plus 6.0图像分析软件测量血管斑块位置的血管腔面积,内膜、中膜面积等指标以计算血管狭窄率和内膜/中膜比值。4、免疫组化分析:通过增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的免疫组化分析测定血管再狭窄位置内膜和中膜增殖细胞的数目和去分化情况。PCNA指数根据以下公式计算:PCNA阳性细胞数/总细胞计数×100%5、免疫荧光染色:通过αa-平滑肌肌动蛋白(alpha smooth muscle actin,α-SMA)的免疫荧光染色观察VSMCs从中膜向内膜迁移和分化的情况。二、统计学分析所有的数据应用SPSS20.0统计软件进行处理。组间的比较采用了单因素方差分析,所有的数据均以均数±标准差(x±s)的方式表示,P0.05被认为组间差异有统计学意义。结果一、动物成模及血糖情况45只新西兰白兔在糖尿病模型建立过程中,5只死亡,10只血糖水平未达标,AS及再狭窄模型建立过程中全部成功。各组实验动物的血糖在整个实验过程中始终保持在300mg/dl(16.7mmol/L)的水平之上。二、血脂指标检测结果.与28天再狭窄组以及对照组相比,28天阿托伐他汀组的TG、TC和LDL-C水平出现下降(P0.05)。而未用药的各再狭窄组(7天再狭窄组、14天再狭窄组、28天再狭窄组)与对照组之间TG、TC和LDL-C水平未见显著统计学差异(P0.05)。上述所有实验组之间的HDL-C水平均未见显著统计学差异(P0.05)。三、不同时间点再狭窄进展情况1、血管形态学检测:HE染色:与对照组相比,各再狭窄组管腔明显变窄,内膜增厚。Masson染色:增厚的内膜主要是平滑肌细胞及基质。EVG染色:增厚的内膜主要是弹力纤维,内弹力板出现断裂,中膜比例缩小以及中膜弹力纤维断裂。随着时间的推移,从PTA术后第7天到第28天,上述病理改变更加典型,程度逐渐加深。2、血管狭窄率和内膜/中膜比值计算显示:与对照组相比,各再狭窄组的狭窄率明显增加,差别具有统计学意义(P0.05)。各再狭窄组的狭窄率,从第7天到14天到28天逐渐增加,差别具有统计学意义(P0.05),而且从第7天到第14天增加速度更显著。各再狭窄组的内膜厚度以及内膜/中膜比的增加趋势与狭窄率一致,差别同样具有统计学意义(P0.05)。结果表明再狭窄的发生是逐渐进展,而且最初的进展速度更快。3、α-SMA的免疫荧光染色显示:对照组的动脉中膜中a-SMA强阳性表达而各再狭窄组的动脉中膜中α-SMA的阳性表达不连续。对照组动脉内膜中未见a-SMA阳性细胞,各再狭窄组的动脉内膜中可见a-SMA阳性细胞。从PTA术后第7天、第14天到第28天,α-SMA阳性细胞逐渐增多,VSMCs的增加趋势与狭窄率一致。4、PCNA免疫组化分析显示:与上述指标不同,PCNA指数在7天再狭窄组最大。14天再狭窄组与7天再狭窄组相比PCNA指数出现下降,14天再狭窄组与28天再狭窄组相比未见显著统计学差异(P0.05)。表明内膜细胞增殖在第7天得到最大程度的诱导,随后下降并稳定在一定水平。虽然继续保持增殖的趋势,但增殖的速度在第7天后有所下降并逐渐保持稳定。四、常规剂量阿托伐他汀对再狭窄的影响1、血管狭窄率和内膜/中膜比值计算显示:与28天再狭窄组相比,28天阿托伐他汀组的狭窄率和内膜/中膜比没有明显的下降,未见显著统计差异(P0.05)。常规剂量的阿托伐他汀治疗没有明显抑制再狭窄的发生。2、α-SMA的免疫荧光染色显示:与28天再狭窄组相比,28天阿托伐他汀组的内膜中仍然观察到一定数量的VSMCs,两组之间未见明显差别。3、PCNA免疫组化分析显示:与28天再狭窄组相比,28天阿托伐他汀组的PCNA指数未见显著统计学差异(P0.05)。上述结果显示:常规剂量的阿托伐他汀在实验动物体内没有明显抑制VSMCs向内膜的迁移和增殖,进一步验证了常规剂量阿托伐他汀的治疗没有明显抑制PTA术后再狭窄的发生。结论一、本研究通过球囊拉伤术先建立糖尿病动物AS模型,然后在AS基础上进行PTA手术,进而建立再狭窄动物模型,更好的模拟了糖尿病下肢AS患者PTA术后再狭窄的发生过程,通过双次球囊拉伤术/扩张术成功建立了理想的PTA术后再狭窄动物模型。二、在动物模型中,PTA术后再狭窄的发生发展呈现时间相关性。从第7天到第14天,再狭窄程度进展迅速;从第14天到第28天,再狭窄的进展速度有所减缓,但狭窄程度已经非常严重。内膜中VSMCs的增殖在第7天得到最大程度的诱导,随后增殖速度有所减缓,但第28天仍有一定程度的增殖。三、他汀类药物虽然能够防治AS,而且部分体外和体内实验观察到他汀类药物可以抑制VSMCs增殖、迁移,但是在我们的实验动物体内,常规剂量的阿托伐他汀对VSMCs向内膜的迁移和增殖以及再狭窄的发生发展无显著影响。目的:我们第一部分的实验研究结果表明:常规剂量的阿托伐他汀对VSMCs在动物模型体内的迁移、增殖以及再狭窄的发生发展均无显著影响。在第一部分实验的讨论中,我们对出现阴性结果的可能原因进行了总结。其中血药浓度不足可能是导致阴性结果的重要原因之一。血药浓度不足分为绝对不足和相对不足。血药浓度绝对不足是由于他汀类药物作用的剂量依赖性。剂量依赖性是指可根据药物剂量调整在一定范围内提高疗效。有研究结果表明,他汀类药物对VSMCs的增殖、迁移,抑制冠状动脉PCI术后再狭窄,抑制再狭窄的部位炎症反应,保护损伤内皮等作用均呈剂量依赖性。血药浓度相对不足与不同动物模型相关。很多再狭窄的研究采用单次球囊拉伤模型,但这种模型是损伤正常血管造成的再狭窄,程度较轻,不够贴近人体内再狭窄的过程。而我们的双次损伤模型是在已经发生AS的血管上造成的再狭窄,程度较重,更贴近人体内再狭窄的过程。血药浓度可能因为严重的狭窄而相对不足,因此需要进行强化剂量的阿托伐他汀治疗才有可能获得理想的效果。论文第二部分的研究目的可以概括为:通过应用不同剂量的阿托伐他汀,观察强化剂量阿托伐他汀是否会对血管平滑肌细胞的迁移、增殖以及再狭窄程度产生与常规剂量阿托伐他汀不一样的效果,阿托伐他汀对再狭窄的影响是否呈现剂量依赖性。方法一、建模与分组选取雄性新西兰白兔(体重约1.7kg),适应性喂养1周后,给予高脂饲料喂养至实验终止。1、再狭窄模型建立(1)糖尿病模型建立:耳缘静脉注射四氧嘧啶(剂量为80mg/kg),1周后测量血糖确认糖尿病模型的建立。(2)AS模型建立:糖尿病模型建立后1周对髂动脉行球囊拉伤术,拉伤术后4周通过超声检查确认髂动脉AS模型的建立。(3)再狭窄模型建立:在AS形成的血管部位行PTA手术以建立再狭窄模型。2、实验分组与处理将造模成功的再狭窄模型动物随机分为三组:(1)空白对照组(简称对照组)PTA手术当天开始给予与常规组相同体积的生理盐水灌胃至PTA术后第28天。(2)常规剂量阿托伐他汀组(简称常规组):PTA手术当天开始给予常规剂量阿托伐他汀(2.5mg/kg/d)灌胃至PTA术后第28天。(3)强化剂量阿托伐他汀组(简称强化组):PTA手术当天开始给予强化剂量阿托伐他汀(l0mg/kg/d)灌胃至PTA术后第28天。二、标本留取及指标检测1、PTA术后第28天处死动物,留取血液样本及髂动脉标本。2、血液指标检测:TC,TG,LDL-C和HDL-C。3、血管形态学检测:髂动脉段固定在4%多聚甲醛中,脱水后包埋于石蜡中切片,进行以下检测(1)通过Masson染色观察血管平滑肌的形态及胶原蛋白(2)通过EVG染色观察弹力纤维形态。(3)应用Image-Pro Plus 6.0图像分析软件测量血管斑块位置的血管腔面积,内膜、中膜面积等指标以计算血管狭窄率和内膜/中膜比值。4、PCNA免疫组化分析:测定血管再狭窄位置内膜和中膜增殖细胞的数目和去分化情况。PCNA指数根据以下公式计算:PCNA阳性细胞数/总细胞计数×100%5、a-SMA免疫组化染色:观察VSMCs从中膜向内膜迁移和分化的情况。三、统计学分析所有的数据应用SPSS 20.0统计软件进行处理。组间的比较采用了单因素方差分析,所有的数据均以均数土标准差(x±s)的方式表示,P0.05被认为组间差异有统计学意义。结果一、动物成模及血糖情况30只新西兰白兔在糖尿病模型建立过程中:3只死亡,9只血糖水平未达标,18只成为糖尿病兔。18只糖尿病兔建立再狭窄模型过程中有3只死亡,15只造模成功。各组实验动物的血糖在整个实验过程中始终保持在300mg/dl(16.7mmol/L)的水平之上。二、血脂检测结果与对照组相比,常规组和强化组的TG、TC和LDL-C水平出现下降(P0.05)。常规组与对照组之间的HDL-C水平未见显著统计学差异(P0.05),与前两组相比,强化组HDL-C水平升高,有统计学差异(P0.05)。三、不同剂量阿托伐他汀对再狭窄的影响1、血管狭窄率和内膜/中膜比值计算显示:与对照组相比,常规组和强化组的狭窄率和内膜/中膜比均无明显下降,未见显著统计差异(P0.05)。常规剂量和强化剂量阿托伐他汀的治疗都没能明显抑制再狭窄的发生。2、α-SMA的免疫组化染色显示:与对照组相比,常规组和强化组内膜中的α-SMA阳性细胞均未见明显减少的趋势。常规剂量和强化剂量阿托伐他汀的治疗均没能明显减少内膜中分化型VSMCs的数量,即没能抑制抑制VSMCs向内膜的迁移与增殖。3、PCNA免疫组化分析显示:与对照组相比,常规组和强化组的PCNA指数均无明显下降,未见显著统计学差异(P0.05)。常规剂量和强化剂量阿托伐他汀的治疗没能明显降低再狭窄部位增殖细胞的数量,即使强化治疗也没能抑制再狭窄部位细胞的增殖与去分化。结论强化剂量的阿托伐他汀与常规剂量的阿托伐他汀相似,对VSMCs在体内的迁移、增殖、去分化以及再狭窄发生发展的影响均无显著效果。在我们的糖尿病下肢动脉硬化PTA术后再狭窄的模型中,阿托伐他汀对再狭窄的影响未呈现剂量依赖性。
[Abstract]:Objective the prevalence of atherosclerosis (atherosclerosis, AS) is high in diabetic patients, and is prone to occur in aorta, coronary artery, peripheral artery and other parts. The coronary artery stent implantation is often used at present, but the peripheral AS of the extremities is often dominated by lower extremity artery disease, especially the subgenu artery and its branches. The treatment of the inferior genicular artery has been a difficult problem because of the low internal diameter and low pressure. The new balloon catheter has the advantages of low profile, low pressure, long length, and good compliance, which greatly increases the feasibility of the interventional treatment of the inferior genicular artery. Therefore, percutaneous transluminal angioplasty (percutaneous transluminal angioplasty, PTA) has become an important way to treat diabetic lower extremity artery, especially below knee AS. However, the incidence of restenosis after PTA is high, with an average of about 30%, which seriously affects the long-term effect of PTA. How to prevent and control restenosis after PTA is the key problem that affects the AS treatment of diabetic lower extremity. The statins are the hydroxyl methylamyl two acyl coenzyme A (hydroxy-methylglutaryl-CoA, HMG-CoA) reductase inhibitor. Experimental studies show that besides lowering cholesterol, they also have non lipid lowering anti AS effects: inhibiting inflammatory reaction of vascular endothelium, stabilizing atherosclerotic plaques, improving vascular endothelial function and inhibiting intimal hyperplasia. For the above reasons, statins are clinically used for the prevention of restenosis after PTA in AS patients. However, the mechanism of restenosis after PTA is different from that of AS. The proliferation and migration of vascular smooth muscle cells (VSMCs) play a key role in the process of restenosis. Although some statins in vitro inhibit the proliferation, migration and apoptosis of VSMCs, clinical trials have found that statins inhibit the restenosis. Does statins inhibit restenosis, especially in diabetic lower extremity AS after PTA restenosis? This is a problem that is not yet clear. The limitations of clinical acquisition of vascular specimens make it necessary for us to provide more intuitive and accurate evidence in animal experiments. In many studies, a single balloon injury is used to establish an animal model of restenosis. The disadvantage of this model is that the establishment of restenosis models directly on normal blood vessels rather than AS vessels is quite different from that in human vascular restenosis. In order to solve this problem, the aim of our study is summarized as follows: firstly, we experiment through the first balloon injury of rabbit iliac artery to establish diabetic AS model, and then based on the PTA operation, better simulate the process of restenosis in diabetic patients with lower extremity arteriosclerosis after PTA surgery, double balloon injury / surgery application for the expansion of restenosis on the establishment of animal model is more accurate. Next, we observed the progress and characteristics of restenosis after PTA after observing the stenosis rate, intimal hyperplasia and distribution of VSMCs in different time points after PTA. Third, we observed the effects of statins on the migration, proliferation and restenosis degree of vascular smooth muscle cells, so as to determine whether they could inhibit the occurrence of restenosis after PTA. Methods 1. Model and group selected male New Zealand white rabbits (weight about 1.7kg). After 1 weeks of adaptive feeding, high fat diet was given to the experiment to terminate. 1, double balloon injury / dilation and restenosis model was established. (1) diabetes model was established: four yuan (four 80mg/kg) was injected into the ear vein, and blood glucose was measured after 1 weeks to confirm the establishment of the diabetes model. (2) AS model: 1 weeks after Duiqia artery balloon injury was established by diabetes model, strain 4 weeks after surgery by ultrasound examination confirmed the iliac artery AS model. (3) restenosis model was established: a restenosis model was established by PTA operation at the vascular site of AS. 2. Animal models of restenosis were randomly divided into four groups: 7 day restenosis group, 14 day restenosis group, 28 day restenosis group, 28 days atorvastatin group, and another control group. The treatment of each group (1) 7 days restenosis group: the same volume of saline was given to the stomach and was executed seventh days after PTA. (2) 14 day restenosis group: the same volume of normal saline was given to the stomach, and fourteenth days after PTA was executed (3) for 28 days. The restenosis group was given the same volume of normal saline, and PTA was executed twenty-eighth days later. (4) the 28 day atorvastatin group: the routine dose of atorvastatin (2.5mg/kg/d) was given to the stomach for 28 days. (5) the control group: the same surgical operation was performed in the animals of the restenosis model group, but the balloon dilation was not carried out, and the same volume of physiological saline was given to the stomach. Two. Specimens were collected and tested for indicators 1. According to the experimental grouping, animals were killed at different time points (seventh days, 14 days, 28 days after PTA), and blood samples and iliac artery specimens were left. 2, detection of serum lipids: (1) serum total cholesterol (TC); (2) triglyceride (Triglyceride, TG); (3) low density lipoprotein (low density lipoprotein cholesterin; LDL-C); (4) high density lipoprotein (high, TG). 3, detection of vascular morphology: iliac artery segment was fixed in 4% paraformaldehyde, dehydrated after embedding in paraffin sections, the following test (1) the general morphological observation of blood vessels stained by HE (2) to observe the morphology and collagen vascular smooth muscle stained by Masson (3) to observe the elastic fiber morphology by EVG staining. (4) using Image-Pro Plus 6 image analysis software to measure vascular plaque area, intima and middle membrane area to calculate vessel stenosis rate and intima / middle membrane ratio. 4. Immunohistochemical analysis: the number and differentiation of intimal and mesangial proliferating cells were measured by immunohistochemical analysis of proliferating cell nuclear antigen (PCNA).
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R587.2

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