Rcn2对动脉损伤后内膜增生的影响及机制研究(硕士)(论文40000字)
摘 要
目的：动脉粥样硬化是冠心病，缺血性中风和外周动脉疾病的主要原因。动脉粥样硬化病变可能直接或间接造成动脉狭窄或闭塞，影响重要器官的血供，并引起相应的缺血症状。介入经皮血管成形术及支架置入术是治疗闭塞性动脉疾病的主要方法。随着介入器械的发展及技术的进步，介入腔内治疗闭塞性动脉疾病有了长远的进步，但术后再狭窄问题仍然没有彻底解决。新生内膜增生是术后再狭窄的主要病理过程，新生内膜病变主要来源于由中膜向内膜异常增殖和迁移的血管平滑肌细胞。最近的一些研究指出血管再狭窄可能受到饮食因素及遗传因素等的影响。前期研究中我们发现C3H和C57小鼠在Apoe敲除后动脉硬化易感性差异显著。通过数量性状基因座（QTL）的方法发现网钙结合蛋白2（Rcn2）可能是导致这一差异的关键基因。本研究将首先通过动物实验明确Rcn2在高脂饮食加剧动脉损伤后内膜增生过程中的作用，并进一步探讨敲除Rcn2能够抑制内膜增生。其次通过细胞实验明确沉默Rcn2能够抑制血管平滑肌细胞增殖迁移及由ox-LDL诱发的炎症反应。最后在下肢动脉硬化闭塞症患者血浆中检测Rcn2表达水平，分析Rcn2能够成为预测支架后再狭窄的生物学标志物。
研究方法：1、动物实验中选取8-12周龄、体重20-25g的C57BL/6 Apoe-/-小鼠（C57）、C57BL/6 Apoe-/-Rcn2-/-小鼠（Rcn2），建立颈动脉结扎损伤模型。一组小鼠维持普通饮食。另一组在颈动脉损伤前1周开始喂食高脂饮食并一直维持高脂饮食至实验结束。分别于术后1天，3天，以及1,2和4周获得颈动脉标本。病理学检查比较损伤段颈动脉管腔面积，内膜面积，内膜与中膜面积比值等形态学指标。免疫组化染色鉴定内膜增生病变的细胞成分。ElISA检测动物血清促炎因子分泌水平及血脂。2、细胞实验利用原代培养C57 Apoe-/-小鼠主动脉平滑肌细胞，选择用不同浓度的ox-LDL（50ug/ml,100ug/ml,150ug/ml,200ug/ml）刺激血管平滑肌细胞后，检测Rcn2在 mRNA水平和蛋白水平表达情况。利用siRNA转染血管平滑肌细胞，划痕试验检测血管平滑肌细胞的迁移能力。MTT检测血管平滑肌细胞的增殖能力。ELISA法检测细胞上清液中MCP-1及VCAM-1的含量。3、收集2012年1月至2014年12月共筛选了258例连续的股腘动脉CTO病变患者的临床随访资料。其中180例患者纳入本研究，检测了其中62例患者留存血浆中的Rcn2表达水平，并与15例健康对照比较分析。同时还分析了患者中性粒细胞与淋巴细胞比值（NLR）与再狭窄等的相关性。
结果：1、普通饮食组C57小鼠发生了轻度高脂血症，总胆固醇水平为308.5±60.3mg/dl，而高脂饮食组的总胆固醇水平为858.5±88.4mg/dl，为严重高脂血症（p<0.001）。两组HDL胆固醇水平都很低，甘油三酯水平相当（159.3±37.1 mg/dl和149.5±19.2mg/dl，p>0.05）。高脂饮食组在术后1周后出现内膜增生，高脂饮食组在术后2周出现内膜增生。随着时间的增加，两组内膜病灶均增大。术后4周时，高脂饮食组颈动脉平均内膜增生面积大约为普通饮食组的4倍（分别为112,987±11,395μm2和29,539±7,001μm2; n = 5,P< 0.001）。组织学上，高脂饮食术后1周后和普通饮食术后2周的内膜病变主要由泡沫细胞组成。在高脂饮食术后2周，内膜增生是典型的纤维斑块，其含有被纤维帽覆盖的许多泡沫细胞。在4周时，两个饮食组的内膜病变发展到更晚期的阶段，包含新生血管和动脉管腔明显变窄。Rcn2在普通饮食组的内膜病变中低表达，而在高脂饮食组的内膜病变中高表达。Rcn2在高脂饮食组血浆中浓度明显高于普通饮食组。（18.93±1.76ng/ml, 33.03±15.01ng/ml,n=5, p=0.029）。高脂饮食Rcn2小鼠发生了轻度高脂血症，总胆固醇水平为明显低于C57小鼠（264±56mg/dl和858.5±88.4mg/dl，n=5，p<0.001），HDL胆固醇和甘油三酯在两组中均无差异。Rcn2敲除明显抑制高脂饮食条件下，C57 Apoe-/-小鼠颈动脉损伤后内膜增生。主要抑制血管平滑肌细胞由中层向内膜下迁移，抑制血管壁细胞MCP-1的表达。术后4周，在Rcn2小鼠血浆中MCP-1（202.3±39.6ng/ml, 349.3±67.1ng/ml,n=5, p=0.009）及VCAM-1（110.6±20.7ng/ml, 240.3±50.1ng/ml,n=5, p=0.002）浓度明显低于C57小鼠。2、选择用不同浓度的ox-LDL（50ug/ml,100ug/ml,150ug/ml,200ug/ml）刺激血管平滑肌细胞4小时，Rcn2的mRNA表达和蛋白水平在150ug/ml时表达最高。用si Rcn2转染血管平滑肌细胞后用ox-LDL 刺激24小时，MTT测试显示，沉默Rcn2可抑制ox-LDL诱导的平滑肌细胞增殖（p<0.05）。用平板划痕实验检测发现沉默Rcn2后，穿过划痕的血管平滑肌细胞数量显著减少（p<0.05）。ELISA检测细胞上清液中MCP-1及VCAM-1浓度显示沉默Rcn2可抑制ox-LDL诱导的平滑肌细胞分泌MCP-1及VCAM-1（p<0.05）。3、共180例接受支架植入的股腘动脉CTO患者（男性，141例[78.3％];女性，39例[21.7％]）纳入本研究。根据支架再狭窄（ISR）的发生情况和ISR发生的时间将患者分为3组：非ISR组、早期ISR组（1年内）及晚期ISR组。非ISR组和早期ISR组的基线NLR，糖尿病，高脂血症，平均病变长度，流出道数量存在显著差异。在早期ISR组，基线NLR显着高于晚ISR组相应的值（P = 0.03），且两组在处理ISR过程中球囊预扩张使用率（P <0.01），远端栓塞发生率（P <0.01）存在差异。单变量分析显示，基线NLR≥3.62与早期ISR相关，基线NLR <3.62和NLR≥3.62组的早期ISR率分别为16％和32.72％（P <0.01，Table 2）。 多变量logistic分析提示，基线NLR≥3.62是股腘动脉CTO患者支架置入术后早期ISR的独立预测因子。共检测62例患者及15例健康对照的血浆Rcn2浓度。依据患者在随访1年时是否出现再狭窄分为未再狭窄组和再狭窄组。采用单因素方差分析组间差异，发现三组组Rcn2浓度具有统计学差异（F=29.59，P<0.001）。Rcn2浓度与NLR明显正相关,而且Rcn2浓度与低密度脂蛋白胆固醇及缺血程度均相关。受试者操作特征曲线分析（ROC）用Rcn2和早期ISR来定义Rcn2截止值。ROC曲线下面积为0.901，Rcn2浓度为80.43 ng/ml，敏感性和特异性分别为79.92％和83.72％（p<0.001）。
结论：1、高脂饮食明显加剧C57小鼠颈动脉损伤后内膜增生。增生的内膜病变主要包括血管平滑肌细胞、泡沫细胞以及新生血管等。Rcn2在高脂饮食诱导的C57小鼠动脉内膜病变中高表达。Rcn2敲除能够明显抑制高血脂诱导动脉损伤后的内膜增生。主要抑制血管平滑肌细胞的增殖迁移及炎症反应。2、ox-LDL能够诱导血管平滑肌细胞Rcn2表达增加。沉默Rcn2能够有效抑制ox-LDL诱导的血管平滑肌细胞增殖及迁移。沉默Rcn2能够有效抑制ox-LDL诱导的血管平滑肌细胞促炎因子分泌。3、基线NLR≥3.62是股腘动脉CTO患者支架置入术后早期ISR的独立预测因子。基线NLR与 ISR再次介入的难易程度和远端栓塞发生有关。Rcn2在股腘动脉CTO患者中表达明显升高。Rcn2 与NLR呈正相关，并与患者下肢缺血程度及低密度脂蛋白胆固醇水平相关。Rcn2有望成为下肢动脉硬化闭塞症介入术后再狭窄的生物学标志物。
关键词：网钙结合蛋白2；血管平滑肌细胞；新生内膜病变；再狭窄；下肢动脉硬化闭塞症
Abstract
Objective: atherosclerosis is the main cause of coronary heart disease, ischemic stroke and peripheral artery disease. Atherosclerotic lesions may directly or indirectly cause arterial stenosis or occlusion, affect the blood supply of important organs, and cause corresponding ischemic symptoms. Percutaneous transluminal angioplasty and stent placement are the main methods for the treatment of occlusive arterial diseases. With the development of interventional devices and the progress of technology, endovascular treatment of occlusive arterial disease has made long-term progress. However, the problem of restenosis has not yet been completely solved. Neointimal hyperplasia is the main pathological process of restenosis after operation. Neointimal lesions are mainly derived from vascular smooth muscle cells, which proliferate and migrate from the medial membrane to the intima. Recent studies have suggested that vascular restenosis may be affected by dietary factors and genetic factors. In previous studies, we found significant differences in the susceptibility to arteriosclerosis in C3H and C57 mice after Apoe knockout. Through quantitative trait loci (QTL) method, it is found that the net calcium binding protein 2 (Rcn2) may be the key gene that causes this difference. In this study, we first identified the role of Rcn2 in the process of intimal hyperplasia after high-fat diet aggravating arterial injury, and further explored that knockout of Rcn2 could inhibit intimal hyperplasia through animal experiments. Secondly, it is clear that Rcn2 can inhibit the proliferation and migration of vascular smooth muscle cells and the inflammatory response induced by ox-LDL through cell test. Finally, the level of Rcn2 expression was detected in the plasma of the patients with arteriosclerosis obliterans of the lower extremities, and the analysis of Rcn2 could be a biological marker for predicting the restenosis of the stent.
Methods: 1. In animal experiments, 8-12 week old, 20-25g weight C57BL/6 Apoe-/- mice (C57) and C57BL/6 Apoe-/-Rcn2-/- mice (Rcn2) were selected to establish carotid artery ligation injury model. One group of mice maintained an ordinary diet. The other group fed a high - fat diet 1 weeks before the carotid artery injury and maintained a high - fat diet until the end of the experiment. Carotid artery specimens were obtained at 1 days, 3 days after operation, and 1,2 and 4 weeks respectively. Pathological examination compared the area of the carotid artery, the area of the intima, the ratio of the intima and the area of the middle membrane. Immunohistochemical staining was used to identify the cell components of endometrial hyperplasia. The serum levels of pro-inflammatory factors and blood lipids were detected by ElISA. 2, in cell experiments, primary cultured C57 Apoe-/- mouse aortic smooth muscle cells were selected and stimulated with different concentrations of ox-LDL (50ug/ml, 100ug/ml, 150ug/ml, 200ug/ml) to stimulate vascular smooth muscle cells, and Rcn2 expression at mRNA level and protein level was detected. Vascular smooth muscle cells were transfected by siRNA, and the migration ability of vascular smooth muscle cells was detected by scratch test. MTT was used to detect the proliferation of vascular smooth muscle cells. The content of MCP-1 and VCAM-1 in cell supernatant was detected by ELISA. 3, from January 2012 to December 2014 were selected from 258 consecutive cases of femoral popliteal artery lesions in patients with clinical follow-up data CTO. 180 of the patients were included in the study, and the level of Rcn2 expression in the remaining plasma of 62 patients was detected and compared with 15 healthy controls. The correlation between the ratio of neutrophil to lymphocyte (NLR) and restenosis was also analyzed.
Results: 1. In the general diet group, C57 mice developed mild hyperlipidemia, the total cholesterol level was 308.5 + 60.3mg/dl, while the total cholesterol level in the high-fat diet group was 858.5 + 88.4mg/dl, which was severe hyperlipidemia (p<0.001). The levels of HDL cholesterol in the two groups were very low, and the levels of triglycerides were equal (159.3 + 37.1 mg/dl and 149.5 + 19.2mg/dl, p>0.05). The hyperlipidemia group appeared intimal hyperplasia 1 weeks after the operation, and the high fat diet group appeared intimal hyperplasia at the 2 week after the operation. With the increase of time, the two groups of endometrium lesions were all increased. At the 4 week after operation, the average intimal hyperplasia area of the carotid artery in the high-fat diet group was about 4 times higher than that in the ordinary diet group (112987, 11395, M2 and 29539 + 7001 m2, respectively); n = 5, P< 0.001). Histologically, 1 weeks after the high fat diet and 2 weeks after the common diet, the endometrium was mainly composed of foam cells. At 2 weeks after the high fat diet, endometrial hyperplasia is a typical fibrous plaque, which contains many foam cells covered by a fibrous cap. At 4 weeks, the endarterosis of the two diet groups developed to a more advanced stage, including the narrowing of the neovascularization and the arterial lumen. The expression of Rcn2 is low in the endocardial lesions of the common diet group, and is highly expressed in the endintimal lesions of the high fat diet group. The plasma concentration of Rcn2 in the high fat diet group was significantly higher than that in the normal diet group. (18.93 + 1.76ng/ml, 33.03 + 15.01ng/ml, n=5, p=0.029). The high-fat diet Rcn2 mice had mild hyperlipidemia, the total cholesterol level was significantly lower than that of C57 mice (264 + 56mg/dl and 858.5 + 88.4mg/dl, n=5, p<0.001), HDL cholesterol and triglyceride were not different in the two groups. Rcn2 knockout obviously inhibited the intimal hyperplasia of the carotid artery in C57 Apoe-/- mice under the high fat diet. It mainly inhibits the migration of vascular smooth muscle cells from the middle layer to the intima, and inhibits the expression of MCP-1 in the vascular wall cells. At 4 weeks after operation, the concentrations of MCP-1 (202.3 + 39.6ng/ml, 349.3 + 67.1ng/ml, n=5, p=0.009) and VCAM-1 (110.6 + 20.7ng/ml, 240.3 + 50.1ng/ml, n=5, p=0.002) in plasma of Rcn2 mice were significantly lower than those of the mice. 2, we choose different concentrations of ox-LDL (50ug/ml, 100ug/ml, 150ug/ml, 200ug/ml) to stimulate vascular smooth muscle cells for 4 hours, the mRNA expression and protein level of Rcn2 are highest at 150ug/ml. After transfection of Si Rcn2 into vascular smooth muscle cells, ox-LDL was stimulated for 24 hours. MTT test showed that Rcn2 could inhibit ox-LDL induced smooth muscle cell proliferation (p<0.05). The number of vascular smooth muscle cells passing through scratches was significantly reduced (p<0.05) after Rcn2 was detected by a flat scratch test. The concentration of MCP-1 and VCAM-1 in the supernatant of ELISA showed that silent Rcn2 inhibited the secretion of MCP-1 and VCAM-1 (p<0.05) by ox-LDL induced smooth muscle cells. The pressure of 3, a total of 180 patients who underwent stent implantation of the femoral popliteal artery in patients with CTO (male 141 cases, female 39 cases, [78.3%]; [21.7%]) were included in this study. According to the occurrence of ISR and the time of ISR, the patients were divided into 3 groups: non ISR group, early ISR group (1 years) and late ISR group. There were significant differences in baseline NLR, diabetes, hyperlipidemia, mean lesion length, and the number of outflow channels in the non ISR group and the early ISR group. In the early ISR group, baseline NLR was significantly higher than that in the late ISR group (P = 0.03), and there was a difference in the rate of balloon dilation (P <0.01) and the incidence of distal embolization (P <0.01) between the two groups during the treatment of ISR. Univariate analysis showed that baseline NLR is more than 3.62 and the early ISR, NLR <3.62 and NLR = 3.62 of baseline group of early ISR rates were 16% and 32.72% (P <0.01, Table 2). Multivariate logistic analysis showed that the baseline NLR = 3.62 is a independent predictor of early ISR popliteal artery stent implantation in patients with CTO. The plasma concentrations of Rcn2 were measured in 62 patients and 15 healthy controls. Restenosis was divided into no restenosis group and restenosis group based on the 1 years of follow-up. It was found that the concentration of Rcn2 in the three groups was statistically different (F=29.59, P<0.001) by single factor analysis of variance. The concentration of Rcn2 was positively correlated with NLR, and the concentration of Rcn2 was associated with low density lipoprotein cholesterol and the degree of ischemia. The operator's operation characteristic curve analysis (ROC) uses Rcn2 and early ISR to define the Rcn2 cut-off value. The area under the ROC curve was 0.901, the concentration of Rcn2 was 80.43 ng/ml, and the sensitivity and specificity were 79.92% and 83.72% respectively (p<0.001).
Conclusion: 1. High fat diet obviously aggravate the intimal hyperplasia of C57 mice after carotid artery injury. Hyperplastic endometrial lesions mainly include vascular smooth muscle cells, foam cells and neovascularization. Rcn2 is highly expressed in the intima lesions of C57 mice induced by high fat diet. Rcn2 knockout can obviously inhibit the hyperplasia of intima after hyperlipidemia induced arterial injury. It mainly inhibits the proliferation, migration and inflammation of vascular smooth muscle cells. 2, ox-LDL can induce the increase of Rcn2 expression in vascular smooth muscle cells. Rcn2 silencing can effectively inhibit the proliferation and migration of vascular smooth muscle cells induced by ox-LDL. Rcn2 silencing can effectively inhibit the secretion of proinflammatory cytokines in vascular smooth muscle cells induced by ox-LDL. 3, baseline NLR more than 3.62 independent predictors of early ISR femoral popliteal artery stent implantation in patients with CTO. The degree of difficulty in the re intervention of baseline NLR and ISR is associated with the occurrence of distal embolism. The expression of Rcn2 in femoral popliteal artery in patients with CTO increased significantly. Rcn2 has a positive correlation with NLR and is related to the degree of lower limb ischemia and low density lipoprotein cholesterol levels in the patients. Rcn2 is expected to be a biological marker for restenosis after interventional therapy for arteriosclerosis obliterans of the lower extremities.
Keywords: Rcn2; Vascular smooth muscle cells; Neointimal lesion; Restenosis. Arteriosclerosis obliterans
英文缩略语
英文缩写 英文全称 中文全称
AS Arteriosclerosis 动脉粥样硬化
PTA Percutaneous transluminal angioplasty 血管成形术
DCB Drug-coated balloon 药物涂层球囊
DES Drug-eluting stent 药物洗脱支架
ISR In-stent restenosis 支架再狭窄
VSMC Vascular smooth muscle cells 血管平滑肌细胞
Apoe Apolipoprotein E 载脂蛋白E
QTL Quantitative trait locus 数量性状基因座
Rcn2 Reticulocalbin-2 网钙结合蛋白-2
Ox-LDL Oxidized low density lipoprotein 氧化低密度脂蛋白
LDL Low density lipoprotein 低密度脂蛋白
VCAM-1 Vascular cell adhesion molecule -1 血管细胞粘附因子-1
MCP-1 Monocyte chemoattractant protein-1 单核细胞趋化蛋白-1
DMEM dulbecco’s modified eagle’s medium 达尔伯克氏改良伊格尔氏培养基
FBS Fetal bovine serum 胎牛血清
BSA bovine serum albumin 牛血清白蛋白
PBS phosphate buffer solution 磷酸盐缓冲液
α-SMA α-Smooth muscle actin α-平滑肌肌动蛋白
vWF von Willebrand Factor 血管性血友病因子
MMP matrix metalloprotein 基质金属蛋白酶
FITC fluorescein isothiocyanate 异硫氰酸荧光素
IL interleukin 白细胞介素
LPS Lipopolysaccharide 脂多糖
PAGE polyacryrlamide gel electropho- resis 聚丙烯酰胺凝胶电泳
CM chylomicra 乳糜颗粒
VLD L very low 极低密度脂蛋白
HDL High density lipoprotein HDL
TNF-β Tumor Necrosis Factor-β 肿瘤坏死因子-β
PDGF-BB Platelet-Derived Growth Factor BB 血小板源性生长因子
ASO Atherosclerotic occlusive disease 下肢动脉闭塞硬化症
NLR Neutrophil to lymphocyte ratio 中性粒细胞与淋巴细胞比值
CTO Chronic Total Occlusion 慢性完全闭塞
ABI ankle brachial index 踝肱指数
FPG Fasting plasma glucose 空腹血糖
TC Total cholesterol 总胆固醇
TG Triglyceride 甘油三酯
LDL-C Low density lipoprotein 低密度脂蛋白-胆固醇
HDL-C High density lipoprotein
高密度脂蛋白-胆固醇
CRP C-reactive protein C反应蛋白
ECM extracellular matrix 细胞外基质
VEGF Vascular endothelial growth factor 血管内皮生长因子
TGF Transfer growth factor 转移生长因子
PDGF platelet derived growth factor 血小板衍生因子
EPCs Endothelial progenitor cells 内皮祖细胞
SMPCs Vascular smooth muscle progenitor cells 血管平滑肌祖细胞
G-CSF Granulocyte colony stimulating factor 粒细胞集落刺激因子
ET-1 endothelin-1 内皮素 -1
目录
目 录 13
第一部分：Rcn2对动脉损伤后内膜增生的影响 13
1 前言……………………………………………… …… … …………1 13
2 材料与方法……………………………………………… …… ……4 13
3 结果………………………………………………… …… ……21 14
4 讨论………………………………… …… …… …… …… ……… 32 14
5 结论……………………………………………………… …… …… 33 14
第二部分Rcn2对血管平滑肌细胞增殖、迁移功能的影响 14
1 前言…………………………………………………………………45 14
2 材料与方法……………………………………… …… …… ……48 14
3 结果………………………………………………… …… ……21 14
4 讨论………………………………… …… …… …… …… ……… 32 14
5 结论……………………………………………………… …… …… 33 15
第三部分Rcn2与股腘动脉慢性完全闭塞支架再狭窄的相关性研究 15
1 前言…………………………………………………………………45 15
2 材料与方法……………………………………… …… …… ……48 15
3 结果………………………………………………… …… ……21 15
4 讨论………………………………… …… …… …… …… ……… 32 15
5 结论……………………………………………………… …… …… 33 15
6 孔细胞培养板购自Corning 公司； 19
2.1.2研究对象 21
2.2.3小鼠颈动脉结扎损伤模型的建立 22
2.2.4小鼠颈总动脉取材 22
2.2.5 HE染色 22
2.2.6病理形态学分析 23
2.2.7 免疫组化染色 23
2.2.8 ElISA检测血清促炎因子浓度及血脂 24
2.2.9统计学分析 25
3 结果 26
3.1高脂饮食加剧C57 Apoe-/-小鼠血脂升高 26
3.2高血脂促进C57 Apoe-/-小鼠颈动脉损伤后内膜增生 26
3.3 Rcn2在高脂饮食条件下增生内膜中表达增高 32
3.4 Rcn2在高脂饮食条件下血浆中的浓度升高 32
3.5 Rcn2敲除抑制高脂饮食诱发的高血脂 32
3.6 Rcn2敲除抑制C57 Apoe-/-小鼠颈动脉损伤后内膜增生 33
3.7 Rcn2敲除抑制血清MCP-1、VCAM-1水平升高 34
4 讨论 34
5 结论 59
第三部分：Rcn2与股腘动脉慢性完全闭塞支架再狭窄的相关性研究 60
1 前言 60
2 材料和方法 62
3 结果 66
4 讨论 74
5 结论 77
本研究创新性的自我评价 81
参考文献 82
综 述 89
1.炎症反应与下肢动脉介入术后再狭窄关系 89
2.下肢动脉介入术后再狭窄的细胞机制 90
2.1内皮细胞在再狭窄中的作用 90
2.2血管平滑肌细胞在再狭窄中的作用 92
2.3单核 /巨噬细胞在再狭窄形成中的作用 92
2.4骨髓干细胞在血管修复及再狭窄过程中的作用 93
2.5血管局部炎症与干细胞动员 95
3.炎症因子与下肢动脉介入术后再狭窄的相关性研究 95
4.干预炎症在治疗再狭窄中的作用 97
4.1抗血小板药物 97
4.2皮质类固醇 97
4.3他汀类药物 98
4.4药物涂层球囊及药物洗脱支架 98
参考文献 99
致谢 106 |
