Nampt在动脉粥样硬化发生发展中的作用及机制
发布时间：2018-07-25 17:22
【摘要】：动脉粥样硬化(Atherosclerosis,AS)是脑梗,冠心病,外周血管病等多种心血管疾病的主要诱因。随着生活水平的不断提高,AS的发病率也在迅猛增加。AS病变基础是脂质代谢障碍,斑块是由病变的血管细胞和凋亡细胞累积形成,斑块一旦破裂会造成血管栓塞进而引发脑卒中,危及人的生命健康。AS的发病机制尚未明确,因此临床上并没有特异性好且有效的动脉粥样硬化治疗药物。烟酰胺磷酸核糖转移酶(nicotinamide phosphoribosyltransferase,NAMPT)是一种由脂肪细胞分泌的脂肪因子,在细胞内和细胞外都具有活性。Nampt是烟酰胺腺嘌呤二核苷酸(NAD)合成的限速酶,可将底物烟酰胺(NAM)转化成产物烟酰胺单核苷酸(NMN),NMN在催化条件下,生成NAD。NAD称作为辅酶I,是参与线粒体呼吸链氧化还原反应所必需的辅酶。NAD的生物学功能极其重要,可能能够直接决定着细胞的命运。本课题组前期基本证实了Nampt在脑缺血后发挥神经保护作用。动脉粥样硬化被认为是脑卒中的发病因素,但Nampt在AS中确切的作用尚未被证实。因此,我们采用体外补充Nampt酶产物NMN以及Nampt转基因小鼠与Apo E-/-杂交的Apo E-/-;NamptTg鼠来研究Nampt对动脉粥样硬化发生发展的影响,初步确定了Nampt可促进动脉粥样硬化的发生发展。本课题进一步研究了抑制TNF-α对动脉粥样硬化发生发展的影响,初步探讨了Nampt发挥作用的机制,从而为动脉粥样硬化的治疗提供了新靶点。实验方法:1.将8只8周龄左右的Apo E-/-雄性小鼠分为两组,对照组和实验组各4只,同时西方高脂饲料喂养,两个月后,实验组NMN饮水给药,给药剂量为300 mg/kg/天,对照组则继续普通饮水。观察NMN给药即体外补充Nampt酶产物NMN对AS小鼠主动脉上脂质沉积的影响。此外,采用油红O染色,HE染色,观察体外补充Nampt酶产物NMN,对AS小鼠斑块厚度及尺寸的影响,F4/80免疫组化染色观察主动脉根部斑块的巨噬细胞浸润情况,TNF-α免疫组化染色确定体外补充NMN对主动脉根部斑块的炎症因子表达情况的影响。2.将Nampt转基因鼠与世界上公认的动脉粥样硬化模型鼠Apo E-/-进行杂交,获得Apo E-/-;NamptTg鼠,作为实验组,以Apo E-/-小鼠作为对照组,西方高脂饲料喂养4个月。对小鼠进行主动脉油红O染色,观察Nampt转基因对AS斑块的影响。采用油红O染色,HE染色,观察Nampt转基因对动脉粥样硬化斑块的厚度和尺寸的影响;F4/80免疫组化检测巨噬细胞浸润情况,观察Nampt转基因对动脉粥样硬化斑块巨噬细胞浸润的影响;Masson染色,天狼星红染色,观察Nampt转基因对AS小鼠斑块纤维帽和胶原情况的影响;TNF-α,IL-1β免疫组化染色,观察Nampt转基因对AS小鼠斑块炎症因子表达水平的影响;ICAM-1,VCAM-1免疫组化染色,观察Nampt转基因对AS小鼠斑块中粘附及趋化因子的表达水平的影响;TUNEL实验,观察Nampt转基因对AS斑块中细胞凋亡数目的影响;此外,还进行了CD47免疫组化染色,初步探索了参与AS发生发展的可能机制。3.将6只Apo E-/-8周龄左右雄性小鼠,分为两组,对照组和实验组各3只,6只Apo E-/-;NamptTg 8周龄左右雄性鼠,分为两组,对照组和实验组各3只。同时西方高脂饮食4个月。实验组采用腹腔注射TNF-α抗体,剂量为5 mg/kg/天,给药一个月,随后进行了主动脉根部切片油红O染色,F4/80免疫组化染色,CD47免疫组化染色,α-SMA免疫组化染色,TUNEL细胞凋亡检测,进一步探索了Nampt对动脉粥样硬化发生发展影响的作用机制。实验结果:1.NMN饮水给药组与对照组(正常饮水)相比NMN给药组主动脉上脂质沉积较多,斑块面积和厚度较大,巨噬细胞浸润程度较高,炎症(TNF-α)表达水平升高。NMN促进了AS斑块增多和炎症浸润。2.Apo E-/-和Apo E-/-;NamptTg的主动脉油红O染色结果可以看出,相较于Apo E-/-组,Apo E-/-;NamptTg组的主动脉脂质沉积较多。Nampt转基因会使得斑块面积和厚度变大,巨噬细胞(F4/80)浸润程度加重,坏死中心扩大,III型胶原增多,并且炎症因子(TNF-α,IL-1β)表达水平升高,粘附和趋化因子(ICAM-1,VCAM-1)表达水平升高,斑块中细胞凋亡数量增多。3.Nampt转基因小鼠AS进程加快的同时也伴随着CD47表达水平的上调。抑制TNF-α不会对Apo E-/-和Apo E-/-;NamptTg AS小鼠的血脂和斑块面积产生影响,但F4/80和α-SMA的表达水平下降,即斑块炎症和纤维化得到了改善,此外,抑制TNF-α,CD47的表达水平也随之下调,斑块细胞凋亡也得到了抑制。实验结论:1.通过体外补充Nampt的酶产物NMN,可促进AS的发生发展,体现为斑块面积的显著增加和炎症的增强。2.和野生型小鼠相比较,在Nampt转基因小鼠上使用高脂饮食诱导的AS发生发展进程明显加快,主要体现为斑块面积的增加;此外,Nampt转基因小鼠的斑块组织中的炎症细胞浸润、炎症因子的表达均增加,提示斑块的稳定性明显变差。3.使用中和抗体注射抑制TNF-α,可以延缓Nampt转基因鼠的AS进程;Nampt转基因鼠AS斑块中CD47表达水平升高,而当TNF-α被中和抗体抑制后,CD47的表达水平也随之降低。提示TNF-α-CD47通路可能参与了Nampt促进AS发生发展的作用。
[Abstract]:Atherosclerosis (AS) is the main cause of a variety of cardiovascular diseases such as cerebral infarction, coronary heart disease and peripheral vascular disease. With the continuous improvement of living standards, the incidence of AS is also increasing rapidly on the basis of.AS lesion, which is caused by the accumulation of pathological blood tube cells and apoptotic cells, and once the plaque breaks down. Nicotinamide phosphoribosyltransferase (NAMPT) is a kind of fat factor secreted by fat cells, which is a kind of fat factor secreted by adipocytes. Both intracellular and extracellular active.Nampt are the speed limiting enzymes in the synthesis of nicotinamide adenine dinucleotide (NAD), which can convert nicotinamide (NAM) into product nicotinamide single nucleotide (NMN). NMN is used as a coenzyme I in the catalytic condition, and is a necessary biological.NAD in respiratory chain redox reaction. The function is extremely important and may directly determine the fate of the cells. This group has confirmed that Nampt plays neuroprotective effect after cerebral ischemia. Atherosclerosis is considered to be a factor in stroke, but the exact role of Nampt in AS has not been confirmed. Therefore, we use the Nampt enzyme product NMN and Nam in vitro. PT transgenic mice and Apo E-/- hybrid Apo E-/-; NamptTg mice to study the effect of Nampt on the development of atherosclerosis and preliminarily determine that Nampt can promote the development of atherosclerosis. This subject further studies the effect of the inhibition of TNF- alpha on the development of atherosclerosis and preliminarily discusses the mechanism of the action of Nampt. In order to provide new targets for the treatment of atherosclerosis, 1. Apo E-/- male mice of 8 8 weeks old were divided into two groups, the control group and the experimental group were 4, while the western high fat feed was fed, and after two months, the experimental group NMN drinking water was given, the dosage was 300 mg/kg/ days, and the control group continued the common drinking water. Observe NMN to the control group. The effect of Nampt enzyme product NMN on the lipid deposition in the aorta of AS mice was supplemented in vitro. In addition, the effects of Nampt enzyme product NMN on the plaque thickness and size of AS mice were observed by oil red O staining and HE staining. The infiltration of macrophages in the aortic root plaque was observed by F4/80 immunohistochemical staining. TNF- alpha immunohistochemical staining was true. The effect of NMN on the expression of inflammatory factors in the aortic root plaque in vitro.2. hybridized the Nampt transgenic mice with the recognized atherosclerotic model rat Apo E-/- in the world, and obtained the Apo E-/-; NamptTg mice, as the experimental group, the Apo E-/- mice were used as the control group, and the western high fat feed was fed for 4 months. Pulse oil red O staining was used to observe the effect of Nampt transgenic on AS plaque. The influence of Nampt transgenic on the thickness and size of atherosclerotic plaques was observed by oil red O staining and HE staining. The influence of macrophage infiltration by F4/80 immunohistochemistry and the effect of Nampt transgenic on the infiltration of atherosclerotic plaque macrophages was observed; Masson staining, The effect of Nampt transgenic on the condition of fibrous cap and collagen in AS mice was observed by Sirius red. TNF- alpha and IL-1 beta immunohistochemical staining were used to observe the effect of Nampt transgenic on the expression level of inflammatory factors in the plaque of AS mice. ICAM-1, VCAM-1 immunhistochemical staining was used to observe the expression of Nampt transgene on the adhesion and chemokines in the plaque of AS mice. The effect of leveling; TUNEL experiment was carried out to observe the effect of Nampt transgenic on the number of apoptotic cells in AS patches; in addition, CD47 immunohistochemical staining was also carried out to explore a possible mechanism to participate in the development of AS by.3., which divided 6 male and right male mice of Apo E-/-8 weeks into two groups, 3 in each group and in the experimental group, 6 Apo E-/-; NamptTg 8 weeks of age. Male rats were divided into two groups, 3 in the control group and in the experimental group. At the same time, the western high fat diet was given for 4 months. The experimental group was injected with TNF- alpha antibody in the abdominal cavity, the dose was 5 mg/kg/ days, and the drug was given for one month. Then the aorta root section was stained with oil red O, F4/80 immunohistochemical staining, CD47 immunohistochemical staining, alpha -SMA immunohistochemical staining, TUNEL cell withering. The mechanism of the effect of Nampt on the occurrence and development of atherosclerosis was further explored. The results of experimental results: compared with the control group (normal drinking water), the 1.NMN drinking water supply group had more lipid deposition in the NMN administration group, the area and thickness of the plaque was higher, the degree of macrophage infiltration was higher, and the expression level of inflammation (TNF- alpha) increased by.NMN. The increase of AS plaques and inflammatory infiltration of.2.Apo E-/- and Apo E-/-; the result of the O staining of the aorta of NamptTg can be seen, compared to the Apo E-/- group, Apo E-/-, the lipid deposition in the aorta of the NamptTg group will increase the area and thickness of the plaque, the infiltration degree of the macrophage is aggravated, the necrotic center enlarges, and the collagen type is enlarged. Increased expression of inflammatory factors (TNF- a, IL-1 beta), increased expression of adhesion and chemokines (ICAM-1, VCAM-1), increased number of apoptotic cells in the plaque, increased the AS process in.3.Nampt transgenic mice and up regulation of CD47 expression level. The inhibition of TNF- a not to Apo E-/- and Apo E-/-. Influence of patch area, but the expression level of F4/80 and alpha -SMA decreased, that is, plaque inflammation and fibrosis improved. In addition, inhibition of TNF- alpha, CD47 expression level also decreased, and plaque cell apoptosis was inhibited. Experimental conclusion: 1. supplementation of Nampt enzyme product NMN in vitro can promote the occurrence and development of AS, which is reflected as plaque. Compared with the enhanced.2. and the wild type of inflammation, the development process of AS induced by high fat diet in Nampt transgenic mice was obviously accelerated, which was mainly reflected in the increase of patch area. In addition, the inflammatory cell infiltration in the plaque tissues of the Nampt transgenic mice increased, and the expression of inflammatory factors increased. The stability of the plaque was significantly worse.3. using neutralization antibody injection to inhibit TNF- alpha, which could delay the AS process in Nampt transgenic mice; the expression level of CD47 in the AS plaques of Nampt transgenic mice increased, and the expression level of CD47 decreased when TNF- a was neutralized by neutralizing antibodies. It suggested that TNF- alpha -CD47 pathway may be involved in the development of Nampt. The role.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R543.5
本文编号：2144512
[Abstract]:Atherosclerosis (AS) is the main cause of a variety of cardiovascular diseases such as cerebral infarction, coronary heart disease and peripheral vascular disease. With the continuous improvement of living standards, the incidence of AS is also increasing rapidly on the basis of.AS lesion, which is caused by the accumulation of pathological blood tube cells and apoptotic cells, and once the plaque breaks down. Nicotinamide phosphoribosyltransferase (NAMPT) is a kind of fat factor secreted by fat cells, which is a kind of fat factor secreted by adipocytes. Both intracellular and extracellular active.Nampt are the speed limiting enzymes in the synthesis of nicotinamide adenine dinucleotide (NAD), which can convert nicotinamide (NAM) into product nicotinamide single nucleotide (NMN). NMN is used as a coenzyme I in the catalytic condition, and is a necessary biological.NAD in respiratory chain redox reaction. The function is extremely important and may directly determine the fate of the cells. This group has confirmed that Nampt plays neuroprotective effect after cerebral ischemia. Atherosclerosis is considered to be a factor in stroke, but the exact role of Nampt in AS has not been confirmed. Therefore, we use the Nampt enzyme product NMN and Nam in vitro. PT transgenic mice and Apo E-/- hybrid Apo E-/-; NamptTg mice to study the effect of Nampt on the development of atherosclerosis and preliminarily determine that Nampt can promote the development of atherosclerosis. This subject further studies the effect of the inhibition of TNF- alpha on the development of atherosclerosis and preliminarily discusses the mechanism of the action of Nampt. In order to provide new targets for the treatment of atherosclerosis, 1. Apo E-/- male mice of 8 8 weeks old were divided into two groups, the control group and the experimental group were 4, while the western high fat feed was fed, and after two months, the experimental group NMN drinking water was given, the dosage was 300 mg/kg/ days, and the control group continued the common drinking water. Observe NMN to the control group. The effect of Nampt enzyme product NMN on the lipid deposition in the aorta of AS mice was supplemented in vitro. In addition, the effects of Nampt enzyme product NMN on the plaque thickness and size of AS mice were observed by oil red O staining and HE staining. The infiltration of macrophages in the aortic root plaque was observed by F4/80 immunohistochemical staining. TNF- alpha immunohistochemical staining was true. The effect of NMN on the expression of inflammatory factors in the aortic root plaque in vitro.2. hybridized the Nampt transgenic mice with the recognized atherosclerotic model rat Apo E-/- in the world, and obtained the Apo E-/-; NamptTg mice, as the experimental group, the Apo E-/- mice were used as the control group, and the western high fat feed was fed for 4 months. Pulse oil red O staining was used to observe the effect of Nampt transgenic on AS plaque. The influence of Nampt transgenic on the thickness and size of atherosclerotic plaques was observed by oil red O staining and HE staining. The influence of macrophage infiltration by F4/80 immunohistochemistry and the effect of Nampt transgenic on the infiltration of atherosclerotic plaque macrophages was observed; Masson staining, The effect of Nampt transgenic on the condition of fibrous cap and collagen in AS mice was observed by Sirius red. TNF- alpha and IL-1 beta immunohistochemical staining were used to observe the effect of Nampt transgenic on the expression level of inflammatory factors in the plaque of AS mice. ICAM-1, VCAM-1 immunhistochemical staining was used to observe the expression of Nampt transgene on the adhesion and chemokines in the plaque of AS mice. The effect of leveling; TUNEL experiment was carried out to observe the effect of Nampt transgenic on the number of apoptotic cells in AS patches; in addition, CD47 immunohistochemical staining was also carried out to explore a possible mechanism to participate in the development of AS by.3., which divided 6 male and right male mice of Apo E-/-8 weeks into two groups, 3 in each group and in the experimental group, 6 Apo E-/-; NamptTg 8 weeks of age. Male rats were divided into two groups, 3 in the control group and in the experimental group. At the same time, the western high fat diet was given for 4 months. The experimental group was injected with TNF- alpha antibody in the abdominal cavity, the dose was 5 mg/kg/ days, and the drug was given for one month. Then the aorta root section was stained with oil red O, F4/80 immunohistochemical staining, CD47 immunohistochemical staining, alpha -SMA immunohistochemical staining, TUNEL cell withering. The mechanism of the effect of Nampt on the occurrence and development of atherosclerosis was further explored. The results of experimental results: compared with the control group (normal drinking water), the 1.NMN drinking water supply group had more lipid deposition in the NMN administration group, the area and thickness of the plaque was higher, the degree of macrophage infiltration was higher, and the expression level of inflammation (TNF- alpha) increased by.NMN. The increase of AS plaques and inflammatory infiltration of.2.Apo E-/- and Apo E-/-; the result of the O staining of the aorta of NamptTg can be seen, compared to the Apo E-/- group, Apo E-/-, the lipid deposition in the aorta of the NamptTg group will increase the area and thickness of the plaque, the infiltration degree of the macrophage is aggravated, the necrotic center enlarges, and the collagen type is enlarged. Increased expression of inflammatory factors (TNF- a, IL-1 beta), increased expression of adhesion and chemokines (ICAM-1, VCAM-1), increased number of apoptotic cells in the plaque, increased the AS process in.3.Nampt transgenic mice and up regulation of CD47 expression level. The inhibition of TNF- a not to Apo E-/- and Apo E-/-. Influence of patch area, but the expression level of F4/80 and alpha -SMA decreased, that is, plaque inflammation and fibrosis improved. In addition, inhibition of TNF- alpha, CD47 expression level also decreased, and plaque cell apoptosis was inhibited. Experimental conclusion: 1. supplementation of Nampt enzyme product NMN in vitro can promote the occurrence and development of AS, which is reflected as plaque. Compared with the enhanced.2. and the wild type of inflammation, the development process of AS induced by high fat diet in Nampt transgenic mice was obviously accelerated, which was mainly reflected in the increase of patch area. In addition, the inflammatory cell infiltration in the plaque tissues of the Nampt transgenic mice increased, and the expression of inflammatory factors increased. The stability of the plaque was significantly worse.3. using neutralization antibody injection to inhibit TNF- alpha, which could delay the AS process in Nampt transgenic mice; the expression level of CD47 in the AS plaques of Nampt transgenic mice increased, and the expression level of CD47 decreased when TNF- a was neutralized by neutralizing antibodies. It suggested that TNF- alpha -CD47 pathway may be involved in the development of Nampt. The role.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R543.5
【参考文献】
相关期刊论文 前1条
1 刘俊田;;动脉粥样硬化发病的炎症机制的研究进展[J];西安交通大学学报(医学版);2015年02期
，本文编号：2144512
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