Nampt在動(dòng)脈粥樣硬化發(fā)生發(fā)展中的作用及機(jī)制
發(fā)布時(shí)間：2018-07-25 17:22
【摘要】：動(dòng)脈粥樣硬化(Atherosclerosis,AS)是腦梗,冠心病,外周血管病等多種心血管疾病的主要誘因。隨著生活水平的不斷提高,AS的發(fā)病率也在迅猛增加。AS病變基礎(chǔ)是脂質(zhì)代謝障礙,斑塊是由病變的血管細(xì)胞和凋亡細(xì)胞累積形成,斑塊一旦破裂會(huì)造成血管栓塞進(jìn)而引發(fā)腦卒中,危及人的生命健康。AS的發(fā)病機(jī)制尚未明確,因此臨床上并沒有特異性好且有效的動(dòng)脈粥樣硬化治療藥物。煙酰胺磷酸核糖轉(zhuǎn)移酶(nicotinamide phosphoribosyltransferase,NAMPT)是一種由脂肪細(xì)胞分泌的脂肪因子,在細(xì)胞內(nèi)和細(xì)胞外都具有活性。Nampt是煙酰胺腺嘌呤二核苷酸(NAD)合成的限速酶,可將底物煙酰胺(NAM)轉(zhuǎn)化成產(chǎn)物煙酰胺單核苷酸(NMN),NMN在催化條件下,生成NAD。NAD稱作為輔酶I,是參與線粒體呼吸鏈氧化還原反應(yīng)所必需的輔酶。NAD的生物學(xué)功能極其重要,可能能夠直接決定著細(xì)胞的命運(yùn)。本課題組前期基本證實(shí)了Nampt在腦缺血后發(fā)揮神經(jīng)保護(hù)作用。動(dòng)脈粥樣硬化被認(rèn)為是腦卒中的發(fā)病因素,但Nampt在AS中確切的作用尚未被證實(shí)。因此,我們采用體外補(bǔ)充Nampt酶產(chǎn)物NMN以及Nampt轉(zhuǎn)基因小鼠與Apo E-/-雜交的Apo E-/-;NamptTg鼠來(lái)研究Nampt對(duì)動(dòng)脈粥樣硬化發(fā)生發(fā)展的影響,初步確定了Nampt可促進(jìn)動(dòng)脈粥樣硬化的發(fā)生發(fā)展。本課題進(jìn)一步研究了抑制TNF-α對(duì)動(dòng)脈粥樣硬化發(fā)生發(fā)展的影響,初步探討了Nampt發(fā)揮作用的機(jī)制,從而為動(dòng)脈粥樣硬化的治療提供了新靶點(diǎn)。實(shí)驗(yàn)方法:1.將8只8周齡左右的Apo E-/-雄性小鼠分為兩組,對(duì)照組和實(shí)驗(yàn)組各4只,同時(shí)西方高脂飼料喂養(yǎng),兩個(gè)月后,實(shí)驗(yàn)組NMN飲水給藥,給藥劑量為300 mg/kg/天,對(duì)照組則繼續(xù)普通飲水。觀察NMN給藥即體外補(bǔ)充Nampt酶產(chǎn)物NMN對(duì)AS小鼠主動(dòng)脈上脂質(zhì)沉積的影響。此外,采用油紅O染色,HE染色,觀察體外補(bǔ)充Nampt酶產(chǎn)物NMN,對(duì)AS小鼠斑塊厚度及尺寸的影響,F4/80免疫組化染色觀察主動(dòng)脈根部斑塊的巨噬細(xì)胞浸潤(rùn)情況,TNF-α免疫組化染色確定體外補(bǔ)充NMN對(duì)主動(dòng)脈根部斑塊的炎癥因子表達(dá)情況的影響。2.將Nampt轉(zhuǎn)基因鼠與世界上公認(rèn)的動(dòng)脈粥樣硬化模型鼠Apo E-/-進(jìn)行雜交,獲得Apo E-/-;NamptTg鼠,作為實(shí)驗(yàn)組,以Apo E-/-小鼠作為對(duì)照組,西方高脂飼料喂養(yǎng)4個(gè)月。對(duì)小鼠進(jìn)行主動(dòng)脈油紅O染色,觀察Nampt轉(zhuǎn)基因?qū)S斑塊的影響。采用油紅O染色,HE染色,觀察Nampt轉(zhuǎn)基因?qū)?dòng)脈粥樣硬化斑塊的厚度和尺寸的影響;F4/80免疫組化檢測(cè)巨噬細(xì)胞浸潤(rùn)情況,觀察Nampt轉(zhuǎn)基因?qū)?dòng)脈粥樣硬化斑塊巨噬細(xì)胞浸潤(rùn)的影響;Masson染色,天狼星紅染色,觀察Nampt轉(zhuǎn)基因?qū)S小鼠斑塊纖維帽和膠原情況的影響;TNF-α,IL-1β免疫組化染色,觀察Nampt轉(zhuǎn)基因?qū)S小鼠斑塊炎癥因子表達(dá)水平的影響;ICAM-1,VCAM-1免疫組化染色,觀察Nampt轉(zhuǎn)基因?qū)S小鼠斑塊中粘附及趨化因子的表達(dá)水平的影響;TUNEL實(shí)驗(yàn),觀察Nampt轉(zhuǎn)基因?qū)S斑塊中細(xì)胞凋亡數(shù)目的影響;此外,還進(jìn)行了CD47免疫組化染色,初步探索了參與AS發(fā)生發(fā)展的可能機(jī)制。3.將6只Apo E-/-8周齡左右雄性小鼠,分為兩組,對(duì)照組和實(shí)驗(yàn)組各3只,6只Apo E-/-;NamptTg 8周齡左右雄性鼠,分為兩組,對(duì)照組和實(shí)驗(yàn)組各3只。同時(shí)西方高脂飲食4個(gè)月。實(shí)驗(yàn)組采用腹腔注射TNF-α抗體,劑量為5 mg/kg/天,給藥一個(gè)月,隨后進(jìn)行了主動(dòng)脈根部切片油紅O染色,F4/80免疫組化染色,CD47免疫組化染色,α-SMA免疫組化染色,TUNEL細(xì)胞凋亡檢測(cè),進(jìn)一步探索了Nampt對(duì)動(dòng)脈粥樣硬化發(fā)生發(fā)展影響的作用機(jī)制。實(shí)驗(yàn)結(jié)果:1.NMN飲水給藥組與對(duì)照組(正常飲水)相比NMN給藥組主動(dòng)脈上脂質(zhì)沉積較多,斑塊面積和厚度較大,巨噬細(xì)胞浸潤(rùn)程度較高,炎癥(TNF-α)表達(dá)水平升高。NMN促進(jìn)了AS斑塊增多和炎癥浸潤(rùn)。2.Apo E-/-和Apo E-/-;NamptTg的主動(dòng)脈油紅O染色結(jié)果可以看出,相較于Apo E-/-組,Apo E-/-;NamptTg組的主動(dòng)脈脂質(zhì)沉積較多。Nampt轉(zhuǎn)基因會(huì)使得斑塊面積和厚度變大,巨噬細(xì)胞(F4/80)浸潤(rùn)程度加重,壞死中心擴(kuò)大,III型膠原增多,并且炎癥因子(TNF-α,IL-1β)表達(dá)水平升高,粘附和趨化因子(ICAM-1,VCAM-1)表達(dá)水平升高,斑塊中細(xì)胞凋亡數(shù)量增多。3.Nampt轉(zhuǎn)基因小鼠AS進(jìn)程加快的同時(shí)也伴隨著CD47表達(dá)水平的上調(diào)。抑制TNF-α不會(huì)對(duì)Apo E-/-和Apo E-/-;NamptTg AS小鼠的血脂和斑塊面積產(chǎn)生影響,但F4/80和α-SMA的表達(dá)水平下降,即斑塊炎癥和纖維化得到了改善,此外,抑制TNF-α,CD47的表達(dá)水平也隨之下調(diào),斑塊細(xì)胞凋亡也得到了抑制。實(shí)驗(yàn)結(jié)論:1.通過(guò)體外補(bǔ)充Nampt的酶產(chǎn)物NMN,可促進(jìn)AS的發(fā)生發(fā)展,體現(xiàn)為斑塊面積的顯著增加和炎癥的增強(qiáng)。2.和野生型小鼠相比較,在Nampt轉(zhuǎn)基因小鼠上使用高脂飲食誘導(dǎo)的AS發(fā)生發(fā)展進(jìn)程明顯加快,主要體現(xiàn)為斑塊面積的增加;此外,Nampt轉(zhuǎn)基因小鼠的斑塊組織中的炎癥細(xì)胞浸潤(rùn)、炎癥因子的表達(dá)均增加,提示斑塊的穩(wěn)定性明顯變差。3.使用中和抗體注射抑制TNF-α,可以延緩Nampt轉(zhuǎn)基因鼠的AS進(jìn)程;Nampt轉(zhuǎn)基因鼠AS斑塊中CD47表達(dá)水平升高,而當(dāng)TNF-α被中和抗體抑制后,CD47的表達(dá)水平也隨之降低。提示TNF-α-CD47通路可能參與了Nampt促進(jìn)AS發(fā)生發(fā)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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R543.5
本文編號(hào)：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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R543.5
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 劉俊田;;動(dòng)脈粥樣硬化發(fā)病的炎癥機(jī)制的研究進(jìn)展[J];西安交通大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2015年02期
，本文編號(hào)：2144512
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