本文選題：Salusin-β + 糖尿病心肌病�。� 參考：《南京醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景糖尿病心肌病(Diabetic Cardiomyopathy,DCM)是指發(fā)生于糖尿病患者,而不能用高血壓性心臟病、冠心病、心臟瓣膜病以及動(dòng)脈粥樣硬化性疾病來(lái)解釋的心臟結(jié)構(gòu)和功能異常性疾病�；颊咴谔悄虿〉幕A(chǔ)上,出現(xiàn)心臟微血管病變和心肌代謝紊亂,進(jìn)而發(fā)生心臟廣泛局灶性壞死,重癥患者甚至猝死。糖尿病心肌病的病理特點(diǎn)主要包括心肌炎癥、心肌肥大、心肌纖維化、心肌凋亡和自噬以及心功能障礙等。糖尿病是心力衰竭的一個(gè)獨(dú)立危險(xiǎn)因素,男性糖尿病患者發(fā)生心力衰竭的的風(fēng)險(xiǎn)高出同性別正常人兩倍,而女性患者則高出同性別正常人五倍。糖尿病使心力衰竭的患病率提高約2.5倍,15%-35%的糖尿病患者發(fā)生心力衰竭,66%的糖尿病患者死于心臟疾病。Salusins是2003年從編碼人類扭轉(zhuǎn)應(yīng)力障礙基因TOR2A進(jìn)行選擇性剪切而來(lái)的產(chǎn)物,包括salusin-α和salusin-β,分別由28個(gè)和20個(gè)氨基酸殘基組成。Salusin-β是一種血管活性肽,在人、大鼠和小鼠的心血管系統(tǒng)、免疫系統(tǒng)以及中樞神經(jīng)系統(tǒng)中均有廣泛表達(dá)。我們實(shí)驗(yàn)室前期研究發(fā)現(xiàn)活性氧(Reactive oxygen species,ROS)的產(chǎn)生介導(dǎo)了 salusin-β誘導(dǎo)的血管平滑肌細(xì)胞(vascular smooth muscle cells,VSMCs)的泡沫細(xì)胞形成和單核細(xì)胞粘附,并涉及血管損傷引起的VSMCs的遷移和內(nèi)膜增生。干涉salusin-β表達(dá)能有效降低血管內(nèi)ROS的產(chǎn)生。糖尿病患者血漿salusin-β水平升高,抑制內(nèi)源性salusin-β改善心肌缺血引起的心肌重塑。然而salusin-β在糖尿病心肌病中是否發(fā)揮作用尚不明確,本研究主要探討salusin-β在糖尿病心肌病中的作用及其分子機(jī)制。目的1.探討salusin-β在糖尿病心肌病的心肌炎癥和氧化應(yīng)激中的作用與機(jī)制;2.探討salusin-β干涉能否改善糖尿病心肌病的心肌炎癥和氧化應(yīng)激。方法聯(lián)合應(yīng)用高脂飲食(high fat diet,HFD)和小劑量鏈脲霉素(streptozocin,STZ)來(lái)誘導(dǎo)2型糖尿病模型:將SD大鼠隨機(jī)分為4組,每組6只。其中一組用來(lái)做為對(duì)照組,另外3組用來(lái)誘導(dǎo)2型糖尿病。2型糖尿病具體誘導(dǎo)方法是:先給予高脂飲食(34.5%脂肪,17.5%蛋白質(zhì)、48%碳水化合物)喂養(yǎng)4周,在禁食12h后,腹腔單次注射小劑量鏈脲霉素(27.5mg/kg體重,溶解于檸檬酸鹽緩沖液中,pH=4.5)。在注射鏈脲霉素12周后,檢測(cè)大鼠空腹血糖(禁食12h),血糖大于11.lmmol/L的大鼠視為糖尿病大鼠。這3組糖尿病大鼠通過(guò)尾靜脈分別注射磷酸鹽緩沖溶液(PBS)、空病毒以及salusin-β干涉病毒(1.0 × 1011 PFU),2周后,重復(fù)此項(xiàng)操作一次。大鼠的心肌細(xì)胞系H9c2細(xì)胞在含體積分?jǐn)?shù)為10%胎牛血清(FBS)的DMEM培養(yǎng)基中進(jìn)行連續(xù)傳代培養(yǎng)。用酶消化法和差速離心法從出生后1-3天內(nèi)的新生大鼠的心肌內(nèi)獲得原代心肌細(xì)胞。用含糖量為33.3mmol/L的培養(yǎng)基來(lái)模擬體外高糖環(huán)境,培養(yǎng)H9c2細(xì)胞和原代心肌細(xì)胞,正常組的H9c2細(xì)胞和原代心肌細(xì)胞使用含糖量為5.5mmol/L的培養(yǎng)基培養(yǎng)。大鼠禁食4h后,腹腔注射胰島素(1單位/kg體重),檢測(cè)胰島素抵抗。禁食12h后,腹腔注射葡萄糖(lg/kg體重),檢測(cè)糖耐量。超聲心動(dòng)圖檢測(cè)大鼠心功能。酶聯(lián)免疫吸附測(cè)定法(enzyme-linked immunosorbent assay,ELISA)檢測(cè)大鼠心肌、血漿和細(xì)胞中salusin-β、白介素-1β(interleukin-1β,IL-lβ)、白介素-6(interleukin-6,IL-6)、以及腫瘤壞死因子(tumor necrosis factor-α,TNF-α)的含量。蛋白質(zhì)免疫分析技術(shù)(Western blotting)檢測(cè)心肌組織及細(xì)胞中prosalusin、VCAM-1、NOX2、NOX4、4-HNE、p65、IL-lβ、IL-6、和 TNF-α 的蛋白含量。用 real-time PCR(RT-PCR)法檢測(cè)細(xì)胞及心肌組織中 salusin-β、VCAM-1、NOX2、NOX4、4-HNE、p65、IL-1β、IL-6、和 TNF-α 的基因表達(dá)水平。Dihydroethidium(DHE)作為超氧化物陰離子熒光探針用來(lái)檢測(cè)心肌組織的活性氧水平。免疫熒光染色(immunofluorescence,IF)用來(lái)檢測(cè)大鼠心肌組織中salusin-β的表達(dá)水平。結(jié)果1.Salusin-β增強(qiáng)H9c2細(xì)胞的炎癥和氧化應(yīng)激。2.NADPH氧化酶抑制劑二苯乙內(nèi)酰脲(diphenyleneiodonium,DPI)和抗氧化劑N-乙酰半胱氨酸(N-acetylcysteine,NAC)都能阻斷salusin-β和高糖誘導(dǎo)的H9c2細(xì)胞炎癥和氧化應(yīng)激以及NFκB的激活。3.NFκB抑制劑Bay11-7082能夠阻斷salusin-β及高糖誘導(dǎo)的H9c2細(xì)胞炎癥,但不能阻斷salusin-β及高糖誘導(dǎo)的H9c2細(xì)胞氧化應(yīng)激。4干涉salusin-β改善高糖誘導(dǎo)的心肌細(xì)胞炎癥、氧化應(yīng)激以及NFκB激活。5糖尿病大鼠的血漿以及心肌中salusin-β表達(dá)增加。6干涉salusin-β改善糖尿病大鼠的心肌炎癥、氧化應(yīng)激、NFκB激活以及心功能障礙。結(jié)論1.Salusin-β通過(guò)NOX2/ROS/NFKB引起糖尿病心肌病的心肌炎癥;2.干涉salusin-β減輕糖尿病的心肌炎癥和氧化應(yīng)激,并改善心功能。
[Abstract]:Diabetic Cardiomyopathy (DCM) is an abnormal cardiac and functional disorder that occurs in patients with diabetes and cannot be explained by hypertensive heart disease, coronary heart disease, valvular heart disease, and atherosclerotic diseases. The pathological features of diabetic cardiomyopathy mainly include myocarditis, myocardial hypertrophy, myocardial fibrosis, myocardial apoptosis and autophagy, and cardiac dysfunction. Diabetes is an independent risk factor for heart failure, and heart failure in male diabetic patients. The risk was two times higher than normal homosexual people, while women were five times higher than normal homosexual people. Diabetes increased the incidence of heart failure about 2.5 times, 15%-35% diabetic patients had heart failure, and 66% of diabetic patients died from heart disease.Salusins in 2003 from the coding of human torsion stress disorder gene TOR2A The products of sexual shear, including salusin- alpha and salusin- beta, are composed of 28 and 20 amino acid residues,.Salusin- beta, a vasoactive peptide, which are widely expressed in the cardiovascular system, the immune system and the central nervous system in human, rat and mice. We found reactive oxygen species (Reactive oxygen species) in the early laboratory. The production of ROS) mediates the formation of vascular smooth muscle cells (VSMCs) foams and monocyte adhesion to salusin- beta induced vascular smooth muscle cells, and involves the migration of VSMCs and intimal hyperplasia caused by vascular damage. Interference of salusin- beta expression can effectively reduce the production of ROS in the blood tube. The plasma salusin- beta level of diabetic patients Increase, inhibit endogenous salusin- beta to improve myocardial remodeling caused by myocardial ischemia. However, it is not clear whether salusin- beta plays a role in diabetic cardiomyopathy. This study mainly discusses the role and molecular mechanism of salusin- beta in diabetic cardiomyopathy. Objective 1. to explore the myocarditis and oxidation of salusin- beta in diabetic cardiomyopathy. 2. to explore whether salusin- beta interference can improve myocarditis and oxidative stress in diabetic cardiomyopathy. Methods combined use of high fat diet (high fat diet, HFD) and small dose of streptozotocin (streptozocin, STZ) to induce type 2 diabetes mellitus model: the rats were randomly divided into 4 groups, each group was 6. One group was used as a control. In the other 3 groups, the other 3 groups used to induce type 2 diabetes mellitus (type 2 diabetes) to induce the specific induction of diabetes: first feeding the high fat diet (34.5% fat, 17.5% protein, 48% carbohydrate) for 4 weeks. After fasting, the abdominal cavity was injected with small dose of streptozotocin (27.5mg/kg weight, dissolved in citrate buffer, pH=4.5). After injection of streptozotocin for 12 weeks, Rats with fasting blood glucose (fasting 12h) and rats with blood glucose greater than 11.lmmol/L were treated as diabetic rats. The 3 groups of diabetic rats were injected with phosphate buffer solution (PBS), empty virus and salusin- beta interfero virus (1 x 1011 PFU) in the tail vein. After 2 weeks, this operation was repeated. The rat's myocardial cell line H9c2 cells were contained in the volume. Continuous subculture was carried out in the DMEM medium of 10% fetal bovine serum (FBS). Primary cardiomyocytes were obtained by enzyme digestion and differential centrifugation from the myocardium of newborn rats at 1-3 days after birth. The high glucose environment in vitro was simulated with the medium containing sugar content of 33.3mmol/L, cultured H9c2 cells and primary cardiomyocytes, and H9c2 of the normal group. Cells and primary cardiomyocytes were cultured in medium with sugar content of 5.5mmol/L. After fasting 4h, rats were intraperitoneally injected with insulin (1 units of /kg body weight) to detect insulin resistance. After fasting 12h, glucose tolerance was detected by intraperitoneal injection of glucose (lg/kg weight). Cardiac function of rats was detected by echocardiography. Enzyme linked immunosorbent assay (enzyme-linked IMM) Unosorbent assay, ELISA) detection of rat myocardium, plasma and cell salusin- beta, interleukins -1 beta (interleukin-1 beta, IL-l beta), interleukins -6 (interleukin-6, IL-6), and tumor necrosis factor (tumor necrosis alpha, alpha). Protein immunoassay technique for detection of myocardial tissue and cells 1, NOX2, NOX4,4-HNE, p65, IL-l beta, IL-6, and TNF- alpha protein content. Use real-time PCR (RT-PCR) method to detect the gene expression level of salusin- beta, VCAM-1, NOX2, and alpha, as a superoxide anion fluorescent probe used to detect reactive oxygen species in myocardial tissue. Level. Immunofluorescence (IF) was used to detect the expression level of salusin- beta in rat myocardial tissue. Results 1.Salusin- beta enhanced H9c2 cell inflammation and oxidative stress.2.NADPH oxidase inhibitor two Phenylacetyl urea (diphenyleneiodonium, DPI) and the antioxidant N- acetyl cysteine (N-acetylcysteine, NAC). Blocking salusin- beta and high glucose induced H9c2 cell inflammation and oxidative stress, and the activation of.3.NF kappa B inhibitor Bay11-7082 in NF kappa B can block salusin- beta and high glucose induced H9c2 cell inflammation, but can not block salusin- beta and high glucose induced H9c2 cell oxidative stress.4 interfere with the high glucose induced cardiomyocyte inflammation and oxidation Stress and NF kappa B activated the plasma of.5 diabetic rats and the expression of salusin- beta in myocardium increased.6 interference salusin- beta to improve myocarditis, oxidative stress, activation of NF kappa B and cardiac dysfunction in diabetic rats. Conclusion 1.Salusin- beta induces myocarditis in diabetic cardiomyopathy by NOX2/ROS/NFKB; 2. interfering with salusin- beta to alleviate diabetes Cardiac myositis and oxidative stress and the improvement of cardiac function.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R587.2;R542.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Loren E WOLD;Asli F CEYLAN-ISIK;Jun REN;;Oxidative stress and stress signaling: menace of diabetic cardiomyopathy[J];Acta Pharmacologica Sinica;2005年08期

，

本文編號(hào)：1864340