【摘要】：目的:近年文獻報道2型糖尿病(type 2 diabetes mellitus,T2DM)患者數量呈逐年上升的趨勢,糖尿病相關心臟并發(fā)癥是2型糖尿病患者晚期最主要的死亡原因。臨床及動物實驗發(fā)現(xiàn),食物中補充n-3系多不飽和脂肪酸(n-3 Polyunsaturated fatty acids,n-3 PUFAs),可以對抗心肌肥大,改善心功能。本課題組前期研究發(fā)現(xiàn)高脂飲食并注射小劑量STZ(鏈脲佐菌素)誘導的2型糖尿病模型大鼠心肌n-3 PUFAs二十二碳六烯酸(docosahexaenoic acid,DHA)含量明顯下降,并伴有左心肥大,左心室心肌收縮功能明顯下降,而飲食中補充DHA的2型糖尿病大鼠上述情況均有所改善。細胞凋亡是指為維持內環(huán)境的穩(wěn)態(tài)由基因控制的細胞自主有序的死亡,與細胞壞死不同,凋亡涉及一系列基因的激活,表達及調控。前期實驗已發(fā)現(xiàn)在整體水平2型糖尿病大鼠心肌內細胞凋亡增加,飲食補充DHA的2型糖尿病大鼠細胞凋亡有所改善。但DHA及其它n-3 PUFAs是否確可對抗心肌細胞凋亡,作用及機制又如何?需要在細胞水平進行研究。細胞骨架是指真核細胞中的蛋白纖維網絡結構。細胞骨架不僅在維持細胞形態(tài),承受外力,保持內部結構有序性方面起重要作用,還參與許多重要的生命活動。在心肌細胞中,細胞骨架和它的結合蛋白組成動力系統(tǒng)。文獻報道,在心肌肥大和心力衰竭的動物的心肌細胞中可見細胞骨架改變。前期實驗已發(fā)現(xiàn)T2DM大鼠肌原纖維紋理模糊,纖維束排列不規(guī)則,出現(xiàn)斷裂溶解,補充DHA可阻止T2DM大鼠心肌肌原纖維的斷裂和溶解。DHA等n-3 PUFAs確對心肌細胞骨架有什么影響嗎?需在細胞水平證實�；谏鲜瞿康�,本實驗以H9C2大鼠心肌細胞株作為研究對象,給予高濃度軟脂酸模擬糖尿病心肌所處的高脂環(huán)境,造成細胞損傷后,補充DHA等n-3 PUFAs,觀察H9C2心肌細胞凋亡情況及細胞骨架改變,探究DHA的保護機制。方法:1 MTS實驗對細胞活性進行檢測,確定可使細胞活力下降的C16:0濃度以及dha的保護濃度。2westernblot檢測細胞凋亡相關的cleavedcaspase3,cleavedparp,parp和fadd蛋白表達。3realtime-rtpcr檢測細胞凋亡相關基因caspase3,9,12,炎癥相關基因tnf-α以及氧化應激相關基因ho-1等的mrna表達量。4tunel法檢測不同濃度c16:0,不同濃度n-3系pufas以及不同濃度n-6系aa處理后的細胞凋亡情況。5鬼筆環(huán)肽染色法觀察c16:0對h9c2心肌細胞骨架影響,以及給予dha等n-3系pufas或者n-6系的aa后能否逆轉c16:0導致的細胞骨架的改變。6westernblot檢測高c16:0對肌鈣蛋白tnnt2,肌鈣蛋白酶calpain1,calpain2,calpains1的蛋白量的影響,以及補充dha后tnnt2,calpain1,calpain2,calpains1蛋白量的變化。7realtime-rtpcr檢測補充dha之后細胞calpain1mrna表達量的變化。8結果數據以x±sd表示,采用spss17.0統(tǒng)計學軟件統(tǒng)計學分析,兩組之間的差異采用配對t檢驗進行分析,以p0.05為差異有統(tǒng)計學意義。結果:1脂質超載引起了h9c2心肌細胞的活力下降,dha則可以改善脂質超載損傷的細胞活力給予150-400μmol/l濃度范圍內的c16:0處理細胞24小時后,細胞活性明顯低于bsa溶劑對照組(p0.05),并且c16:0濃度越高h9c2心肌細胞活性越低。單純dha處理細胞24小時后,細胞活性與對照組無統(tǒng)計學差別,但在c16:0200μmol/l基礎上補充10、100μmol/ldha后,c16:0導致的細胞活力的下降得以改善。2c16:0引起的心肌細胞活力下降與氧化應激及炎癥反應的增加有關隨著c16:0濃度的增加,h9c2心肌細胞氧化應激相關因子ho-1和炎癥相關因子tnf-α的mrna表達量明顯增加,表明c16:0引起的心肌細胞活力下降與心肌細胞氧化應激及炎癥反應的發(fā)生有關。3脂超載引起的細胞活力下降與內質網凋亡途徑介導的凋亡有關c16:0處理h9c2細胞24小時后,隨著c16:0濃度的逐漸升高,細胞凋亡的數量也出現(xiàn)增加。與此同時,細胞凋亡關的caspase3的mrna表達量,以及cleavedcaspase3和cleavedparp蛋白量明顯增加,而細胞壞死的標志性蛋白fadd的表達量卻沒有出現(xiàn)明顯變化,表明c16:0引起心肌細胞活性降低主要是凋亡而不是壞死所致。因為內質網凋亡途徑的重要起始子caspase12而不是細胞線粒體凋亡途徑的上游分子caspase9的mrna表達量表達增加,表明c16:0超載引起的細胞凋亡與內質網凋亡途徑有關。4dha等n-3pufas改善脂超載損傷的心肌細胞活力與其降低細胞凋亡有關給予200μmol/l16:0處理24小時后,凋亡細胞數量增多,但同時給予1、10、100μmol/ldha、epa和lna處理后,則可降低由c16:0上調的凋亡細胞的數量;dha、epa、lna均可以逆轉c16:0導致的cleavedcaspase3和cleavedparp蛋白量的增加。但是,n-6pufas花生四烯酸aa本身卻可增加細胞凋亡。5脂超載引起h9c2心肌細胞活下降,與其引起心肌細胞細胞骨架改變有關隨著c16:0濃度增加,心肌細胞內的肌絲增粗,折疊,致使細胞由規(guī)則的長梭形變成有多處凹陷的不規(guī)則多邊形。隨著細胞肌絲形態(tài)的改變,細胞的肌鈣蛋白酶calpain1,calpain2和calpains1蛋白表達量增加,而肌鈣蛋白troponint(tnnt2)的蛋白量逐漸下降。表明c16:0超載引起的細胞活性下降與其肌鈣蛋白酶calpain表達增加,從而引起肌鈣蛋白troponint的降解,導致細胞骨架形態(tài)改變有關。6dha等n-3pufas能夠抑制脂超載引起的細胞骨架的改變,進而改善脂超載所致的心肌細胞活力下降給h9c2補充1、10、100μmol/ldha、epa和lna后,可逆轉c16:0所致的h9c2細胞骨架改變。但是n-6pufa花生四烯酸aa本身即可造成細胞骨架翻折,并且隨著aa濃度的增加,形態(tài)異常細胞數量逐漸增加。給h9c2補充dha能降低c16:0導致的肌鈣蛋白酶calpain1,2,S1表達上升和TNNT2蛋白量的下降。補充100μmol/L DHA后,C16:0引起的Calpain 1 mRNA的表達上升,被緩解。結論:1 DHA可以改善C16:0脂超載導致的H9C2心肌細胞細胞活力下降。2 DHA可能的作用機制:(1)通過抑制內質網凋亡途徑從而抑制細胞凋亡;(2)通過下調肌鈣蛋白酶Calpain表達,減少肌鈣蛋白TNNT2降解,進而改善細胞骨架的損傷。
[Abstract]:Objective: Recent literatures have reported that the number of type 2 diabetes mellitus (T2DM) patients is increasing year by year. Diabetes-related cardiac complications are the leading cause of death in patients with advanced type 2 diabetes mellitus. Our previous study found that the content of n-3 PUFAs docosahexaenoic acid (DHA) in myocardium of type 2 diabetic rats induced by high-fat diet and low-dose STZ (streptozotocin) significantly decreased, accompanied by left ventricular hypertrophy, and left ventricular systolic function significantly decreased. Apoptosis refers to the autonomous and orderly death of cells controlled by genes in order to maintain homeostasis of the internal environment. Unlike cell necrosis, apoptosis involves the activation, expression and regulation of a series of genes. Previous experiments have found that the heart of type 2 diabetic rats at the overall level is involved. Intramuscular apoptosis was increased in type 2 diabetic rats fed with DHA. However, whether DHA and other n-3 PUFAs can indeed prevent cardiomyocyte apoptosis, and what are their effects and mechanisms? Cellular level studies are needed. Cytoskeleton refers to the structure of protein fiber network in eukaryotic cells. Cytoskeleton is not only in maintaining fine structure. Cytoskeleton and its binding proteins constitute a dynamic system in cardiomyocytes. Cytoskeleton alterations have been reported in cardiomyocytes of hypertrophic and heart failure animals. Previous experiments have found that T2D is involved in many important life activities. Does n-3 PUFAs, such as DHA, have any effect on the cytoskeleton of cardiomyocytes in T2DM rats? It needs to be confirmed at the cellular level. The apoptosis and cytoskeleton of H9C2 myocardial cells were observed and the protective mechanism of DHA was explored after high concentration palmitic acid was given to simulate the hyperlipidemic environment of diabetic myocardium. Methods: 1 MTS assay was used to detect the cell viability and to determine the concentration of C16:0 which could decrease the cell viability and the protection of dha. 2 Western blot was used to detect the expression of cleaved caspase 3, cleaved parp, PARP and FADD protein. 3 realtime-rtpcr was used to detect the expression of caspase 3, 9, 12, inflammation-related gene TNF-a and oxidative stress-related gene ho-1. 5 ghost-pencil peptide staining was used to observe the effect of c16:0 on the cytoskeleton of H9c2 myocardial cells and whether the changes of cytoskeleton induced by c16:0 could be reversed by DHA and other n-3 PUFAs or n-6 aa. The changes of tnnt 2, calpain 1, calpain 2 and calpain 1 protein after DHA supplementation were observed. 7 realtime-rtpcr was used to detect the changes of calpain 1 mRNA expression after DHA supplementation. Results: 1. Lipid overload caused the decrease of H9c2 myocardial cell viability, DHA could improve the cell viability of lipid overload injury, and the activity of H9c2 myocardial cells treated with c16:0 in the concentration range of 150-400 micromol/l for 24 hours was significantly lower than that of BSA solvent control group (p0.05). The higher the concentration of c16:0, the lower the activity of H9c2 myocardial cells. There was no significant difference in cell viability between the control group and the DHA treated cells 24 hours later. However, the decrease of cell viability induced by c16:0 was ameliorated by supplementation of 10,100 micromol/l DHA with c16:0200 micromol/l. The decrease of cardiomyocyte viability induced by c16:0 was related to the increase of oxidative stress and inflammatory reaction with the increase of c16:0 concentration. The increase of the degree of H9c2 myocardial cell oxidative stress-related factors HO-1 and inflammation-related factors TNF-a mRNA expression increased significantly, indicating that c16:0-induced decline in cardiomyocyte viability is related to oxidative stress and inflammation. 3 lipid overload-induced cell viability decline is related to endoplasmic reticulum apoptosis pathway-mediated apoptosis At the same time, the expression of Caspase-3 mrna, cleaved caspase-3 and cleaved PARP protein were significantly increased, while the expression of fadd, a marker of cell necrosis, did not change significantly. 6:0 decreased cardiomyocyte viability was mainly due to apoptosis rather than necrosis, because the expression of caspase 12, an important initiator of the endoplasmic reticulum apoptosis pathway, was higher than that of caspase 9, an upstream molecule of the mitochondrial apoptosis pathway, suggesting that apoptosis induced by c16:0 overload was related to the apoptosis pathway of endoplasmic reticulum. The viability of cardiomyocytes injured by hyperlipidemia was related to the decrease of apoptosis. The number of apoptotic cells increased 24 hours after treatment with 200 micromol/l16:0, but the number of apoptotic cells increased by 1,10,100 micromol/ldha, EPA and LNA at the same time could be decreased by treatment with 1,10,100 micromol/ldha, EPA and lna. However, n-6 PUFAs arachidonic acid AA itself can increase cell apoptosis. 5 lipid overload leads to the decrease of H9c2 cardiomyocyte viability, which is related to the cytoskeleton change of cardiomyocyte. with the increase of c16:0 concentration, the myofilament in cardiomyocyte thickens and folds, resulting in the cells from regular long shuttle to multiple depressions. The expression of calpain-1, calpain-2 and calpain-1 protein increased with the change of myofibril morphology, while the expression of troponin-2 protein decreased gradually. It indicated that the decrease of cell activity and the increase of calpain expression induced by c16:0 overload could result in troponin troponin expression. 6 DHA and other n-3 PUFAs can inhibit the changes of cytoskeleton induced by lipid overload, and then improve the decrease of cardiomyocyte viability induced by lipid overload. When H9c2 is supplemented with 1,10,100 micromol/ldha, EPA and lna, the cytoskeleton changes of H9c2 induced by c16:0 can be reversed. The expression of calpain 1,2,S1 and the decrease of TNNT2 protein induced by c16:0 could be decreased by DHA supplementation. The expression of calpain 1 mRNA induced by C16:0 increased and was alleviated by 100 micromol/L DHA supplementation. To improve the activity of H9C2 cardiomyocytes induced by C16:0 lipid overload, the possible mechanisms of 2-DHA are: (1) inhibiting apoptosis by inhibiting the endoplasmic reticulum apoptosis pathway; (2) reducing the expression of calpain and the degradation of troponin TNNT2, thereby improving the cytoskeleton damage.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R587.2;R54

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相關博士學位論文 前1條

1 侯連國;DHA保護2型糖尿病心肌的分子機制研究[D];河北醫(yī)科大學;2013年

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本文編號：2234492