【摘要】：研究目的觀察曲美他嗪(Trimetazidine,TMZ)預(yù)處理能否有效減輕心肌細(xì)胞缺氧/復(fù)氧(Hypoxia/reoxygenation,H/R)損傷,并探索心肌細(xì)胞缺氧/復(fù)氧期間,曲美他嗪預(yù)處理是否經(jīng)過單磷酸腺苷活化蛋白激酶(AMP-activated protein kinase,AMPK)通路調(diào)控自噬。研究方法第一部分實(shí)驗(yàn):觀察H/R過程中曲美他嗪的保護(hù)作用,并確定最有效濃度。構(gòu)建新生大鼠心肌細(xì)胞H/R模型,研究對象隨機(jī)分為對照組、H/R組、H/R+TMZ(10μM)組、H/R+TMZ(50μM)組、H/R+TMZ(100μM)組。采用MTS檢測細(xì)胞活性,收集培養(yǎng)液并通過試劑盒檢測LDH水平以觀察細(xì)胞損傷程度,比較各濃度療效,確定最有效濃度。第二部分實(shí)驗(yàn):探明曲美他嗪對H/R過程中自噬的影響及干預(yù)自噬表達(dá)對曲美他嗪療效影響本部分實(shí)驗(yàn)主要采用氯喹(chloroquine,Cq)抑制自噬流,實(shí)驗(yàn)分組為對照組、對照+TMZ 組、對照+Cq 組、H/R 組、H/R+TMZ 組、H/R+Cq 組、H/R+TMZ+Cq組。MTS檢測細(xì)胞活性,試劑盒檢測培養(yǎng)液LDH水平,并通過Western blot半定量分析自噬相關(guān)蛋白Beclin-1、LC3和p62的表達(dá),轉(zhuǎn)染病毒mRFP-GFP-LC3并通過共聚焦顯微鏡以測定細(xì)胞自噬相關(guān)蛋白分布情況,透射電鏡直接觀察各組自噬體數(shù)量,TUNEL染色檢測心肌細(xì)胞凋亡水平。第三部分實(shí)驗(yàn):探明H/R過程中干預(yù)AMPK對曲美他嗪所調(diào)控的自噬的影響本部分實(shí)驗(yàn)主要使用compound C(com C)抑制AMPK信號,實(shí)驗(yàn)分組為對照組、對照 + com C 組、H/R 組、H/R+TMZ 組、H/R+ com C 組、H/R+TMZ+ com C組。MTS檢測細(xì)胞活性,試劑盒檢測培養(yǎng)液LDH水平,最后通過Western blot半定量分析自噬相關(guān)蛋白LC3和p62及AMPK信號通路蛋白p-AMPK/AMPK和p-mTOR/mTOR的表達(dá)。結(jié)果1.曲美他嗪預(yù)處理減輕心肌細(xì)胞H/R損傷與對照組相比,H/R組的細(xì)胞活性顯著性下降(P0.05);各曲美他嗪預(yù)處理組細(xì)胞活性均比H/R組高(P0.05),H/R+TMZ(50μM)組細(xì)胞活性比H/R+TMZ(10μM)組高(P0.05),但與 H/R+TMZ(100μM)組無統(tǒng)計(jì)學(xué)差異(P0.05)。因此50μM為曲美他嗪的最有效濃度。2.曲美他嗪預(yù)處理在心肌細(xì)胞H/R期間促進(jìn)自噬流H/R處理后各自噬相關(guān)蛋白均表達(dá)上調(diào)(P0.05,C vs.H/R組),然而曲美他嗪預(yù)處理后Beclin-1、LC3-Ⅱ/LC3-Ⅰ比值均進(jìn)一步增強(qiáng),p62減少(P0.05,H/R vs.H/R + TMZ組);透射電鏡結(jié)果顯示,相比對照組,H/R組細(xì)胞漿內(nèi)出現(xiàn)更多自噬體,(P0.05);而相比H/R組,H/R+TMZ組自噬體數(shù)量進(jìn)一步增加(P0.05)。共聚焦顯微鏡下,對照組無明顯LC3聚集現(xiàn)象;H/R處理后,自噬體數(shù)量明顯增加(P0.05,C vs.H/R組);與H/R組相比,H/R+TMZ組的自噬溶酶體更多(P0.05)。3.Cq預(yù)處理抵消曲美他嗪對心肌細(xì)胞的保護(hù)作用Cq 預(yù)處理后,LC3-Ⅱ/LC3-Ⅰ、p62 的表達(dá)均增加(P0.05,H/R + TMZ vs.H/R + TMZ + Cq組);共聚焦顯微鏡下,Cq預(yù)處理顯著減少自噬溶酶體(P0.05,H/R + TMZ vs.H/R + TMZ + Cq 組);與 H/R+TMZ 組相比,H/R+TMZ+Cq組細(xì)胞活性明顯下降(P0.05);4.曲美他嗪預(yù)處理減少H/R中細(xì)胞凋亡TUNEL染色結(jié)果顯示,與對照組相比,H/R組凋亡細(xì)胞顯著性增加(P0.05);曲美他嗪預(yù)處理后,凋亡細(xì)胞數(shù)量明顯減少(P0.05,H/Rvs.H/R + TMZ組);然而H/R + TMZ+ Cq與H/R + TMZ組相比,凋亡細(xì)胞顯著性增加(P0.05)。5.曲美他嗪預(yù)處理通過AMPK-mTOR調(diào)控H/R中的自噬與H/R組相比,曲美他嗪預(yù)處理顯著增加p-AMPK/AMPK的比值以及減少p-mTOR/mTOR 的比值(P0.05,H/Rvs.H/R + TMZ 組);與 H/R + TMZ 組相比,compound C 減少 p-AMPK/AMPK 比值,增加 p-mTOR/mTOR 比值(P0.05);此外,compound C減少LC3-Ⅱ/LC3-Ⅰ比值并增加了 p62的表達(dá);H/R +TMZ+comC與H/R + TMZ組相比,其細(xì)胞活性減少,LDH水平增加(P0.05),結(jié)論曲美他嗪預(yù)處理通過AMPK-mTOR信號通路促進(jìn)自噬流減輕心肌細(xì)胞H/R損傷
[Abstract]:Objective To investigate whether trimetazidine (TMZ) preconditioning can effectively alleviate hypoxia/reoxygenation (H/R) injury in cardiomyocytes and whether trimetazidine preconditioning can be regulated by AMP-activated protein kinase (AMPK) pathway during hypoxia/reoxygenation in cardiomyocytes. METHODS The first part of the experiment was to observe the protective effect of trimetazidine during H/R and determine the most effective concentration.The H/R model of neonatal rat cardiomyocytes was established. The level of LDH was measured to observe the degree of cell injury and compare the therapeutic effects of different concentrations to determine the most effective concentration. Part II: Experiments: To explore the effect of trimetazidine on autophagy in H/R and the effect of autophagy on trimetazidine. This part of the experiment mainly used chloroquine (Cq) to inhibit autophagy. The experiment was divided into control group and control group + TMZ. MTS assay was used to detect cell activity, LDH level in culture medium, and the expression of autophagy-related proteins Beclin-1, LC3 and p62 was semi-quantitatively analyzed by Western blot. The distribution of autophagy-related proteins was detected by confocal microscopy. The number of autophages was observed by transmission electron microscopy and the level of myocardial apoptosis was detected by TUNEL staining. Part III: To explore the effect of AMPK intervention on autophagy regulated by trimetazidine during H/R. In this part, compound C (com C) was used to inhibit AMPK signal. The experiment was divided into control group, control + com C group, H/R group, H/R + TMZ group. Group H/R+com C, H/R+TMZ+com C, H/R+TMZ+com C. MTS was used to detect cell activity and LDH level in culture medium. The expressions of autophagy-associated proteins LC3 and p62, and AMPK signaling pathway proteins p-AMPK/AMPK and p-mTOR/mTOR were analyzed semi-quantitatively by Western blot. Results 1. Trimetazidine pretreatment reduced H/R injury of cardiomyocytes compared with control group. The cell viability of H / R + TMZ group was higher than that of H / R group (P 0.05). The cell viability of H / R + TMZ group was higher than that of H / R + TMZ group (P 0.05), but there was no significant difference between H / R + TMZ group (P 0.05). During R period, the expression of autophagy-related proteins was up-regulated after autophagy H/R treatment (P 0.05, C vs. H/R group). However, after trimetazidine pretreatment, the ratio of Beclin-1, LC3-II/LC3-I was further enhanced and p62 was decreased (P 0.05, H/R vs. H/R + TMZ group). Compared with H/R group, the number of autophages in H/R+TMZ group was further increased (P 0.05). There was no obvious LC3 aggregation in control group under confocal microscope; the number of autophages in H/R group was significantly increased (P 0.05, C vs. H/R group); the number of autophagic lysosomes in H/R+TMZ group was more than that in H/R group (P 0.05). 3. Cq pretreatment counteracted the protective effect of trimetazidine on cardiomyocytes. After pretreatment, the expression of LC3-II/LC3-I and p62 increased (P 0.05, H/R+TMZ vs.H/R+TMZ+Cq group); under confocal microscope, Cq pretreatment significantly decreased autophagy lysosome (P 0.05, H/R+TMZ vs.H/R+TMZ+Cq group); compared with H/R+TMZ+Cq group, the activity of H/R+TMZ+Cq group decreased significantly (P 0.05); Apoptotic TUNEL staining showed that the number of apoptotic cells in H/R group was significantly increased (P 0.05); the number of apoptotic cells was significantly decreased after trimetazidine pretreatment (P 0.05, H/Rvs.H/R+TMZ group); however, the number of apoptotic cells in H/R+TMZ+Cq group was significantly increased compared with that in H/R+TMZ group (P 0.05). 5. Trimetazidine pretreatment regulated H/R by AMPK-mTOR. Compared with H/R group, trimetazidine pretreatment significantly increased the p-AMPK/AMPK ratio and decreased the p-mTOR/mTOR ratio (P 0.05, H/Rvs.H/R+TMZ group); compared with H/R+TMZ group, compound C decreased the p-AMPK/AMPK ratio, increased the p-mTOR/mTOR ratio (P 0.05); in addition, compound C decreased the LC3-II/LC3-I ratio and increased the p62 table. Compared with H/R+TMZ+comC group, H/R+TMZ+comC group showed decreased cell activity and increased LDH level (P 0.05). Conclusion Trimetazidine preconditioning can alleviate H/R injury of cardiomyocytes by promoting autophagy through AMPK-mTOR signaling pathway.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R54

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