本文關(guān)鍵詞： Nrf2 骨骼肌 肌漿網(wǎng) Ca~(2+) 有氧運動　出處：《北京體育大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：研究目的:Nrf2是體內(nèi)重要的核轉(zhuǎn)錄因子,是細胞氧化還原平衡的關(guān)鍵調(diào)節(jié)者。Ca~(2+)在骨骼肌收縮過程中起重要作用。本研究探討Nrf2在骨骼肌肌漿網(wǎng)對Ca~(2+)調(diào)節(jié)過程中的作用,研究Nrf2在有氧訓(xùn)練對小鼠骨骼肌肌漿網(wǎng)Ca~(2+)調(diào)節(jié)中的作用及其機制。研究方法:離體實驗采用小鼠骨骼肌C2C12細胞系,以Nrf2的激動劑t-BHQ和Nrf2的抑制劑Brusatol進行干預(yù)。熒光比色法檢測各組C2C12細胞ROS水平。采用Ca~(2+)染料Fluo-4 AM加載細胞,激光掃描共聚焦顯微鏡檢測在100μM H_2O_2氧化應(yīng)激刺激下C2C12細胞內(nèi)游離Ca~(2+)濃度([Ca~(2+)]i)的變化。Western Blot法檢測各組C2C12細胞Nrf2以及肌漿網(wǎng)鈣調(diào)控蛋白Ry R、SERCA1、SERCA2、FKBP12、Ca M、Ca MKⅡ、PKA蛋白表達。在體實驗采用8周齡Nrf2敲除小鼠(Nrf2 KO)和同周齡野生型小鼠(WT)為研究對象,各隨機分為安靜對照組和有氧運動組:野生安靜組(WC)、野生運動組(WE)、敲除安靜組(KC)和敲除運動組(KE),每組10只。WE組和KE組進行8周跑臺有氧運動訓(xùn)練,方案為:跑速12m/min,運動強度持續(xù)時間60min/d,運動頻率5d/w。熒光比色法檢測各組小鼠骨骼肌ROS水平�？梢姽獗壬珯z測骨骼肌組織鈣離子濃度。熒光定量PCR測定各組小鼠骨骼肌Nrf2、NQO1、HO-1和CAT m RNA的表達。Western Blot法檢測各組小鼠骨骼肌Nrf2、NQO1、HO-1和CAT以及肌漿網(wǎng)鈣調(diào)控蛋白Ry R、SERCA1、SERCA2、FKBP12、Ca M、Ca MKⅡ、PKA蛋白表達。研究結(jié)果:(1)與對照組相比,Nrf2抑制劑組C2C12細胞ROS水平非常顯著增加;(2)在H_2O_2氧化應(yīng)激刺激下,與對照組相比,Nrf2抑制劑組C2C12細胞[Ca~(2+)]i在185.4秒后非常顯著增加,而Nrf2激動劑組[Ca~(2+)]i在61.8秒后顯著降低;(3)與對照組相比,Nrf2抑制劑組Nrf2蛋白表達顯著降低,肌漿網(wǎng)鈣調(diào)節(jié)蛋白Ry R、FKBP12、PKA顯著增加,SERCA1和SERCA2蛋白顯著降低,而Nrf2激動劑組蛋白表達無顯著變化;(4)與WE組相比,KE組ROS水平顯著降低;(5)與WC組相比,KC組Nrf2、NQO1和CAT m RNA表達非常顯著降低,而WE組Nrf2、NQO1和HO-1 m RNA非常顯著增加;與WE組相比,Nrf2、NQO1、HO-1和CAT m RNA表達均顯著降低;(6)與WC組相比,KC組Nrf2、PKA和SERCA1蛋白表達降低,肌漿網(wǎng)鈣調(diào)節(jié)蛋白Ry R、FKBP12、Ca M表達增加;WE組較WC組Ry R(P0.05)和Ca MKⅡ表達增加;與WE組相比,KE組Nrf2、NQO1、HO-1和CAT表達均減少,而肌漿網(wǎng)鈣調(diào)節(jié)蛋白PKA和Ca MKⅡ表達顯著增加;對于Nrf2 KO小鼠,KE組較KC組Ca M表達減少,而PKA、Ca MKⅡ和SERCA1表達顯著增加;(7)各組小鼠骨骼肌組織[Ca~(2+)]無顯著性差異。結(jié)論:(1)Nrf2在H_2O_2介導(dǎo)的C2C12細胞[Ca~(2+)]i異常增加中起到保護作用;(2)Nrf2的缺失可引起小鼠骨骼肌肌漿網(wǎng)鈣釋放作用增強、鈣回收作用減弱;而八周有氧運動訓(xùn)練明顯改善了Nrf2敲除鼠肌漿網(wǎng)的鈣回收功能。
[Abstract]:Objective: Nrf2 is an important nuclear transcription factor in vivo. The Nrf2 plays an important role in the contraction of skeletal muscle. In this study, we studied the relationship between Nrf2 and Ca~(2 in the sarcoplasmic reticulum of skeletal muscle. The role of regulation in the process. To study the role and mechanism of Nrf2 in the regulation of Ca~(2 in mouse skeletal muscle sarcoplasmic reticulum by aerobic training. Methods: mouse skeletal muscle C2C12 cell line was used in vitro. Nrf2 agonist t-BHQ and Nrf2 inhibitor Brusatol were used to intervene. Fluorescence colorimetric assay was used to detect the ROS level of C2C12 cells in each group. Ca~(2 was used. Dye Fluo-4 AM loaded cells. The concentration of free Ca~(2 in C2C12 cells stimulated by oxidative stress was detected by laser scanning confocal microscopy (LSCM). [Ca~(2). Western Blot assay was used to detect the expression of Nrf2 in C2C12 cells and the expression of the sarcoplasmic reticulum calcium regulatory protein (Ry RtSERCA1) in C2C12 cells. SERCA2, FKBP12, Ca MN, Ca MK 鈪，

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