【摘要】：目的：槲皮素甲基化改造得到的五甲基槲皮素（PMQ），對心肌缺血再灌注損傷具有保護作用。本實驗采用原代SD大鼠乳鼠心肌細胞，建立心肌缺氧/復(fù)氧（anoxia/re-oxygenation, A/R）損傷模型，探討五甲基槲皮素預(yù)處理對大鼠心肌細胞缺氧/復(fù)氧損傷的保護作用及其對線粒體功能的影響。 方法：培養(yǎng)原代SD大鼠乳鼠心肌細胞，隨機分為8組，每組實驗重復(fù)3次：（1）正常對照組（Cont）；（2）Cont+DMSO組；（3）缺氧/復(fù)氧組（A/R）；（4）A/R+DMSO組；（5）缺氧預(yù)適應(yīng)的延遲相組（DPC）；（6）10μmol/L PMQ+A/R組；（7）30μmol/L PMQ+A/R組；（8）100μmol/L PMQ+A/R組。用MTT比色法檢測細胞存活率,Beckman生化自動分析儀檢測培養(yǎng)液中乳酸脫氫酶(LDH)活性，，酶聯(lián)免疫檢測儀測定各組脂質(zhì)過氧化物丙二醛（MDA）含量、抗氧化物酶（SOD）活性、谷胱甘肽過氧化物酶（GSH-Px）的活性；流式細胞法檢測線粒體膜電位（Δψm）以及細胞凋亡情況、線粒體腫脹法檢測各組心肌細胞線粒體通透性轉(zhuǎn)換孔(mPTP)開放情況。 結(jié)果：與Cont組相比，A/R組的各項指標均顯示細胞凋亡增加（P0.01）,說明A/R模型構(gòu)建成功；與A/R組相比，DPC組的各項指標均顯示細胞凋亡減少，說明DPC模型構(gòu)建成功（P0.01）；與A/R組相比，不同劑量PMQ (10,30,100μmol/L)預(yù)處理24h組，其呈現(xiàn)劑量依賴性的使LDH活性降低、細胞存活率增加、MDA含量顯著減少、SOD酶活性顯著上升、GSH-Px酶活性顯著上升、細胞凋亡減少（P0.05或P0.01）；與A/R組相比，30,100μmol/L PMQ預(yù)處理24h組，其線粒體膜電位更為穩(wěn)定、mPTP開放減少（P0.05或P0.01）。 結(jié)論：PMQ預(yù)處理24h后，可產(chǎn)生藥理性延遲保護作用，其作用機制與抑制氧化應(yīng)激、穩(wěn)定線粒體膜電位、抑制mPTP開放，進而減少細胞凋亡有關(guān)。
[Abstract]:Aim: to study the protective effect of pentamethylquercetin (PMQ), on myocardial ischemia reperfusion injury. In this study, we established myocardial hypoxia / reoxygenation (anoxia/re-oxygenation, A / R) injury model by primary SD rat neonatal cardiomyocytes, and investigated the protective effect of pentamethylquercetin preconditioning on hypoxia / reoxygenation injury of rat cardiomyocytes and its effect on mitochondrial function. Methods: primary SD rat cardiomyocytes were cultured and randomly divided into 8 groups: (1) normal control group (Cont); (2) Cont DMSO group, (3) anoxic / reoxygenated group (A / R); (4) A / R DMSO group, (5) anoxic preconditioning delayed phase group (DPC); (6) 10 渭 mol/L PMQ A / R group, and (3) anoxia / reoxygenation group (A / R); (4) A / R DMSO group. (7) 30 渭 mol/L PMQ A / R group and (8) 100 渭 mol/L PMQ A / R group. The cell survival rate was detected by MTT colorimetry, the activity of lactate dehydrogenase (LDH) was detected by Beckman biochemical automatic analyzer, the content of lipid peroxide malondialdehyde (MDA) and the activity of antioxidant enzyme (SOD) (SOD) were measured by enzyme-linked immunosorbent assay (Elisa). The activity of glutathione peroxidase (GSH-Px), mitochondrial membrane potential (螖 蠄 m) and apoptosis were detected by flow cytometry, and the opening of mitochondrial permeability transition pore (mPTP) was detected by mitochondrial swelling method. Results: compared with the Cont group, all the indexes in the A / R group showed an increase in apoptosis (P0.01), which indicated that the A / R model was successfully constructed, the DPC group showed a decrease in apoptosis, which indicated that the DPC model was successfully constructed (P0.01), and compared with the A / R group, all the indexes in the DPC group showed that the model was successfully constructed (P0.01). After pretreatment with different doses of PMQ (10 ~ 30100 渭 mol/L) for 24 h, the LDH activity decreased in a dose-dependent manner, the cell survival rate increased, the MDA content decreased, the activity of SOD increased significantly, the activity of GSH-Px increased significantly and the apoptosis of cells decreased in a dose-dependent manner (P0.05 or P0.01). Compared with the A / R group, the mitochondrial membrane potential of 30100 渭 mol/L PMQ pretreated for 24 hours was more stable and the opening of mPTP was decreased (P0.05 or P0.01). Conclusion: after pretreatment with PMQ for 24 h, the pharmacological delayed protective effect can be produced. The mechanism is related to the inhibition of oxidative stress, the stabilization of mitochondrial membrane potential, the inhibition of opening of mPTP, and the reduction of apoptosis.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R96

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