發(fā)布時間：2018-06-07 03:02

  本文選題：H9C2心肌細胞株 + 凋亡��； 參考：《安徽醫(yī)科大學》2016年博士論文

【摘要】：研究背景與目的:缺血再灌注損傷是在血栓性疾病中常見的現(xiàn)象。缺血心肌在得到再灌注的同時,由于激活免疫反應而導致梗塞周圍出現(xiàn)更多的損傷,并與心肌細胞凋亡的增加有關。損傷的結果會導致心肌細胞的丟失和梗塞面積的擴大。因此,研究缺血再灌注損傷所致的心肌細胞凋亡具有重要的臨床意義。有多種信號傳導通路參與心肌細胞的凋亡,TLR4-NF-κB通路在其中起著重要的作用。TLR4是TOLL系列受體之一,能被多種刺激信號激活,如應激、炎癥、壞死等。TLR4主要在心臟表達,將信號傳導至胞內(nèi)并在酶的作用下活化NF-κB,而NF-κB則進入核內(nèi)引起相關基因的表達,促進炎癥因子的產(chǎn)生。β-arrestin被認為是一種多功能受體蛋白,參與GPCR的脫敏。β-arrestin通過結合NF-κB的抑制因子IкBα而調(diào)控NF-κB基因的表達。因此,β-arrestin被認為是TLR4-NF-κB信號通路的負調(diào)控因子。卡維地洛是一種新型的非選擇性β受體阻滯劑,應用于治療高血壓和心絞痛。在慢性心力衰竭患者中,卡維地洛能夠降低其住院率和死亡率。在多項臨床研究中,相對于其他β受體阻滯劑,卡維地洛在抑制左室重構和保持射血分數(shù)方面顯示出顯著的優(yōu)勢。這種作用涉及多種機制,抑制心肌細胞凋亡是其中之一。然而,有關卡維地洛對TLR4-NF-κB介導的心肌細胞凋亡的影響尚不清楚。卡維地洛是否能對β-arrestin產(chǎn)生影響更有少見報道。因此,我們準備兩部分實驗來探討卡維地洛對缺血再灌注損傷心肌細胞模型中TLR4-NF-κB通路的作用以及β-arrestin表達的影響。材料與方法:第一部分H9C2心肌細胞株的培養(yǎng)和缺血/再灌注模型建立方法:培養(yǎng)H9C2心肌細胞株,采用缺氧復氧模型模擬心肌缺血再灌注損傷,缺氧4小時后按照復氧時間不同分為5組:對照組,復氧4h組,復氧6h組,復氧10h組。觀察指標包括熒光顯微鏡下細胞生長形態(tài)變化,mtt值,培養(yǎng)液中l(wèi)dh、mda及sod含量。結果:H9C2心肌細胞株在體外培養(yǎng)成功,并建立缺氧復氧模型。缺氧復氧后細胞形態(tài)鏡下發(fā)生明顯變化,表現(xiàn)為核染色細胞增多。在實驗組中,mtt值顯著下降,并隨著復氧時間延長而更為顯著。ldh和mda含量分別顯著增加,sod則顯著降低,具有顯著性差異。結論:本研究在體外通過缺氧復氧而成功構建H9C2心肌細胞模擬缺血再灌注模型,缺氧4小時復氧6小時可造成顯著心肌細胞損傷,為進一步實驗提供標本。第二部分H9C2心肌細胞凋亡中tlr4-nfκb、β-arrestin的表達和卡維地洛的作用方法:采用不同劑量卡維地洛(1um、5um、10umol/l)、tlr4封閉抗體(20ug/ml)、nf-κb抑制劑pdtc(100umol/l)預孵育H9C2心肌細胞1h后,建立模擬缺血再灌注模型。實驗分為7組:正常對照組(control組),模擬缺血再灌注組(sd組),小劑量組卡維地洛組(car1卡維地洛1umol/l),中劑量組卡維地洛組(car5卡維地洛5umol/l),高劑量組卡維地洛組(car10卡維地洛10umol/l),tlr4封閉抗體組(tlr4-ab組tlr4封閉抗體20ug/ml),nf-κb抑制劑pdtc組(pdtc組pdtc100umol/l)。應用蛋白質(zhì)印跡技術檢測細胞凋亡蛋白bax,bcl-2。通過熒光定量pcr法測定tlr4、核蛋白nf-κb、β-arrestin的表達。結果:H9C2心肌細胞株在i/r后,與正常對照組相比,tunnel檢測細胞凋亡率增高,bax升高,bcl-2下降,tlr4和nf-κb表達增多,β-arrestin表達減少。在經(jīng)過卡維地洛預處理的H9C2i/r模型中,與i/r組相比bcl-2明顯增加,細胞凋亡率和bax顯著降低。通過rt-pcr技術檢測,在卡維地洛預處理組中,tlr4和nf-κb表達量減少,β-arrestin表達增多。上述指標的變化,在中、高劑量卡維地洛組中更為顯著。tlr4抗體和nf-κb阻斷劑(pdtc)預處理H9C2心肌細胞,與i/r組相比,可表現(xiàn)出與卡維地洛組相同的顯著改變。結論:1.TLR4-NF-κB信號通路與H9C2心肌細胞株I/R所致凋亡過程有關2.卡維地洛可以通過抑制TLR4/NF-κB表達而減輕H9C2心肌細胞I/R所致的凋亡,在中、高劑量組中更為顯著3.卡維地洛可能會激活β-arrestin,負調(diào)控TLR4-NF-κB信號。
[Abstract]:Background and purpose: ischemia-reperfusion injury is a common phenomenon in thrombotic diseases. Ischemic myocardium is involved in reperfusion and causes more damage around the infarct due to the activation of the immune response. It is related to the increase of apoptosis of myocardial cells. The result of injury will lead to the loss of myocardial cells and the enlargement of infarct size. Therefore, the study of myocardial apoptosis induced by ischemia-reperfusion injury is of important clinical significance. There are a variety of signal transduction pathways involved in the apoptosis of cardiac myocytes. TLR4-NF- kappa B pathway plays an important role in.TLR4, one of the TOLL series receptors, which can be activated by a variety of stimuli, such as stress, inflammation, and necrosis, mainly in the heart. Dirty expression, transmit the signal to the intracellular and activate NF- kappa B under the action of enzyme, while NF- kappa B enters the nucleus to induce the expression of related genes and promote the production of inflammatory factors. Beta -arrestin is considered to be a kind of multifunctional receptor protein and participates in desensitization of GPCR. The expression of beta -arrestin regulates the expression of NF- kappa gene by binding NF- kappa B inhibitor I B alpha. Therefore, beta -arrestin is considered to be a negative regulator of the TLR4-NF- kappa B signaling pathway. Carvedilol is a new non selective beta blocker, used in the treatment of hypertension and angina. In patients with chronic heart failure, carvedilol can reduce the rate of hospitalization and mortality. In a number of clinical studies, it is relative to other beta receptors. The effect of carvedilol on TLR4-NF- kappa B induced cardiomyocyte apoptosis is not clear. Whether carvedilol can produce a shadow of beta -arrestin, however, is not clear about the effect of carvedilol on the apoptosis of cardiac myocytes. There are more rare reports. Therefore, we prepare two experiments to explore the effect of carvedilol on the TLR4-NF- kappa B pathway in the myocardial cell model of ischemia-reperfusion injury and the effect of the expression of beta -arrestin. Materials and methods: the first part of the culture of H9C2 cardiomyocytes and the establishment of ischemia / reperfusion model: the cultivation of H9C2 cardiac myocytes. A hypoxic reoxygenation model was used to simulate myocardial ischemia reperfusion injury, and 4 hours after hypoxia were divided into 5 groups according to the time of reoxygenation: control group, reoxygenation 4H group, reoxygenation 6h group and reoxygenation 10h group. The observation indexes include the changes of cell growth morphology under fluorescence microscope, MTT value, LDH, MDA and SOD content in the culture medium. Results: H9C2 cardiac myocytes were cultured in vitro. In the experimental group, the MTT value decreased significantly, and the content of.Ldh and MDA increased significantly with the prolonged reoxygenation time, and the sod decreased significantly. Conclusion: This study was in vitro. The model of H9C2 myocardial ischemia reperfusion was successfully constructed by hypoxia and reoxygenation. 4 hours anoxic reoxygenation for 6 hours could cause significant myocardial damage and provide specimens for further experiments. Second part of H9C2 cardiomyocyte apoptosis, the expression of tlr4-nf kappa B, the expression of beta -arrestin and the action method of Carvedilol (1 Um, 5um, 10umol/l), TLR4 closed antibody (20ug/ml), nf- kappa B inhibitor PDTC (100umol/l) preincubated H9C2 myocardial cells 1H, the model of simulated ischemia reperfusion was established. The experiment was divided into 7 groups: normal control group (control group), simulated ischemia reperfusion group, carvedilol group (carvedilol) and medium dose group carvedilol group. 5 carvedilol 5umol/l), high dose carvedilol group (car10 carvedilol 10umol/l), TLR4 closed antibody group (tlr4-ab group TLR4 closed antibody 20ug/ml), nf- kappa B inhibitor PDTC group (PDTC group pdtc100umol/l). N expression. Results: compared with the normal control group, the apoptosis rate of H9C2 cells increased, Bax increased, bcl-2 decreased, TLR4 and nf- kappa B expression increased, and the expression of beta -arrestin decreased. In the H9C2i/r model pretreated by carvedilol, the rate of apoptosis was significantly increased and the rate of apoptosis decreased significantly. After RT-PCR test, the expression of TLR4 and nf- kappa B decreased and the expression of beta -arrestin increased in the carvedilol preconditioning group. In the high dose carvedilol group, a more significant.Tlr4 antibody and nf- kappa B blocker (PDTC) pretreated the H9C2 cardiomyocytes in the high dose carvedilol group. Compared with the I /r group, the same significant changes were shown in the carvedilol group. Conclusion: 1.TLR4-NF- kappa B signaling pathway is related to the apoptosis process induced by H9C2 myocardial cell line I/R. 2. carvedilol can reduce the apoptosis of I/R induced by H9C2 cardiomyocytes by inhibiting the expression of TLR4/NF- kappa B. In the high dose group, more significantly 3. carvedilol may activate beta -arrestin and negatively regulate TLR4-NF- kappa B signal.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R54

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本文編號：1989439