內(nèi)質(zhì)網(wǎng)應(yīng)激在內(nèi)源性心肌細(xì)胞保護(hù)中的作用及其信號(hào)轉(zhuǎn)導(dǎo)機(jī)制研究
發(fā)布時(shí)間：2018-08-26 16:59
【摘要】： 心肌缺血是心肌重構(gòu)和心力衰竭的主要原因，減輕心肌缺血損傷是防治心力衰竭的關(guān)鍵，盡早恢復(fù)缺血心肌血流是減輕心肌缺血損傷的根本措施。但是，經(jīng)過(guò)一定時(shí)間缺血的組織器官在恢復(fù)血液灌注后，會(huì)出現(xiàn)不可逆性再灌注損傷。探索激發(fā)機(jī)體內(nèi)源性保護(hù)機(jī)制，減輕缺血再灌注(ischemia/reperfusion, I/R)損傷是防治心肌缺血的關(guān)鍵。缺血預(yù)處理(ischemic preconditioning, IPC)及缺血后處理(ischemic postconditioning, I-postC)是近年發(fā)現(xiàn)的重要內(nèi)源性保護(hù)機(jī)制，可明顯縮小缺血心肌的梗死面積，減輕I／R損傷。探討IPC和I-postC心臟保護(hù)的共同及不同機(jī)制具有重要的科學(xué)意義，并將為其臨床應(yīng)用提供新的思路。 內(nèi)質(zhì)網(wǎng)(endoplasmic reticulum, ER)是真核細(xì)胞中重要細(xì)胞器，I／R過(guò)程中的多種刺激因素，包括缺氧、酸中毒、ATP耗竭、氧化應(yīng)激、鈣超載等均可導(dǎo)致內(nèi)質(zhì)網(wǎng)功能失調(diào)，即內(nèi)質(zhì)網(wǎng)應(yīng)激(ER stress, ERS)。一定程度的ERS可誘導(dǎo)葡萄糖調(diào)節(jié)蛋白類(glucose-regulated proteins, GRPs)、鈣網(wǎng)蛋白(calreticulin, CRT)、折疊酶等ER伴侶分子表達(dá)上調(diào)，增強(qiáng)ER處理未折疊蛋白的能力，促進(jìn)ER功能恢復(fù)；ERS持續(xù)存在或過(guò)強(qiáng)時(shí)將誘導(dǎo)caspase-12、CHOP／GADD153等促凋亡因子的表達(dá)／活化，觸發(fā)ER相關(guān)性凋亡信號(hào)途徑，誘導(dǎo)細(xì)胞凋亡。持續(xù)而嚴(yán)重的ERS是由I／R誘導(dǎo)并導(dǎo)致組織細(xì)胞損傷的重要機(jī)制。IPC、I-postC是否可通過(guò)調(diào)節(jié)ERS反應(yīng)，增強(qiáng)細(xì)胞對(duì)應(yīng)激因素的耐受能力，緩解ER應(yīng)激程度，從而減輕I／R損傷?本工作采用乳鼠心肌細(xì)胞缺氧／復(fù)氧(hypoxia／reoxygenation, H／R)模型模擬在體I／R損傷，觀察缺氧預(yù)處理(hypoxic preconditioning, HPC)和缺氧后處理(hypoxic postconditioning, H-postC)對(duì)于ER應(yīng)激分子GRP78、CRT表達(dá)及caspase-12活化的影響及其與心肌細(xì)胞保護(hù)的關(guān)系，并探討HPC和H-postC調(diào)節(jié)ERS、介導(dǎo)心肌細(xì)胞保護(hù)的細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)機(jī)制。主要方法與結(jié)果如下： 原代培養(yǎng)的Sprague-Dawley乳鼠心肌細(xì)胞缺氧2 h復(fù)氧14 h復(fù)制H／R模型，HPC組細(xì)胞缺氧20 min復(fù)氧24 h后進(jìn)行H／R操作；H-postC組細(xì)胞缺氧2h后先進(jìn)行3輪5 min復(fù)氧／5 min缺氧的缺氧后處理，再進(jìn)行持續(xù)復(fù)氧14 h結(jié)束實(shí)驗(yàn)。 1．HPC和H-postC對(duì)于心肌細(xì)胞H／R損傷的影響采用臺(tái)盼藍(lán)排斥實(shí)驗(yàn)檢測(cè)細(xì)胞存活率；采用LDH活性測(cè)試盒檢測(cè)細(xì)胞培養(yǎng)液中LDH漏出情況；采用Annexin V-FITC細(xì)胞凋亡檢測(cè)試劑盒檢測(cè)細(xì)胞凋亡率。結(jié)果顯示：HPC、H-postC可明顯提高H／R心肌細(xì)胞的存活率，減輕細(xì)胞凋亡和LDH漏出。 2．HPC、H-postC對(duì)ERS分子表達(dá)／活化的影響采用逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(reverse transcription-polymerase chain reaction, RT-PCR)方法檢測(cè)細(xì)胞GRP 78 mRNA水平；Western blot分析檢測(cè)CRT表達(dá)和caspase-12活化水平。結(jié)果表明：H／R誘導(dǎo)細(xì)胞GRP 78 mRNA、CRT表達(dá)和caspase-12活化上調(diào)，HPC及H-postC使H／R細(xì)胞GRP 78 mRNA上調(diào)程度增加，減輕H／R后CRT過(guò)表達(dá)程度及caspase-12活化水平，但H-postC對(duì)H／R心肌細(xì)胞caspase-12活化的抑制效果較HPC差。 3．HPC、H-postC調(diào)節(jié)ERS的信號(hào)途徑研究于HPC、H-postC前應(yīng)用p38 MAPK特異性抑制劑SB203580及JNK特異性抑制劑SP600125，分別在HPC及H-postC處理前加入細(xì)胞培養(yǎng)液(終濃度分別為5μmol／L、25μmol／L，37℃預(yù)孵育5-10 min)。結(jié)果顯示：(1)HPC前應(yīng)用SB203580可明顯抑制CRT表達(dá)上調(diào)并明顯減弱HPC抑制caspase-12活化、減輕心肌細(xì)胞H／R損傷的作用；而應(yīng)用SP600125對(duì)CRT表達(dá)和caspase-12活化水平和HPC的心肌細(xì)胞保護(hù)作用均無(wú)明顯影響。(2)H-postC前應(yīng)用SB203580可明顯抑制CRT表達(dá)上調(diào)，并降低H-postC對(duì)細(xì)胞H／R損傷的保護(hù)，但僅輕度減弱H-postC對(duì)caspase-12活化的抑制作用；H-postC前應(yīng)用SP600125對(duì)CRT表達(dá)及H-postC細(xì)胞保護(hù)作用未產(chǎn)生明顯影響，但可進(jìn)一步降低caspase-12的活化水平。 結(jié)論：HPC及H-postC均可減輕H／R心肌細(xì)胞的損傷及凋亡，，二者對(duì)細(xì)胞的保護(hù)水平相當(dāng)；其細(xì)胞保護(hù)機(jī)制均涉及p38 MAPK介導(dǎo)的ERS調(diào)節(jié)及ERS相關(guān)細(xì)胞凋亡途徑的抑制。但HPC對(duì)H／R誘導(dǎo)caspase-12活化的抑制效果較H-postC為強(qiáng)。JNK活化可促進(jìn)caspase-12激活，可能參與H／R誘導(dǎo)的過(guò)度ERS反應(yīng)及ERS介導(dǎo)的細(xì)胞凋亡。
[Abstract]:Myocardial ischemia is the main cause of myocardial remodeling and heart failure. Reducing myocardial ischemic injury is the key to prevent and treat heart failure. Early recovery of ischemic myocardial blood flow is the fundamental measure to reduce myocardial ischemic injury. The key to preventing and treating myocardial ischemia is to stimulate the endogenous protective mechanism of organism and to reduce ischemia/reperfusion (I/R) injury. Ischemic preconditioning (IPC) and ischemic postconditioning (I-postC) are important endogenous protective mechanisms discovered in recent years, which can significantly reduce the ischemic myocardium. It is of great scientific significance to explore the common and different mechanisms of IPC and I-postC cardioprotection and to provide new ideas for its clinical application.
Endoplasmic reticulum (ER) is an important organelle in eukaryotic cells. Many stimulating factors in I/R process, including hypoxia, acidosis, ATP depletion, oxidative stress, calcium overload and so on, can lead to endoplasmic reticulum dysfunction, that is, endoplasmic reticulum stress (ERS). The up-regulation of ER chaperone molecules such as Ted proteins, GRPs, calreticulin (CRT) and folding enzyme enhances the ability of ER to treat unfolded proteins and promotes the recovery of ER function; the persistence or excessive presence of ERS induces the expression and activation of pro-apoptotic factors such as caspase-12, CHOP/GAD153, triggers ER-related apoptotic signaling pathways and induces cell apoptosis. Apoptosis. Persistent and severe ERS is an important mechanism by which I/R induces tissue and cell damage. Can IPC and I-postC enhance the tolerance of cells to stress factors and alleviate ER stress so as to reduce I/R injury by regulating the response of ERS? The effects of hypoxic preconditioning (HPC) and hypoxic postconditioning (H-postC) on the expression of ER stress molecules GRP78, CRT and caspase-12 activation and their relationship with myocardial cell protection were observed. The relationship between HPC and H-postC regulating ERS and mediating myocardial cell protection was also discussed. The main methods and results are as follows:
The primary cultured Sprague-Dawley neonatal rat cardiomyocytes were hypoxic for 2 h and reoxygenated for 14 h to replicate the H/R model. The HPC group was hypoxic for 20 min and reoxygenated for 24 h, and the H-postC group was hypoxic for 2 h, followed by 3 rounds of 5 min reoxygenation/5 min hypoxic postconditioning followed by 14 h continuous reoxygenation.
1. The effects of HPC and H-postC on H/R injury of cardiomyocytes were detected by Trypan blue rejection assay; LDH leakage was detected by LDH activity test kit; and apoptosis was detected by Annexin V-FITC cell apoptosis test kit. The results showed that HPC and H-postC could significantly improve H/R myocardial cells. The survival rate alleviated cell apoptosis and LDH leakage.
2. The effects of HPC and H-postC on the expression and activation of ERS were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respectively. The up-regulation of se-12 activation, the up-regulation of GRP 78 mRNA in H/R cells by HPC and H-postC, and the down-regulation of CRT overexpression and caspase-12 activation after H/R were alleviated. However, the inhibition effect of H-postC on the activation of caspase-12 in H/R myocardial cells was worse than that of HPC.
3. The signal pathway of regulating ERS by HPC and H-postC was studied before HPC and H-postC were treated with SB203580, a p38 MAPK specific inhibitor, and SP600125, a JNK specific inhibitor. Cell culture medium was added before HPC and H-postC treatment (the final concentration was 5 micromol/L, 25 micromol/L, pre-incubated at 37 C for 5-10 minutes). The results showed that: (1) SB203580 could be used before HPC treatment. SP600125 had no significant effect on CRT expression, caspase-12 activation level and myocardial protective effect of HPC. (2) SB203580 before H-postC significantly inhibited the up-regulation of CRT expression and decreased H/postC on myocardial cells. The protective effect of H-postC on the activation of caspase-12 was slightly weakened, but the protective effect of SP600125 on the expression of CRT and the cytoprotection of H-postC before H-postC was not obvious, but the activation level of caspase-12 was further decreased.
CONCLUSION: Both HPC and H-postC can reduce the injury and apoptosis of H/R cardiomyocytes, and both of them have the same level of protection to H/R cardiomyocytes. The mechanism of cell protection involves the regulation of ERS mediated by p38 MAPK and the inhibition of ERS-related apoptosis pathway. 12 activation may be involved in H / R induced excessive ERS response and ERS mediated apoptosis.
【學(xué)位授予單位】：中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R363.2
本文編號(hào)：2205568
[Abstract]:Myocardial ischemia is the main cause of myocardial remodeling and heart failure. Reducing myocardial ischemic injury is the key to prevent and treat heart failure. Early recovery of ischemic myocardial blood flow is the fundamental measure to reduce myocardial ischemic injury. The key to preventing and treating myocardial ischemia is to stimulate the endogenous protective mechanism of organism and to reduce ischemia/reperfusion (I/R) injury. Ischemic preconditioning (IPC) and ischemic postconditioning (I-postC) are important endogenous protective mechanisms discovered in recent years, which can significantly reduce the ischemic myocardium. It is of great scientific significance to explore the common and different mechanisms of IPC and I-postC cardioprotection and to provide new ideas for its clinical application.
Endoplasmic reticulum (ER) is an important organelle in eukaryotic cells. Many stimulating factors in I/R process, including hypoxia, acidosis, ATP depletion, oxidative stress, calcium overload and so on, can lead to endoplasmic reticulum dysfunction, that is, endoplasmic reticulum stress (ERS). The up-regulation of ER chaperone molecules such as Ted proteins, GRPs, calreticulin (CRT) and folding enzyme enhances the ability of ER to treat unfolded proteins and promotes the recovery of ER function; the persistence or excessive presence of ERS induces the expression and activation of pro-apoptotic factors such as caspase-12, CHOP/GAD153, triggers ER-related apoptotic signaling pathways and induces cell apoptosis. Apoptosis. Persistent and severe ERS is an important mechanism by which I/R induces tissue and cell damage. Can IPC and I-postC enhance the tolerance of cells to stress factors and alleviate ER stress so as to reduce I/R injury by regulating the response of ERS? The effects of hypoxic preconditioning (HPC) and hypoxic postconditioning (H-postC) on the expression of ER stress molecules GRP78, CRT and caspase-12 activation and their relationship with myocardial cell protection were observed. The relationship between HPC and H-postC regulating ERS and mediating myocardial cell protection was also discussed. The main methods and results are as follows:
The primary cultured Sprague-Dawley neonatal rat cardiomyocytes were hypoxic for 2 h and reoxygenated for 14 h to replicate the H/R model. The HPC group was hypoxic for 20 min and reoxygenated for 24 h, and the H-postC group was hypoxic for 2 h, followed by 3 rounds of 5 min reoxygenation/5 min hypoxic postconditioning followed by 14 h continuous reoxygenation.
1. The effects of HPC and H-postC on H/R injury of cardiomyocytes were detected by Trypan blue rejection assay; LDH leakage was detected by LDH activity test kit; and apoptosis was detected by Annexin V-FITC cell apoptosis test kit. The results showed that HPC and H-postC could significantly improve H/R myocardial cells. The survival rate alleviated cell apoptosis and LDH leakage.
2. The effects of HPC and H-postC on the expression and activation of ERS were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respectively. The up-regulation of se-12 activation, the up-regulation of GRP 78 mRNA in H/R cells by HPC and H-postC, and the down-regulation of CRT overexpression and caspase-12 activation after H/R were alleviated. However, the inhibition effect of H-postC on the activation of caspase-12 in H/R myocardial cells was worse than that of HPC.
3. The signal pathway of regulating ERS by HPC and H-postC was studied before HPC and H-postC were treated with SB203580, a p38 MAPK specific inhibitor, and SP600125, a JNK specific inhibitor. Cell culture medium was added before HPC and H-postC treatment (the final concentration was 5 micromol/L, 25 micromol/L, pre-incubated at 37 C for 5-10 minutes). The results showed that: (1) SB203580 could be used before HPC treatment. SP600125 had no significant effect on CRT expression, caspase-12 activation level and myocardial protective effect of HPC. (2) SB203580 before H-postC significantly inhibited the up-regulation of CRT expression and decreased H/postC on myocardial cells. The protective effect of H-postC on the activation of caspase-12 was slightly weakened, but the protective effect of SP600125 on the expression of CRT and the cytoprotection of H-postC before H-postC was not obvious, but the activation level of caspase-12 was further decreased.
CONCLUSION: Both HPC and H-postC can reduce the injury and apoptosis of H/R cardiomyocytes, and both of them have the same level of protection to H/R cardiomyocytes. The mechanism of cell protection involves the regulation of ERS mediated by p38 MAPK and the inhibition of ERS-related apoptosis pathway. 12 activation may be involved in H / R induced excessive ERS response and ERS mediated apoptosis.
【學(xué)位授予單位】：中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R363.2
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