本文選題：心肌細(xì)胞 + 缺血-再灌注�。� 參考：《川北醫(yī)學(xué)院》2012年碩士論文

【摘要】：目的： calpain在心肌缺血-再灌注（ischemia-reperfusion，I-R）損傷中發(fā)揮重要作用。μ-和m-calpain是哺乳動(dòng)物組織普遍表達(dá)的calpain亞型，它們?cè)谛募-R過程中是否都發(fā)生活化呢？目前仍不清楚。持續(xù)心肌缺血引起顯著的細(xì)胞壞死，再灌注時(shí)由于能量代謝恢復(fù)反而加強(qiáng)細(xì)胞凋亡。線粒體通透性轉(zhuǎn)換孔（mitochondrial permeability transition pore, mPTP）開放被認(rèn)為是線粒體凋亡途徑激活的關(guān)鍵步驟�；罨腸alpain是否通過誘導(dǎo)mPTP開放導(dǎo)致I-R心肌細(xì)胞凋亡呢？未見相關(guān)文獻(xiàn)報(bào)道。本研究擬通過分析心肌I-R過程中μ-和m-calpain的活化情況及calpain活性與mPTP開放的關(guān)系，揭示calpain在I-R心肌細(xì)胞凋亡發(fā)生中的作用及機(jī)制。 方法： 原代培養(yǎng)C57BL/6乳鼠心肌細(xì)胞，通過去糖、缺氧模擬缺血，復(fù)糖、復(fù)氧模擬再灌注，建立I-R損傷模型。為研究calpain的活化情況，將培養(yǎng)2-3天的心肌細(xì)胞隨機(jī)分為正常組、缺血6小時(shí)（hour，h）組及缺血6 h后再灌注3 h、6 h、12 h組，采用臺(tái)盼藍(lán)拒染法檢測(cè)細(xì)胞存活率；蛋白質(zhì)印跡技術(shù)（Western Blot）檢測(cè)fodrin降解產(chǎn)物（fodrin breakdownproduct，F(xiàn)BDP）及μ-和m-calpain催化亞基N末端蛋白表達(dá)。為研究calpain活性與mPTP開放的關(guān)系，將培養(yǎng)2-3天的心肌細(xì)胞隨機(jī)分為正常對(duì)照組（Control）、PD150606處理組（PD）、I-R組以及PD+I/R組。采用Western Blot檢測(cè)FBDP蛋白表達(dá)；臺(tái)盼藍(lán)拒染試驗(yàn)檢測(cè)細(xì)胞存活率；原位末端轉(zhuǎn)移酶標(biāo)記技術(shù)（terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay，TUNEL）檢測(cè)細(xì)胞凋亡；分光光度法檢測(cè)Caspase-3活性；鈣黃綠素-鈷共孵育法檢測(cè)mPTP開放情況；以JC-1為熒光探針結(jié)合激光掃描共聚焦技術(shù)檢測(cè)線粒體膜電位（Δψm）水平。 結(jié)果： 缺血6 h后再灌注12 h組細(xì)胞存活率顯著低于缺血6 h組（65.68±3.78% vs. 81.70±3.91%, p0.05）。與正常組比較，，缺血6 h組FBDP蛋白表達(dá)無明顯改變；缺血6 h后再灌注3 h、6 h、12 h組FBDP蛋白表達(dá)顯著升高（p0.05）。正常組、缺血6 h組未檢測(cè)到μ-calpain催化亞基N末端自動(dòng)裂解片段；缺血6 h后再灌注3 h、6 h、12 h組檢測(cè)到μ-calpain催化亞基N末端自動(dòng)裂解片段；所有分組均未檢測(cè)到m-calpain催化亞基N末端自動(dòng)裂解片段。I-R組細(xì)胞活力低于Control和PD組（p0.01），而PD+I-R組高于I-R組（p0.05）；I-R組TUNEL陽性核比率、Caspase-3活性高于Control和PD組（p0.01），而PD+I-R組低于I-R組（p0.05）；I-R引起顯著的mPTP開放和Δψm下降（I-R與Control和PD組比較，p0.01），PD150606預(yù)處理可抑制這種效應(yīng)（PD+I-R與I-R組比較，p0.05）。 結(jié)論： 心肌I-R誘導(dǎo)μ-calpain活化，抑制calpain活性防止I-R介導(dǎo)的mPTP開放和Δψm降低，從而減少心肌細(xì)胞凋亡。本研究提示calpain可能通過誘導(dǎo)mPTP開放致I-R心肌細(xì)胞凋亡。
[Abstract]:Objective: calpain plays an important role in myocardial ischemia-reperfusion (I-R) injury. 渭-and m-calpain are commonly expressed calpain subtypes in mammalian tissues. Are they all activated during myocardial I-R? It is still unclear. Sustained myocardial ischemia resulted in significant cell necrosis and increased cell apoptosis during reperfusion due to recovery of energy metabolism. The opening of mitochondrial permeability transition pore (mitochondrial permeability transition pore, mPTP is considered to be a key step in activation of mitochondrial apoptosis pathway. Does activated calpain induce apoptosis of I-R cardiomyocytes by inducing mPTP opening? No related literature was reported. By analyzing the activation of 渭-and m-calpain and the relationship between the activity of calpain and the opening of mPTP in myocardial I-R, the role and mechanism of calpain in apoptosis of I-R cardiomyocytes were revealed. Methods: C57BL / 6 neonatal rat cardiomyocytes were cultured in primary culture. I-R injury model was established by hypoxia-simulated ischemia, resucrose, reoxygenation and reperfusion. In order to study the activation of calpain, cardiomyocytes cultured for 2-3 days were randomly divided into normal group, ischemia 6 hour group and reperfusion group 3 h and 6 h after ischemia for 12 h. The cell survival rate was measured by trypan blue exclusion method. Western blot was used to detect the expression of fodrin breakdown product (FBP), 渭-and m-calpain catalytic subunit N-terminal protein. To study the relationship between the activity of calpain and the opening of mPTP, cardiomyocytes cultured for 2-3 days were randomly divided into normal control group (control) treated with PD150606 (PD) I-R group and PD I / R group. The expression of FBDP protein was detected by Western blot, the cell survival rate was detected by trypan blue exclusion assay, the apoptosis was detected by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling Tunel, the activity of Caspase-3 was detected by spectrophotometry. The level of mitochondrial membrane potential (螖 蠄 m) was detected by co-incubation of calcium xanthine and cobalt, using JC-1 as fluorescence probe and laser scanning confocal technique. Results: the cell survival rate in 12 h reperfusion group was significantly lower than that in 6 h ischemia group (65.68 鹵3.78% vs 81.70 鹵3.91, p0.05). Compared with the normal group, the expression of FBDP protein did not change significantly in the 6h ischemia group, but the FBDP protein expression increased significantly (p0.05) in the 12h group at 3h after reperfusion (p0.05). In the normal group, the N-terminal fragment of the catalytic subunit of 渭 -calpain was not detected in the 6h ischemia group, but the N-terminal fragment was detected at the end of the N-terminal of the catalytic subunit of 渭 -calpain after reperfusion for 3 h and 6 h / 12 h after ischemia for 6 h after ischemia. In all groups, the activity of Caspase-3 was higher in PD I-R group than that in control and PD group (p0.01), but the activity of Caspase-3 in PD I-R group was higher than that in control and PD group (p0.01), but the activity of Caspase-3 in PD I-R group was lower than that in I-R group (p0.05). The activity of Caspase-3 in PD I-R group was higher than that in control and PD group (p0.01). The activity of Caspase-3 in PD I-R group was higher than that in Control and PD group (p0.01). I-R induced significant mPTP opening and 螖 蠄 m decrease (P 0.01 compared with control and PD groups). Pretreatment with PD150606 could inhibit this effect (p0.05 compared with I-R group and I-R group). Conclusion: myocardial I-R induces the activation of 渭 -calpain, inhibits the activity of calpain, prevents I-R-mediated mPTP opening and 螖 蠄 m decrease, and thus reduces the apoptosis of cardiac myocytes. This study suggests that calpain may induce apoptosis of I-R cardiomyocytes by mPTP.
【學(xué)位授予單位】：川北醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

【共引文獻(xiàn)】

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本文編號(hào)：2096243