Calpain介导缺血—再灌注心肌细胞凋亡机制研究

发布时间：2018-07-04 13:37

本文选题：心肌细胞 + 缺血-再灌注　；参考：《川北医学院》2012年硕士论文

【摘要】：目的： calpain在心肌缺血-再灌注（ischemia-reperfusion，I-R）损伤中发挥重要作用。μ-和m-calpain是哺乳动物组织普遍表达的calpain亚型，它们在心肌I-R过程中是否都发生活化呢？目前仍不清楚。持续心肌缺血引起显著的细胞坏死，再灌注时由于能量代谢恢复反而加强细胞凋亡。线粒体通透性转换孔（mitochondrial permeability transition pore, mPTP）开放被认为是线粒体凋亡途径激活的关键步骤。活化的calpain是否通过诱导mPTP开放导致I-R心肌细胞凋亡呢？未见相关文献报道。本研究拟通过分析心肌I-R过程中μ-和m-calpain的活化情况及calpain活性与mPTP开放的关系，揭示calpain在I-R心肌细胞凋亡发生中的作用及机制。方法：原代培养C57BL/6乳鼠心肌细胞，通过去糖、缺氧模拟缺血，复糖、复氧模拟再灌注，建立I-R损伤模型。为研究calpain的活化情况，将培养2-3天的心肌细胞随机分为正常组、缺血6小时（hour，h）组及缺血6 h后再灌注3 h、6 h、12 h组，采用台盼蓝拒染法检测细胞存活率；蛋白质印迹技术（Western Blot）检测fodrin降解产物（fodrin breakdownproduct，FBDP）及μ-和m-calpain催化亚基N末端蛋白表达。为研究calpain活性与mPTP开放的关系，将培养2-3天的心肌细胞随机分为正常对照组（Control）、PD150606处理组（PD）、I-R组以及PD+I/R组。采用Western Blot检测FBDP蛋白表达；台盼蓝拒染试验检测细胞存活率；原位末端转移酶标记技术（terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay，TUNEL）检测细胞凋亡；分光光度法检测Caspase-3活性；钙黄绿素-钴共孵育法检测mPTP开放情况；以JC-1为荧光探针结合激光扫描共聚焦技术检测线粒体膜电位（Δψm）水平。结果：缺血6 h后再灌注12 h组细胞存活率显著低于缺血6 h组（65.68±3.78% vs. 81.70±3.91%, p0.05）。与正常组比较，，缺血6 h组FBDP蛋白表达无明显改变；缺血6 h后再灌注3 h、6 h、12 h组FBDP蛋白表达显著升高（p0.05）。正常组、缺血6 h组未检测到μ-calpain催化亚基N末端自动裂解片段；缺血6 h后再灌注3 h、6 h、12 h组检测到μ-calpain催化亚基N末端自动裂解片段；所有分组均未检测到m-calpain催化亚基N末端自动裂解片段。I-R组细胞活力低于Control和PD组（p0.01），而PD+I-R组高于I-R组（p0.05）；I-R组TUNEL阳性核比率、Caspase-3活性高于Control和PD组（p0.01），而PD+I-R组低于I-R组（p0.05）；I-R引起显著的mPTP开放和Δψm下降（I-R与Control和PD组比较，p0.01），PD150606预处理可抑制这种效应（PD+I-R与I-R组比较，p0.05）。结论：心肌I-R诱导μ-calpain活化，抑制calpain活性防止I-R介导的mPTP开放和Δψm降低，从而减少心肌细胞凋亡。本研究提示calpain可能通过诱导mPTP开放致I-R心肌细胞凋亡。
[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 卤3.78% vs 81.70 卤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.
【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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