低温缺氧-复氧心肌成纤维细胞源性外泌体对心肌细胞损伤的机制研究

Investigation on mechanism of myocardial cell injury induced by exosomes from hypothermic anoxia-reoxygenated cardiac fibroblasts

  • 摘要:
    背景 低温缺氧-复氧大鼠心肌成纤维细胞(rat cardiac fibroblasts,RCF)对H9c2心肌细胞有损伤作用,其调控机制尚不明确。
    目的 观察低温缺氧-复氧RCF分泌的外泌体对H9c2心肌细胞及缝隙链接蛋白43 (connexin 43,Cx43)的影响。
    方法 分别提取并鉴定常氧(95%空气 + 5% CO2,37℃条件下培养5 h)和低温缺氧-复氧(95% N2 + 5% CO2,4℃条件下培养1 h后,95%空气 + 5% CO2,37℃条件下培养4 h)条件下RCF培养液中的外泌体,常氧和低温缺氧-复氧条件下提取的外泌体分别标记为N-exo和I/R-exo。采用随机数字表法将体外培养的大鼠H9c2心肌细胞分为C组(常规条件下培养12 h)、N组(与N-exo共孵育12 h)和I/R组(与I/R-exo共孵育12 h),共孵育结束后,收集心肌细胞,流式细胞术测定H9c2心肌细胞的凋亡率;Western blot检测心肌细胞Cx43和磷酸化Cx43 (p-Cx43)的表达;划痕实验检测心肌细胞的迁移能力。
    结果 C组与N组心肌细胞凋亡率差异无统计学意义(P>0.05),C组和N组心肌细胞凋亡率均低于I/R组(P<0.05);相较于C组,I/R组Cx43及P-Cx43表达下调、细胞迁移能力降低(P<0.05),N组Cx43及P-Cx43表达上调、细胞迁移能力升高(P<0.05)。
    结论 低温缺氧-复氧心肌成纤维细胞释放的外泌体可增加心肌细胞的凋亡率,降低心肌细胞迁移能力,下调心肌细胞Cx43及P-Cx43的表达。

     

    Abstract:
    Background Exosomes derived from hypothermic anoxia-reoxygenated rat cardiac fibroblasts (RCF) cause damage to H9c2 myocardial cells, but the regulatory mechanism remains unclear.
    Objective To examine the impact of exosomes secreted by hypothermic anoxia-reoxygenated RCF on the injury of H9c2 myocardial cells and its effect on the gap junction protein Connexin 43 (Cx43).
    Methods Exosomes were isolated and identified from RCF culture medium under normoxic conditions (cultured in 95% air + 5% CO2 at 37℃ for 5 hours) and hypothermic anoxia-reoxygenation conditions (cultured in 95% N2 + 5% CO2 at 4℃ for 1 hour followed by 95% air + 5% CO2 at 37℃ for 4 hours). The exosomes from the respective conditions were labeled as N-exo and I/R-exo. Using a random number table, in vitro cultured rat H9c2 myocardial cells were divided into three groups: C group (cultured under standard conditions for 12 hours), N group (co-incubated with N-exo for 12 hours), and I/R group (co-incubated with I/R-exo for 12 hours). After co-incubation, myocardial cells were harvested, and flow cytometry was used to determine the apoptosis rate of H9c2 myocardial cells. Western blotting was utilized to detect the expression of Cx43 and phosphorylated Cx43 (p-Cx43), and scratch assay was conducted to assess the migration capability of myocardial cells.
    Results No significant difference in apoptosis rates was observed between the C and N groups (P>0.05), but both were lower than the I/R group (P<0.05). Compared to the C group, the I/R group exhibited decreased expression of Cx43 and p-Cx43 and reduced cell migration ability (P<0.05), whereas the N group showed increased expression of Cx43 and p-Cx43 and enhanced cell migration ability (P<0.05).
    Conclusion Exosomes released by hypothermic anoxia-reoxygenated cardiac fibroblasts can elevate the apoptosis rate of myocardial cells, diminish myocardial cell migration capability, and downregulate the expression of Cx43 and p-Cx43 in myocardial cells.

     

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