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糖尿病小鼠血清外泌体在H9C2心肌细胞损伤调控中的作用及机制
作者:侯智为 李玉莹 宋海旭 李佳荫 邢瑞楠 刘丹 刘晶 闫承慧 田孝祥 
单位:121001  辽宁锦州 锦州医科大学心血管内科(侯智为) 110016  沈阳 北部战区总医院心血管内科/全军心血管病研究所(侯智为、李玉莹、宋海旭、李佳荫、邢瑞楠、刘丹、刘晶、闫承慧、田孝祥) 430030  武汉 华中科技大学同济医学院附属同济医院心血管内科(李玉莹) 710032  西安 空军军医大学西京医院心血管内科(宋海旭) 110169  沈阳 东北大学生命科学与健康学院(李佳荫) 116041  辽宁大连 大连医科大学第二临床医学院心血管内科(邢瑞楠) 
关键词:糖尿病 外泌体 心肌细胞 
分类号:R587.1
出版年,卷(期):页码:2019,44(10):837-842
摘要:

 [摘要]  目的  探讨糖尿病小鼠(db/db)血清外泌体在体外培养的H9C2心肌细胞损伤中的作用及机制。方法  采用db/db小鼠(n=10)及其对照组小鼠(db/+n=5)制备糖尿病心肌损伤小鼠模型。提取小鼠血清中的外泌体,定量检测其数量并应用PKH26标记为红色荧光;采用Western blotting检测外泌体相关蛋白及外泌体刺激后H9C2细胞炎性因子的表达情况;采用TUNEL检测细胞凋亡情况;采用Rab1a中和抗体进行阻断实验。结果  db/db小鼠血清外泌体数量(30.95×109/ml)明显多于db/+小鼠(10.45×109/ml),差异有统计学意义(P<0.01)。体外培养的H9C2细胞能够内吞更多db/db小鼠血清来源的外泌体;应用db/db小鼠血清外泌体干预H9C2细胞可以刺激细胞炎性因子表达明显增加,其中IL-6IL-1β分别增加6.2倍和2.6(P<0.01),且H9C2细胞凋亡明显增多。进一步机制研究发现,Rab1adb/db小鼠血清外泌体中的表达明显增多,应用Rab1a中和抗体阻断db/db小鼠外泌体中Rab1a表达后可明显抑制H9C2细胞对外泌体的内吞及细胞凋亡。结论  db/db小鼠血清来源的外泌体可诱导体外培养的心肌细胞凋亡及发生炎症反应,可能参与了糖尿病心肌损伤的演进。抑制外泌体分泌或干预其调控分子可能成为糖尿病心肌损伤治疗新的研究靶点。

 [Abstract]  Objective  To investigate the effect of exosome in cultured in vitro H9C2 myocardial cells injury of diabetic mice and its mechanism. Methods  The mouse model of diabetic myocardial injury was established by using db/db mice (n=10) and their mate mice db/+ (n=5). Serum exosomes were isolated and quantitated using the exosome isolation reagent and EXOCET Quantitation kit. The serum exosomes were labeled with PKH26 (red fluorescent cell linker) to detect the endocytosis in H9C2 cells. The expressions of exocrine associated protein and inflammatory cytokines in H9C2 cells with or without exosome stimulation were detected by Western blotting. TUNEL was used to detect apoptosis. A neutralizing antibody of Rab1a was used for blocking experiment. Results  Db/db mice produced more exosomes than db/+ mice (30.95×109/ml vs. 10.45×109/ml, P<0.01). Moreover, H9C2 cells cultured in vitro could swallow more serum-exosomes derived from db/db mice. Meanwhile, serum exosome from db/ db mice, as used to interfere H9C2 cells, significantly increased the expression of inflammatory cytokines, such as 6.2 folds to IL-6 and 2.6 folds to IL-1β (P<0.01). Furthermore, the apoptosis in H9C2 cells increased compared to those from db/+ mice. Mechanism studies announced that the increased expression of Rab1a in exosomes-derived from db/db mice, and blocking the expression of Rab1a in exocrine of db/db mice with Rab1a neutralizing antibody could significantly inhibit the endocytosis and apoptosis of H9C2 cells. Conclusions  Serum exosomes isolated from db/db mice may trigger inflammation and apoptosis of cardiomyocytes cultured in vitro, which may be involved in the evolution of diabetic myocardial injury. Inhibition of exosome secretion or intervention of its regulatory molecules may become a new research target for the treatment of diabetic myocardial injury.

基金项目:
国家自然科学基金面上项目(81570265、81670276、81770303);辽宁省自然科学基金(20180550368);军事科技领域青年人才托举工程项目(17-JCJQ-QT-028)
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