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目的:心脏干细胞移植治疗急性心梗的机制不明,而最新研究发现外泌体(exosomes)是干细胞与心肌细胞信号交流的重要媒介。本研究通过解析心脏干细胞分泌外泌体的成分,阐明其中参与心肌保护作用的物质以及调控机制。方法:采用体外分离成年小鼠心脏的Sca-1~+心脏干细胞(Sca-1~+CPCs),从Sca-1~+CPCs的培养上清中分离提取其分泌的exosomes,用Nanosight、投射电镜、Western blot等对exosomes进行鉴定,q PCR解析exosomes中的内容物,找出关键调控的mi RNA,并且通过转染以及萤光素酶报告基因的实验证实其调控的靶基因;最后,细胞加入exosomes预保护后,检测其关键mi RNA及其靶蛋白的表达,流式细胞术检测凋亡细胞比例的变化。结果:Sca-1~+CPCs细胞上清中提取的物质是直径在80 nm左右的双分子层囊泡,且表达CD9、CD63和Alix 3种exosome标志蛋白。氧化应激可以促进CPCs分泌exosomes,这种exosomes中mi R-21表达明显增加,并且被心肌细胞摄取,mi R-21通过与PDCD4靶向结合抑制心肌细胞在氧化应激损伤下的凋亡,加入exosomes对细胞进行保护后,在同样环境下可明显上调细胞内mi R-21,且发现PDCD4及cleaved caspase-3的表达明显下调,凋亡细胞的比例明显减少。结论:心脏干细胞通过分泌的exosomes,将其包裹的mi R-21进入到心肌细胞中抑制PDCD4的表达,从而抗心肌细胞凋亡。本研究为阐明干细胞治疗心肌梗死的作用机制提供了新的视野。
OBJECTIVE: The mechanism of cardiac stem cell transplantation in the treatment of acute myocardial infarction is unknown. Recent studies have found that exosomes are important mediators of stem cell and cardiomyocyte signaling. In this study, by analyzing the components of exosomes secreted by cardiac stem cells, we elucidate the substances involved in myocardial protection and their regulatory mechanisms. Methods: Sca-1 ~ + cardiac stem cells (Sca-1 ~ + CPCs) were isolated from the heart of adult mice in vitro. The secreted exosomes were isolated and purified from the culture supernatant of Sca-1 ~ + CPCs. , Western blot and other exosomes were identified, qPCR analysis of the contents of exosomes to identify key mi RNAs, and confirmed by transfection and luciferase reporter gene regulation of the target gene; Finally, cells were added After exosomes were pre-protected, the expression of key mi RNA and its target protein was detected. The proportion of apoptotic cells was detected by flow cytometry. Results: The material extracted from the supernatant of Sca-1 ~ + CPCs was a bilayer vesicle with a diameter of about 80 nm, and expressed exosome marker proteins of CD9, CD63 and Alix. Oxidative stress can promote the secretion of exosomes by CPCs. The expression of mi R-21 in this exosomes is significantly increased and taken up by cardiomyocytes. Mi R-21 inhibits cardiomyocytes apoptosis under oxidative stress by targeting with PDCD4, After the cells were protected by exosomes, mi R-21 was significantly up-regulated in the same environment, and the expressions of PDCD4 and cleaved caspase-3 were significantly down-regulated and the percentage of apoptotic cells was significantly decreased. CONCLUSIONS: Cardiac stem cells, through excreted exosomes, enter into their cardiomyocytes to inhibit the expression of PDCD4 and thereby prevent cardiomyocyte apoptosis. This study provides a new perspective to elucidate the mechanism of action of stem cells in the treatment of myocardial infarction.