脂肪干细胞外泌体提取方法的改良及其对成骨效应的影响  

Improved isolation of exosomes derived from adipose-derived stem cells and effect on osteogenesis

作　　者：张华[1] 郭澍[1] 佟爽[1] 陈义庆 崔梦莹 赵崇如 ZHANG Hua;GUO Shu;TONG Shuang;CHEN Yiqing;CUI Mengying;ZHAO Chongru(Department of Plastic Surgery,The First Hospital,China Medical University,Shenyang 110001,China)

机构地区：[1]中国医科大学附属第一医院整形外科

出　　处：《中国医科大学学报》2020年第2期115-119,128,共6页Journal of China Medical University

基　　金：国家自然科学基金(51272286);辽宁省自然科学基金(20170541040);国家卫生和健康委员会公益性科研专项(2015SQ00049)

摘　　要：目的通过改良后的ExoQuick试剂盒法、超速离心法、ExoQuick试剂盒法提取脂肪干细胞(ADSCs)来源外泌体,探讨提取的外泌体对ADSCs成骨分化诱导的影响,并且对3种提取方法进行比较。方法使用改良后的ExoQuick试剂盒法、超速离心法、ExoQuick试剂盒法提取外泌体,进行形态学观察及特异性表面标志物CD9、CD63蛋白鉴定;分别将3种方法提取的外泌体加入ADSCs成骨分化诱导过程中,通过对照实验检测形态学变化及Runx2、OCN蛋白表达。结果改良后的ExoQuick试剂盒法、ExoQuick试剂盒法提取的样本均能观察到圆形、类圆形膜性囊泡结构,特异性表面标志物CD9、CD63均可表达,且ExoQuick试剂盒改良法蛋白表达量最多。与未加外泌体的过程相比加入外泌体的ADSCs成骨诱导过程Runx2、OCN蛋白表达明显增多,且Exo-Quick试剂盒改良法蛋白表达量最多。结论ExoQuick试剂盒改良法提取ADSCs来源外泌体效率最高,提取的外泌体促进成骨效应最强。Objective Exosomes were isolated from adipose-derived stem cells using the improved ExoQuick kit,ultracentrifugation and original ExoQuick kit methods.The effects of the extracted exosomes on the induction of osteogenic differentiation of adipose-derived stem cells were investigated,and the differences between the three methods were compared.Methods Exosomes were isolated using the improved ExoQuick kit,ultracentrifugation,and original ExoQuick kit methods.The morphology and expression of the CD9 and CD63 marker proteins were examined in the isolated exosomes.Exosomes obtained by the three methods were used for osteoblast induction of adipose-derived stem cells.Morphology and the expression of Runx2 and OCN proteins were compared to the exosome-free control groups.Results Both the original and improved ExoQuick kit methods retained the circular and quasi-circular membranous vesicle structure.All three methods retained expression of the CD9 and CD63 surface markers.The highest expression was obtained with the improved ExoQuick kit method.Compared with the exosome-free control group,the expression of Runx2 and OCN proteins in the three groups containing the exosomes was significantly higher.The improved ExoQuick kit method resulted in the highest expression of Runx2 and OCN proteins.Conclusion The improved ExoQuick kit method was the most efficient in extracting exosomes from adipose-derived stem cells.Exosomes extracted with this method exerted the strongest osteogenic effect.

关 键 词：脂肪干细胞 外泌体 提取 鉴定 成骨 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]