两种提取小鼠血清来源外泌体方法的比较

Comparison of two different methods of extracting exosomes from mouse sera

目的比较超速离心法与纯化试剂盒提取法提取血清外泌体的有效性与实用性,为后续外泌体相关研究提供借鉴。方法将12只6~8周C57BL/6雄性小鼠按照随机数字表法分为两组,每组6只,即超速离心法提取组及纯化试剂盒提取组。分别用两种方法分离小鼠血清中的外泌体。使用纳米粒度及zeta电位仪(Nano ZS90)检测两组外泌体的粒径大小及分布;使用透射电子显微镜观察两种方法提取外泌体的形态结构;采用蛋白质印迹法(Western blot)检测外泌体标志蛋白的表达水平;对两种方法所提取外泌体中的核糖核酸(RNA)分离鉴定,比较两种方法提取外泌体中RNA的异同。结果两组所得的外泌体直径为30~150 nm,形态均为脂质双分子层囊泡样,Western blot法检测结果显示两组外泌体均表达标志蛋白cluster of differentiation 63和tumor susceptibility gene,差异无统计学意义(P>0.05);与纯化试剂盒提取组比较,超速离心法提取组的外泌体浓度更高,差异有统计学意义(P<0.05),且超速离心法提取组中外泌体所含RNA以microRNA为主,而纯化试剂盒提取组的外泌体中所含RNA主要为piRNA。结论超速离心法与纯化试剂盒提取法均能有效提取外泌体,但所得到的外泌体包含不同RNA成分,为外泌体的研究提供更有效的选择方法。

Objective To compare the effectiveness and practicability of extraction of serum exosomes by ultracentrifugation and purification kit,and to provide references for subsequent exosome studies.Methods Twelve C57BL/6 male mice aged 6-8 weeks were divided into two groups by random table method,with 6 mice in each group,that was the ultracentrifugation extraction group and the purification kit extraction group.Exosomes in mouse serum were isolated by two methods.The particle size and distribution of exosomes in the two groups were detected by Nano particle size and zeta potentiometer(Nano ZS90).The morphological structures of exosomes were extracted by transmission electron microscopy.Western blot was used to detect the expression level of exosome marker proteins.Ribonucleic acid(RNA)was isolated and identified from exosomes extracted by the two methods.Results The outside diameter of the proceeds of two groups was 30-150 nm,and morphology was lipid bilayer vesicle.Western blot results showed that the exosomes in both groups expressed the marker protein cluster of differentiation 63 and tumor susceptibility gene,and the difference was not statistically significant(P>0.05).Compared with the purification kit extraction group,the ultracentrifugation extraction group had a higher exosomes concentration,the difference was statistically significant(P<0.05).Moreover,the RNA in the exosomes in the ultracentrifugation extraction group was mainly microRNA,while the RNA in the exosomes in the purification kit extraction group was mainly piRNA.Conclusion The ultracentrifugation and purification kit extraction method can both effectively extract exosomes,but the exosomes obtain different RNA components,which provides a more effective selection method for the study of exosomes.