三种方法提取血清外泌体的比较研究

Comparative Study on Three Methods for Extraction of Serum Exosomes

目的比较超速离心法、沉淀法和尺寸排阻色谱法提取血清外泌体的得率和纯度。方法选取8例健康体检受试者血清,用三种方法提取外泌体,采用纳米流式检测仪、透射电子显微镜和Pierce BCA蛋白检测试剂盒分别检测外泌体颗粒数、粒径分布、形态特征和蛋白浓度;分析外泌体颗粒数。外泌体颗粒数和蛋白总量比值、Triton X-100处理前后颗粒数变化来评估外泌体提取的得率和纯度。结果超速离心法、沉淀法和尺寸排阻色谱法提取外泌体粒径均分布在40~150 nm;透射电子显微镜下均能观察到呈明显茶托状的双层膜结构,但沉淀法提取的外泌体混有聚乙二醇聚合物及蛋白大颗粒;蛋白质印迹结果显示三种方法提取的外泌体均表达外泌体标记蛋白;沉淀法提取的血清外泌体颗粒数高于超速离心法和尺寸排阻色谱法[(1.11±0.07)×10^(11) particley/ml vs(2.54±0.04)×10^(10)particley/ml,(7.51±0.89)×10^(10)particley/ml],差异均有统计学意义(P<0.05),尺寸排阻色谱法提取外泌体颗粒数高于超速离心法(P<0.001);尺寸排阻色谱法提取外泌体纯度高于超速离心法和沉淀法[(5.66±0.45)×10^(7)particles/μg vs(7.65±0.42)×10^(6) particles/μg,(3.18±0.45)×10^(6) particles/μg],超速离心法提取外泌体纯度高于沉淀法,差异均有统计学意义(P<0.05)。结论超速离心法、沉淀法和尺寸排阻色谱法均能提取出血清外泌体,超速离心法步骤繁琐,耗时较长,提取外泌体得率与纯度较低;沉淀法操作简单,耗时短,提取外泌体得率最高,但外泌体纯度最低;尺寸排阻色谱法操作简单,耗时短,提取外泌体的纯度最高;相比而言,尺寸排阻色谱法显然优于超速离心法和沉淀法。

Objective To compare the efficiency and purity of ultracentrifugation,precipitation and size-exclusion chromatography for extracting exosomes from serum.Methods Three methods were used to extract serum exosomes.The number of exosomal particles,particle size distribution,morphological characteristics and protein concentration were detected by using nano flow detector,transmission electron microscope and Pierce BCA protein detection kit.Analyzed the number of exosomal particles,the ratio of the number of exosomal particles to total protein and the changes in the number of exosomal particles before or after Triton X-100 treatment to evaluate the yield and purity of exosomes extraction.Results The particle size of the exosomes which had been extracted by ultracentrifugation,precipitation and size-exclusion chromatography were all distributed in the range of 40~150 nm,and the bilayer membrane with obvious saucer-like structure could be observed under transmission electron microscope,but the exosomes extracted by precipitation were mixed with polyethylene glycol polymers and large protein particles.The results of Western blot showed that the exosomes extracted by all three methods had expressed the tag proteins.The number of exosomal particles extracted by precipitation was higher than that of ultracentrifugation and sizeexclusion chromatography[(1.11±0.07)×10^(11) particles/ml vs(2.54±0.04)×10^(10) particles/ml,(7.51±0.89)×10^(10) particles/ml],the differences were statistically significant(all P<0.05),and the number of exosomal particles extracted by sizeexclusion chromatography was higher than that of ultracentrifugation(P<0.001).The purity of exosomes extracted by sizeexclusion chromatography was higher than that of ultracentrifugation and precipitation[(5.66±0.45)×10^(7) particles/μg vs(7.65±0.42)×10^(6) particles/μg,(3.18±0.45)×10^(6) particles/μg],and the purity of exosomes extracted by ultracentrifugation was higher than that of precipitation,the differences were statistically significant(P<0.05).Conclusion Ultracentrifugation,precipitation and size-exclusion chromatography can extract the exosomes of Xueqing.Ultracentrifugation is cumbersome,time-consuming and the yield and purity of exosomes are low.The precipitation method is simple and time consuming,and the extraction yield of exosomes is the lowest,but the purity of exosomes its the lowest.Size exclusion chromatography is simple and time-consuming,and the purity of secretes is the highest.In contrast,size-exclusion chromatography is obviously better than ultracentrifugation and precipitation.