肝星状细胞来源外泌体不同提取方法的比较

目的采用超速离心法(UC)和优化后的排阻超滤组合法提取肝星状细胞(HSC)来源外泌体,比较两种提取方法所得外泌体的纯度、浓度和总蛋白量。方法分别用UC法和优化后的排阻超滤组合法提取HSC来源外泌体。使用透射电镜观察外泌体形态,纳米颗粒跟踪分析技术(NTA)检测外泌体浓度及大小,二喹啉甲酸(BCA)试剂盒检测外泌体总蛋白浓度,Western blotting法检测外泌体表面标志物肿瘤易感基因101蛋白(TSG101)、分化抗原9(CD9)的表达。结果两种方法均可得到圆盘形的30~150 nm膜性囊泡。优化后的排阻超滤组合法所得外泌体,在电镜下可见明显的“茶托”形结构,背景较干净,纯度较高。UC法和优化后的排阻超滤组合法所得外泌体浓度分别为2×10^9、3.6×10^11 particles/ml,平均粒径分别为112.6、121.7 nm。优化后的排阻超滤组合法所得外泌体总蛋白浓度及TSG101的表达高于UC法。结论优化后的排阻超滤组合法虽然操作繁琐,成本较高,但提取的HSC来源外泌体浓度、总蛋白浓度和纯度较超速离心法高,可以作为外泌体分离的有效方法。

Comparison of different extraction methods of exosomes from hepatic stellate cell

Objective To extract hepatic stellate cell(HSC)-derived exosomes by ultracentrifugation(UC)and optimized filtration combined with size exclusion chromatography(SEC),and to compare the purity,concentration and total protein concentration.Methods HSC-derived exosomes were extracted by UC and optimized filtration combined with SEC method respectively.The morphology of exosomes was observed by transmission electron microscope(TEM).The concentration and particle size distribution were detected by nano particle size tracking instrument.The total protein concentration of exsomes was quantified by bicinchoninic acid(BCA)kit and the surface marker proteins(tumor susceptibility gene 101 protein(TSG101)and differentiation antigen 9(CD9)were detected by Western blotting.Results The exosomes extracted by UC and optimized filtration combined with SEC method were all oval or bowl-shaped with a size range between 30 nm to150 nm analyzed by TEM.The exosomes isolated by optimized filtration combined with SEC showed more typical bowl-shaped structure with a cleaner background and high purity.The concentration of exosomes-obtained by UC and optimized filtration combined with SEC were 2×10^9 particles/ml and 3.6×10^11 particles/ml,respectively.The average particle sizes were 112.6 nm and 121.7 nm,respectively.The total protein concentration and the expression of TSG101 in exosomes isolated by optimized filtration combined with SEC were higher than those in exosomes isolated by UC.Conclusion Although the optimized filtration combined with SEC method is cumbersome and costly,the isolated HSC-derived exosomes have higher purity and concentration compared with HSC-derived exosomes isolated by UC.Therefore,the optimized filtration combined with SEC method could be an efficient way for exosomes isolation.