类风湿关节炎滑膜成纤维细胞来源外泌体提取方法的研究

The comparative study on the extraction methods of exosomes from synovial fibroblasts in patients with rheumatoid arthritis

目的从外泌体分离的常用方法中探索获取高质量人滑膜成纤维细胞外泌体的有效方法。方法分别用超速离心法、尺寸排阻法和改良的聚合物沉淀法3种方法提取人滑膜成纤维细胞外泌体, 采用透射电镜、蛋白质印迹法(Western Blot)、纳米颗粒追踪分析(NTA)鉴定并比较外泌体的形态特征、粒径分布、浓度以及标志蛋白的表达水平。3组差异比较采用单因素方差分析, 组间两两比较采用LSD-t检验, 以P<0.05表示差异具有统计学意义。结果 3种方法均能成功提取得到外泌体, 透射电镜下均能观察到典型的"茶托状"结构, 蛋白质印迹法均能检测到标志蛋白肿瘤易感基因101(TSG101)、多配体聚糖结合蛋白1(Syntenin-1)、CD63的表达, 粒径主峰均符合30~150 nm范围。外泌体纯度方面:超速离心法杂质最少, 尺寸排阻法杂质中等, 而改良的聚合物沉淀法可观察到大量的聚合物颗粒和蛋白杂质。外泌体得率方面:3种方法得到的外泌体颗粒浓度差异有统计学意义(F=9.61, P=0.049), 改良的聚合物沉淀法得到的外泌体颗粒浓度高于超速离心法[(98.0±17.0)×10^(10)个/ml和(11.6±7.7)×10^(10)个/ml, t=-4.34, P=0.023]。结论 RA滑膜成纤维细胞来源的外泌体不难获取, 3种方法均有效, 然而改良聚合物沉淀法得到的外泌体杂质较多, 可能不利于下游分析不推荐使用;超速离心法得到的外泌体纯度高, 但操作繁琐, 且损耗较大, 适合大体积样本和对纯度要求高的研究;尺寸排阻法得到的外泌体纯度和颗粒浓度均较高, 适合小体积样本。

Objective To compare the efficiency and purity of exosomes extracted from synovial fibroblasts of patients with rheumatoid arthritis by ultracentrifugation,size exclusion chromatography and modified polymer precipitation.Methods The exosomes were extracted from human synovial fibroblasts by ultracentrifugation,size exclusion chromatography and modified polymer precipitation.Transmission electron microscopy,particle size detection and western Blot were used to identify the morphological characteristics,particle size distribution,concentration,and expression of marker proteins.One-way analysis of variance(ANOVA)was used for comparison among the three groups,and LSD-t test was used for pairwise comparison.P<0.05 was considered statistically significant.Results Exosomes could be successfully obtained with all three extraction methods.The typical"saucer-like"structure could be observed under transmission electron microscope.The marker proteins of exosomes TSG101,Syntenin-1 and CD63 were all detectable by western blot.The peaks of main particle size were located within 30~150 nm.As for purity,the exosomes obtained by ultracentrifugation showed the highest purity,while modified polymer precipitation was the worst,with a large number of polymer particles and impurities protein.The purity of exosomes obtained by size exclusion chromatography was the moderate.For extraction efficiency,concentrations of exosomes particles obtained by the three methods were different(F=9.61,P=0.049),and modified polymer precipitation was significantly higher than ultracentrifugation in terms of concentration of exosomes particles[(98.0±17.0)×10^(10) particles/ml vs(11.6±7.7)×10^(10) particles/ml,t=-4.34,P=0.023].Conclusion Human synovial fibroblasts derived exosomes canbe obtained by three methods.Ultracentrifugation is time-consuming,but can produce high-purity exosomes,which may be considered in the situation when high purity requirement with large volume samples are needed.Size exclusion chromatography is a good choice with high yield and purity exosomes,and suitable for small volume samples.Modified polymer precipitation is not recommended due to production of lowest purity exosomes.