大鼠血清外泌体不同提取方法的比较
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摘要
目的:比较大鼠血清外泌体的不同提取方法,优化PEG聚沉法。方法:分别采用差速超速离心法、试剂盒法、PEG聚沉法及优化后的PEG聚沉法提取分离血清外泌体。通过Western Blotting检测外泌体标记蛋白;透射电镜观察形态。结果:Western Blotting检测出外泌体的标记蛋白;电镜下观察到差速超速离心法和优化后的PEG聚沉法提取的外泌体粒径在100 nm左右,呈双层膜结构,试剂盒法和PEG聚沉法提取的外泌体形态大小不一,杂质偏多。结论:优化后的PEG聚沉法能成功提取血清外泌体,此方法操作简便、耗时短、成本低,是可靠且有效的外泌体提取方法。
Objective: To compare different extraction methods of rat serum-derived exosomes and optimize PEG coagulation to obtain more efficient and practical extraction techniques. Methods: Serum exosomes were extracted and separated by differential ultracentrifugation method, kit method, PEG coagulation method and optimized PEG coagulation method. The exosomal marker proteins were detected by Western blotting; the morphology was observed by transmission electron microscope.Results: The expression of exosome labeled protein HSP70, CD63 and TSG101 in exosomes protein obtained by all four extraction methods could detected by Western blotting. The exosomes extracted by differential ultracentrifugation method and optimized PEG coagulation method show a particle size of about 100 nm and a double-layer membrane structure under transmission electron microscope. The exosomes extracted by the kit method and PEG coagulation method were impurities and not uniform in size. Conclusion: The optimized PEG coagulation method was successfully used to extract and separate rat serum-derived exosomes. Compared with the existing methods, this method has the advantages of easy operation, short time-consuming, low-cost and effective.
Objective: To compare different extraction methods of rat serum-derived exosomes and optimize PEG coagulation to obtain more efficient and practical extraction techniques. Methods: Serum exosomes were extracted and separated by differential ultracentrifugation method, kit method, PEG coagulation method and optimized PEG coagulation method. The exosomal marker proteins were detected by Western blotting; the morphology was observed by transmission electron microscope.Results: The expression of exosome labeled protein HSP70, CD63 and TSG101 in exosomes protein obtained by all four extraction methods could detected by Western blotting. The exosomes extracted by differential ultracentrifugation method and optimized PEG coagulation method show a particle size of about 100 nm and a double-layer membrane structure under transmission electron microscope. The exosomes extracted by the kit method and PEG coagulation method were impurities and not uniform in size. Conclusion: The optimized PEG coagulation method was successfully used to extract and separate rat serum-derived exosomes. Compared with the existing methods, this method has the advantages of easy operation, short time-consuming, low-cost and effective.
引文
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[2]L?tvall J,Hill A F,Hochberg F,et al.Minimal experimental requirements for definition of extracellular vesicles and their functions:a position statement from the International Society for Extracellular Vesicles[J].Journal of Extracell Vesicles,2014,3(26913):26913.
[3]Tucci M,Passarelli A,Mannavola F,et al.Serum exosomes as predictors of clinical response to ipilimumab in metastatic melanoma[J].Oncoimmunology,2018,7(2):e1387706.
[4]Repiská G,Kone?ná B,Shelke G V,et al.Is the DNA of placental origin packaged in exosomes isolated from plasma and serum of pregnant women?[J].Clinical Chemistry&Laboratory Medicine(CCLM),2018,56(6):e150-e153.
[5]Thind A,Wilson C.Exosomal miRNAs as cancer biomarkers and therapeutic targets[J].J Extracell Vesicles,2016,5:31292.
[6]卢婉,杨人强,王伶.外泌体的研究进展[J].生命的化学,2013,51(4):438-442.
[7]Mayuri R,Vidya S,Zhu C,et al.Chromatin Dynamics and the RNAExosome Function in Concert to Regulate Transcriptional Homeostasis[J].Cell Reports,2015,13(8):1610-1622.