發(fā)布時間：2018-05-29 20:55

  本文選題：MARC-細胞 + 外泌體　； 參考：《畜牧獸醫(yī)學報》2017年07期

【摘要】：旨在尋找有效方法,能夠從PRRSV感染的MARC-145細胞中分離細胞產(chǎn)生的外泌體,并為探索外泌體在PRRSV感染細胞時的作用奠定基礎(chǔ)。使用試劑盒提取MARC-145細胞產(chǎn)生的外泌體,利用電鏡負染色法和Western blot技術(shù)對MARC-145細胞產(chǎn)生的外泌體進行鑒定;采用碘克沙醇速率區(qū)帶離心分離MARC-145細胞產(chǎn)生的外泌體,并用Western blot技術(shù)檢測外分泌體的分布區(qū)間。用PRRSV感染MARC-145細胞后,使用試劑盒對MARC-145細胞產(chǎn)生的外泌體進行初步分離后,采用免疫磁珠捕獲技術(shù)進行進一步純化,并使用Western blot進行驗證。Western blot結(jié)果顯示MARC-145細胞產(chǎn)生的外泌體存在特異性標記蛋白CD63、Tsg101和Alix,不存在外泌體非標記蛋白EEA1、GRP94和Cytochrome C;電鏡負染色結(jié)果顯示MARC-145細胞產(chǎn)生的外泌體尺寸大小處于30~100nm,符合外泌體特征。利用Western blot對碘克沙醇速率區(qū)帶離心中收集到的樣品進行分析,結(jié)果顯示MARC-145細胞產(chǎn)生的外泌體分布于7.2%~18%碘克沙醇密度區(qū)間。Western blot結(jié)果顯示免疫磁珠捕獲法獲得的對照組不存在外泌體標記蛋白Tsg-101。以上結(jié)果表明MARC-145細胞能夠產(chǎn)生外泌體,并且通過免疫磁珠捕獲法能夠從PRRSV感染的MARC-145細胞中將其分離。
[Abstract]:The aim of this study was to find an effective method to isolate exocrine from MARC-145 cells infected with PRRSV and to lay a foundation for exploring the role of exocrine in PRRSV infected cells. The exocrine produced by MARC-145 cells was extracted by the kit, the exocrine produced by MARC-145 cells was identified by electron microscopy negative staining and Western blot technique, and the exocrine of MARC-145 cells was isolated by centrifugation with iodoxanol rate zone. The distribution of exocrine was detected by Western blot technique. After infecting MARC-145 cells with PRRSV, the exocrine produced by MARC-145 cells was isolated with kit, and further purified by immunomagnetic bead capture technique. Western blot showed that the exocrine produced by MARC-145 cells had specific marker proteins CD63Tsg101 and Alix, and there was no exocrine unlabeled protein EEA1GRP94 or Cytochrome C.The negative staining of electron microscope showed that exocrine size produced by MARC-145 cells was negative. The size of an inch is at 30 ~ 100 nm, which is in line with the characteristics of exocrine body. The samples collected in the rate zone centrifugation of iodoxanol were analyzed by Western blot. The results showed that the exosomes produced by MARC-145 cells were distributed in the density interval of 18% iodoxanol. The results of Western blot showed that there was no exososomal marker protein Tsg-101 in the control group obtained by the method of immunomagnetic bead capture. These results suggest that MARC-145 cells can produce exocrine bodies and can be isolated from MARC-145 cells infected with PRRSV by immunomagnetic bead capture method.
【作者單位】： 山西農(nóng)業(yè)大學動物科技學院;
【基金】：山西省科技攻關(guān)項目(20100312016)
【分類號】：S852.651

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