试剂盒法与差速超速离心法所提取的人牙髓细胞外泌体的鉴定与比较
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摘要
目的:采用试剂盒法以及差速超速离心法提取的人牙髓细胞外泌体(hDPC-DEs),并进行鉴定与比较。方法:培养原代人牙髓细胞(hDPCs)并鉴定,收集hDPCs培养上清,分别采用试剂盒法和差速超速离心法提取hDPC-DEs,在透射电镜下进行形态学观察,利用免疫印记(Western blotting)法对外泌体表面标记蛋白CD9的表达进行检测,并通过酶联免疫吸附试验(ELISA)法测定外泌体表面标记蛋白CD63的浓度。结果:透射电镜下试剂盒法与差速超速离心法所提hDPC-DEs均表现为典型的茶托状双层膜结构,直径在50～120nm,试剂盒组背景杂质较多,差速离心组背景较为清晰。两种方法提取的hDPCDEs特异性标志蛋白CD9均呈阳性表达。试剂盒组外泌体标志蛋白CD63的浓度高于差速超速离心组(P<0.05)。结论:试剂盒法与差速超速离心法均可获得hDPC-DEs,前者操作简便,所提取的hDPC-DEs的浓度较高,但杂质可能比较多。
Objective:To identify and compare human dental pulp cells derived exosomes(hDPC-DEs) extracted by ExoQuick kit and ultra-centrifugation.Methods:Human dental pulp cells(hDPCs) were primarily cultured and identified,and the hDPC-DEs were extracted by ExoQuick kit or ultra-centrifugation.The morphology of extracted exosomes was observed under transmission electron microscopy.The expression of exosomal marker protein CD9 was detected by western blotting,and the concentration of exosomal marker protein CD63 was determined by ELISA.Results:HDPC-DEs obtained by ExoQuick kit and ultra-centrifugation methods showed typical saucer-like double-layer membrane structure,with the diameter of about 50-120 nm under electronic microscopy.There were more impurities in ExoQuick kit group.The exosomal marker protein CD9 in ExoQuick kit and ultra-centrifugation groups were positively expressed,and the concentration of the exosomal marker protein CD63 in the ExoQuick kit group was significantly higher than that in the ultra-centrifugation group(P<0.05).Conclusion:The hDPC-DEs could be successfully extracted by both ultra-centrifugation and ExoQuick kit.The latter method was easy to operate,and could obtain higher concentration of hDPC-DEs,but more impurities.
Objective:To identify and compare human dental pulp cells derived exosomes(hDPC-DEs) extracted by ExoQuick kit and ultra-centrifugation.Methods:Human dental pulp cells(hDPCs) were primarily cultured and identified,and the hDPC-DEs were extracted by ExoQuick kit or ultra-centrifugation.The morphology of extracted exosomes was observed under transmission electron microscopy.The expression of exosomal marker protein CD9 was detected by western blotting,and the concentration of exosomal marker protein CD63 was determined by ELISA.Results:HDPC-DEs obtained by ExoQuick kit and ultra-centrifugation methods showed typical saucer-like double-layer membrane structure,with the diameter of about 50-120 nm under electronic microscopy.There were more impurities in ExoQuick kit group.The exosomal marker protein CD9 in ExoQuick kit and ultra-centrifugation groups were positively expressed,and the concentration of the exosomal marker protein CD63 in the ExoQuick kit group was significantly higher than that in the ultra-centrifugation group(P<0.05).Conclusion:The hDPC-DEs could be successfully extracted by both ultra-centrifugation and ExoQuick kit.The latter method was easy to operate,and could obtain higher concentration of hDPC-DEs,but more impurities.
引文
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[3]JANEBODIN K,ZENG Y,BURANAPHATTHANAW,et al.VEGFR2-dependent angiogenic capacity of pericyte-like dental pulp stem cells[J].J Dent Res,2013,92(6):524-531.
[4]DISSANAYAKA W L,ZHAN X,ZHANG C,et al.Coculture of dental pulp stem cells with endothelial cells enhances osteo-/odontogenic and angiogenic potential in vitro[J].J Endod,2012,38(4):454-463.
[5]AMARNATH S,FOLEY J E,FARTHINGDE,et al.Bone marrow derived mesenchymal stromal cells harnesspurinergenic signaling to tolerize human Th1cells in vivo[J].Stem cells,2015,33(4):1200-1212.
[6]RATAJCZAK J,MIEKUS K,KUCIA M,et al.Embryonic stem cel1.Derived microvesicles reprogram hematopoietic progenitors:evidence for horizontal transfer of mRNA and protein delivery[J].Leukemia,2006,20(5):847-856.
[7]GATTI S,BRUNO S,DEREGIBUS M C,et al.Microvesicles derived from human adult mesenchymal stem cells protect against ischaemia-reperfusion-induced acute and chronic kidney injury[J].Nephrol Dial Transplant,201l,26(5):1474-1483.
[8]VALADI H,EKSTROM K,BOSSIOS A,et al.Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells[J].Nat Cell Biol,2007,9(6):654-659.
[9]PEGTEL D M,COSMOPOULOS K,THORLEY-LA-WSON D A,et al.Functional delivery of viral miRNAs via exosomes[J].Proc Natl Acad Sci USA,2010,107(14):6328-6333.
[10]ZHANG Y,LIU D,CHEN X,et al.Secreted monocytic miR-150enhances targeted endothelial cell migration[J].Mol Cell,2010,39(1):133-144.
[11]KOSAKA N,IGUCHI H,YOSHIOKA Y,et al.Secretory mechanisms and intercellular transfer of microR-NAs in living cells[J].Biol Chem,2010,285(23):17442-17452.
[12]HUOTARI J,HELENIUS A.Endosome maturation[J].EMBO,2011,30(17):3481-500.
[13]SIMPSON R J,JENSEN S S,LIM J W.Proteomic profiling of exosomes:current perspectives[J].Proteomics,2008(8):4083-4099.
[14]YAMASHITA T,TAKAHASHI Y,NISHIKAWAM,et al.Effect of exosome isolation methods on physicochemical properties of exosomes and clearance of exosomes and clearance of exosomes from the blood circulation[J].European Journal of Pharmaceutics&Biopharmaceutics,2016(98):1-8.
[15]王媛,雷迎峰,丁天兵,等.超速离心机的操作及超速离心技术的应用[J].医学理论与实践,2013(16):2144-2146.