【摘要】 目的通过干预细胞膜阴离子通道(AE)和Na+/K+/2Cl-离子共转运体(NKCC),提高聚偏二氟乙烯(PVDF)膜富集脐血有核红细胞(NRBC)的效率。方法以密度为1.067的分离液分离NRBC,用AE抑制剂维拉帕米(verapamil)和NKCC抑制剂呋噻米(furosemide)联合干预,用流式细胞技术和巢式PCR观察干预前后离心法和过5μm孔径PVDF膜法富集NRBC的效率。结果最佳离心分离密度梯度为1.067。流式细胞仪检测离心分离的NRBC平均纯度为2.54%,干预后离心分离NRBC纯度为9.36%,NRBC富集率提升近3.7倍;过PVDF膜前NRBC为0.83%,过膜后NRBC为6.15%,干预后过膜将NRBC富集率提升了7.4倍。用巢式PCR检测,每ml血样中NRBC为120个时,干预后过膜的扩增条带的密度值由4.48升高至17.78,提升近4倍。结论维拉帕米和呋噻米能够通过改变脐血细胞性状,提高NRBC通过PVDF膜的效率。更多还原

【Abstract】 Objective The effectiveness on the enrichment of nuclear red blood cells(NRBC) in umbilical cord blood by polyvinylidene difluoride membrane was investigated with intervention on membrane ionic channels.Methods NRBC was isolated by the isolation solution with a density of 1.067 g/ml.The rates of the cell enrichment using verapamil and furosemide intervention by discontinuous density centrifugation were examined by flow cytometry.The NRBC that past through PVDF membrane with pore size of 5 μm were checked and quantified by nested-PCR.Results The optimal centrifugation density was 1.067 g/ml.The mean purity of NRBC after centrifugation was 2.54%,yet the purity after intervention increased to 9.36%(3.7-fold increment).The NRBC passing through PVDF membrane was from 0.83% to 6.15%,with enrichment of 7.4 times.When NRBC was 120 cells/ml,the density of the nested PCR products increased by nearly 4 times after intervention(4.48 vs 17.78).Conclusions Verapamil and furosemide could affect the morphology of cells and promote the rate of NRBC enrichment.更多还原