3种获得中性粒细胞方法的比较及应用的探讨  

Comparison and discussion of the application of three methods for neutrophil preparation

作　　者：呼沛然 白苗娜[1] 李萌璐 杨欢[1] 姚智[1] 高颖[1] 

机构地区：[1]天津医科大学基础医学院免疫学系/天津市细胞与分子免疫学重点实验室/国家教育部免疫微环境与疾病重点实验室,天津300070

出　　处：《国际检验医学杂志》2014年第16期2123-2125,共3页International Journal of Laboratory Medicine

基　　金：国家自然科学基金资助项目(31071217)

摘　　要：目的比较诱导髓系白血病细胞株分化法、外周血分离纯化法、诱导造血干细胞定向分化法3种获得中性粒细胞的方法。方法用1μmol/L全反式维甲酸(ATRA)诱导NB4细胞分化;用红细胞裂解法结合免疫磁珠法分离纯化外周血中性粒细胞;用Percoll密度梯度离心法结合免疫磁珠法分离纯化外周血干细胞,然后用GM-CSF和G-CSF诱导粒细胞的产生。3种方法获得的细胞通过MGG染色检测细胞形态及中性粒细胞纯度,利用免疫荧光技术检测CD18蛋白的表达及胞内分布特点。结果诱导NB4细胞分化可获得约40%的中性粒细胞,从外周血分离纯化的中性粒细胞纯度约为90%,通过诱导造血干细胞定向分化可获得70%以上的中性粒细胞。免疫荧光结果显示,3种方法获得的中性粒细胞,CD18蛋白的表达强度无明显不同,也都呈现点状分布。结论红细胞裂解法结合免疫磁珠法分离纯化外周血中性粒细胞,纯度高、活性好,有利于快速检测新鲜血液样本中的中性粒细胞;诱导造血干细胞定向分化获得的中性粒细胞,活性高,可在体外维持长达几天之久,可应用于中性粒细胞长时间的体外试验。Objective To make a comparison for the neutrophils prepared either by induction of differentiation of myeloid leuke-mia cell line,or by separation and purification of peripheral blood cells,or by induction of myeloid differentiation of peripheral blood stem cells.Methods NB4 cells were induced differentiation by 1μmol/L all-trans retinoic acid （ATRA）to mature granulo-cytes;neutrophils were separated and purified from peripheral blood by lysis of red blood cells followed by negative selection using magnetic bead-labeled antibodies;hematopoietic stem cells were separated and purified from peripheral blood by Percoll gradient centrifugation followed by negative selection using magnetic bead-labeled antibodies,and were induced to myeloid differentiation by GM-CSF and G-CSF.Morphology and purity of neutrophils prepared by these three methods were studied by means of MGG stai-ning.CD18 protein expression and subcellular distribution were studied by means of immunofluorescence staining.Results Purity of neutrophil was above 40% by induction of differentiation of NB4 cells,and was about 90% if purified from peripheral blood,and was above 70% if induced by myeloid differentiation of peripheral blood stem cells.There was no obvious difference for CD18 ex-pression in neutrophils prepared by these three methods,and staining of CD18 had a dotted pattern distributed in these cells.Con-clusion Peripheral blood neutrophils prepared by lysis of red blood cells followed by negative selection using magnetic bead-labeled antibodies are with high purity and viability which is suitable for immediate test of neutrophils from fresh blood.Neutrophils pre-pared by myeloid differentiation of hematopoietic stem cell are with high viability and last for days,which can be used in long test for neutrphils.

关 键 词：中性粒细胞 外周血干细胞 诱导分化 免疫磁珠法 

分 类 号：R392[医药卫生—免疫学]