全血白膜层单个核细胞的分离、保存与鉴定  

Preservation, analysis and identification of PBMC isolated from whole blood buffy coats

作　　者：李冬妹[1] 陈小林[1] 徐彬 王明慧[1] 贾延军[1] Li Dongmei;Chen Xiaolin;Xu Bin;Wang Minghui;Jia Yanjun(HLA Laboratory, Beijing Red Cross Blood Center, Beijing 100088, China)

机构地区：[1]北京市红十字血液中心白细胞室

出　　处：《北京医学》2019年第8期726-728,731,共4页Beijing Medical Journal

摘　　要：目的建立分离、冻存及鉴定外周血单个核细胞(peripheral blood mononuclear cell, PBMC)的方法,检测PBMC冻存不同时间后各种细胞的含量和活性。方法选择2016年8月至2018年3月北京市红十字血液中心无偿献血志愿者20人,从志愿者全血白膜层中分离出PBMC并用细胞冻存液冻存于液氮中,应用流式细胞术(fluorescence-activated cell sorter, FACS)检测分离出的及冻存复苏的PBMC中粒细胞、单核细胞、淋巴细胞(T细胞、B细胞和NK细胞)的含量及其死细胞的含量。结果 PBMC中淋巴细胞占大多数[(75.45±13.20)%],其次为单核细胞[(13.08±4.97)%]和粒细胞[(11.47±14.40)%]。与白膜层相比,PBMC中粒细胞、单核细胞和淋巴细胞含量分别减少了94%、62%和58%。PBMC冻存后1周、1个月、3个月、1年后,粒细胞死亡比例明显增加,粒细胞占比明显下降(P<0.05);单核细胞占比明显上升(P<0.05),淋巴细胞总数及T细胞、B细胞、NK细胞占比变化很大。结论本研究所确立的方法将为科研人员更好地处理、保存和利用外周血PBMC提供帮助。Objective To establish a method for isolation, cryopreservation and identification of peripheral blood mononuclear cell(PBMC) from whole blood buffy coats, and to detect the content and activity of PBMCs after different cryopreservation time. Methods PBMCs were isolated from the whole blood buffy coats of 20 unpaid blood donation volunteers,and frozen in liquid nitrogen by mixing with cell cryopreservation solution. The contents of granulocytes, monocytes, lymphocytes(including T cells, B cells and NK cells) as well as different dead cells in fresh isolated and frozen PBMCs were detected by fluorescence-activated cell sorter(FACS). Results Lymphocytes accounted for(75.45±13.20)% of PBMC, followed by monocytes(13.08±4.97)% and granulocytes(11.47±14.40)%. Granulocytes had a 94% reduction in PBMC compared to the content in buffy coats, and a decrease of 62% and 58% of monocytes and lymphocytes, respectively. At one week, one months, three months and one year after cryopreservation, the proportion of granulocytes decreased significantly(P < 0.001),while the proportion of monocytes increased(P < 0.001), but the proportion of lymphocytes, T cells, B cells and NK cells did not change very much. The proportion of granulocytes decreased significantly due to more deaths. Conclusions The method established in this study will help researchers to better handle, preserve and utilize PBMC in peripheral blood.

关 键 词：外周血单个核细胞 白膜层 有核细胞 

分 类 号：R446.11[医药卫生—诊断学]