脐带血单个核细胞诱导多能干细胞来源自然杀伤细胞的生物学特性

Biological characteristics of natural killer cells from cord blood mononuclear cell-derived induced pluripotent stem cells

目的利用脐带血单个核细胞(CBMCs)获得诱导多能干细胞(iPSCs)并进一步诱导分化为自然杀伤(NK)细胞。通过与其他来源NK细胞的比较,探讨iPSCs来源NK细胞的生物学特性。方法获取CBMCs并使用非整合质粒电转法对其进行重编程,利用免疫荧光染色和流式细胞术鉴定获得的iPSCs的多能性因子的表达水平。将CBMCs来源iPSCs(CBiPSCs)诱导分化为NK细胞,并利用流式细胞术对NK细胞表型进行鉴定。利用肿瘤细胞体外杀伤实验,比较CB-iPSCs来源NK(CB-iPSCs-NK)细胞、脐带血来源NK(CB-NK)细胞和外周血来源NK(PB-NK)细胞的杀伤活性。多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。结果CBMCs经过携带重编程因子的非整合质粒转染后,启动多能性基因表达,成功地获得iPSCs克隆,经扩增和筛选,最后获得了可稳定增殖的CB-iPSCs。免疫荧光染色和流式细胞术检测结果显示,获得的CB-iPSCs表达高水平的多能性标志物。然后,利用CB-iPSCs成功分化NK细胞。流式细胞术检测NK受体,包括抑制性受体CD94、细胞毒性受体CD16和Nkp46;与CB-NK和PB-NK比较,CB-iPSCs-NK中CD94(94.17﹪±1.80﹪,94.10﹪±1.32﹪比87.60﹪±2.10﹪)和CD16表达(64.53﹪±2.80﹪,92.40﹪±2.00﹪比9.23﹪±3.37﹪)较低,Nkp46表达(85.30﹪±2.36﹪,29.43﹪±9.08﹪比88.00﹪±3.60﹪)较高(P均<0.05)。肿瘤细胞体外杀伤实验结果显示,三种来源NK细胞对K562、MDA-MB-231细胞均具有较好的杀伤作用;但是,对Raji细胞的杀伤作用,CB-iPSCs-NK与CB-NK相似,均弱于PB-NK(P均<0.05)。结论免疫表型和对不同肿瘤细胞杀伤效果的差异,提示CB-iPSCs-NK细胞具有不同的生物学特性。

Objective To obtain induced pluripotent stem cells(iPSCs)from cord blood mononuclear cells(CBMCs)and further induce them to generate natural killer(NK)cells,then to investigate the biological characteristics of CB-iPSCs-derived NK(CB-iPSCs-NK)cells and compare key traits between CB-iPSCs-NK cells and the other-derived NK cells.Methods CBMCs were obtained and transduced with reprogramming factors by non-integrated plasmids to generate iPSCs.The pluripotency factors expressions of CBMCs-derived iPSCs(CB-iPSCs)were determined by immunofluorescence staining and flow cytometry.CB-iPSCs were induced to generate NK cells,and phenotypes of NK cells were identified by flow cytometry.The cytotoxic effects of CB-iPSCs-NK cells,compared with cord blood derived NK(CB-NK)cells and peripheral blood derived NK(PB-NK)cells,were tested by killing tumor cell assay in vitro.One-way ANOVA was used for comparison of variables among multiple groups,and LSD-t test was used for comparison of variables between groups.Results After transfection with reprogramming factors by non-integrated plasmid,pluripotent gene expression of CBMCs were initiated,and clones of iPSCs were successfully obtained.After proliferation and selection,the stable CB-iPSCs were finally obtained.The results of immunofluorescence staining and flow cytometry showed that CB-iPSCs expressed high levels of pluripotency markers.Then,CB-iPSCs-NK cells were successfully obtained from CB-iPSCs.NK receptors including inhibitory receptor CD94,cytotoxic receptor CD16 and Nkp46 were detected by flow cytometry.Compared with CB-NK and PB-NK,CD94 expression of CB-iPSCs-NK was lower(94.17﹪±1.80﹪,94.10﹪±1.32﹪vs 87.60﹪±2.10﹪),CD16 expression was lower(64.53﹪±2.80﹪,92.40﹪±2.00﹪vs 9.23﹪±3.37﹪),Nkp46 expression was higher(85.30﹪±2.36﹪,29.43﹪±9.08﹪vs 88.00﹪±3.60﹪)(All P<0.05).The results of killing tumor cell assays in vitro showed that three kindsof NK cells hadexcitingcytotoxic effects to K562 and MDA-MB-231 cells.However,compared with PB-NK cells,cytotoxic effects of CB-iPSCs-NK cells and CB-NK cells to Raji cells were weaker(All P<0.05).Cytotoxic effect of CB-iPSCs-NK cells to Raji cells was similar to that of CB-NK cells.Conclusion The differences of immunophenotypes and cytotoxic effects suggested that CB-iPSCs-NK cells have different biological characteristics for killing tumor cells.