外周血非动员干细胞体外培养分析红系特异性表达血型基因

摘要

目的 通过外周血非动员干细胞液培养获得红系有核细胞,以分析红系特异性表达的血型基因.方法 外周血经离心分离和免疫磁珠分选获得CD34+造血干细胞,体外液体定向培养生成红系有核细胞.分别取不同培养期的细胞进行免疫荧光和染色分析,并取培养+12 d的细胞进行Rh和ABO血型基因RNA转录分析.结果 从约50 mL外周血中分离到CD34+细胞(3.19±0.13)×104个(总回收率为67.3％±2.7％).经红系定向培养,大约+9d时细胞数爆增至平台期(7.57±0.78)×106个,扩增约237.1±15.5倍.在培养过程中,标志细胞干性的CD34抗原逐渐下降,红系特异性标志CD235a和CD240D逐渐上升.培养+12 d细胞提取的RNA,均能扩增出RHD/CE和ABO基因,DNA序列分析表明其基因型符合血清学表型.结论 建立了一种源于外周血非动员干细胞体外培养分析红系血型基因表达情况的方法,该技术可用于各种红系特异性表达基因的研究.%Objective To analyze specific expression of blood group genes using nucleated erythroid cells cultured from un-mobilized peripheral stem cells in vitro.Methods Hematopoietic stem cells(HSC) bearing the CD34 antigen were isolated from peripheral blood by centrifugation and magnetic beads sorting,followed by suspension culture in vitro.Cells were collected from medium on various stages and analyzed by immunofluorescence.The RNA transcription of RH and ABO blood group genes was analyzed using culture cells on day 12.Results A total of(3.19 ± 0.13) × 104 CD34 + cells were isolated from about 50 mL peripheral blood with a recovery rate of 67.3％ ± 2.7％.The cells amount in erythroid-lineage culture system on day 9 reached a plateau of a 237.1±15.5-fold amplification of the initial cell input.The stem cell-specific CD34 antigen was dropped off,while the erythroid-specific CD235a and CD240D antigens were increased in culture period.RHD/CE and ABO genes can be amplified using RNA extracted from culture cells on day 12,and genotypes of Rh and ABO systems by DNA sequencing were consistent with their serologic phenotypes.Conclusion A method was established to analyze the gene expression of erythroid blood group derived from un-mobilized peripheral stem cells cultured in vitro.It can be used to study the expression of various erythroid-specific genes.