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  

Analysis of erythroid-specific blood group genes using unmobilized peripheral stem cells cultured in vitro

作　　者：许先国[1] 刘瑛[1] 应燕玲[1] 洪小珍[1] 马开荣[1] 蓝小飞[1] 陈舒[1] 何吉[1] 朱发明[1] 吕杭军[1] 

机构地区：[1]浙江省血液中心、卫生部血液安全研究重点实验室,杭州310006

出　　处：《中华医学遗传学杂志》2014年第4期487-490,共4页Chinese Journal of Medical Genetics

基　　金：浙江省自然科学基金（Y12H080002）,浙江省医药卫生科技计划（2013ZDA007）,浙江省卫生高层次创新人才培养项目

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＋12d的细胞进行Rh和ABO血型基因RNA转录分析。结果从约50mL外周血中分离到CD34^＋细胞（3．19±0．13）×10^4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）×10^6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＋12d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 unmobilized 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））〈 10^4 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 unmobilized peripheral stem cells cultured in vitro. It can be used to study the expression of various erythroid-specific genes.

关 键 词：血型 基因表达 细胞培养 红细胞 

分 类 号：R394[医药卫生—医学遗传学]