CD47在初诊正常核型急性髓系白血病患者中的表达及其临床意义

Expression of CD47 in de novo acute myelogenous leukemia with normal karyotype and its clinical significance

目的 探讨CD47在初诊正常核型急性髓系白血病（AML）患者中的表达水平及临床意义.方法 选取137例初诊正常核型AML患者及3名健康志愿者.采用流式细胞术及实时荧光定量聚合酶链反应（qPCR）对健康志愿者骨髓造血干细胞（HSC）和造血祖细胞（MPP）及AML患者骨髓单个核细胞（MNC）和白血病干细胞（LSC,Lin-CD34＋CD38-CD90-）的CD47表达水平进行检测;采用基因组分析平台检测患者FMS样酪氨酸激酶3内部串联重复（FLT3-ITD）.采用超高速流式分选系统分选CD34＋CD38-CD47lo和CD34＋CD38-CD47hi细胞.将两组细胞均用MethoCult H4445培养液培养接种于含有琼脂糖的甲基纤维素板,12 d后计数MPP集落形成单位（CFU）,同时将1＆#215;105个CD34＋CD38-CD47lo和CD34＋CD38-CD47hi细胞分别移植到经280 cGy照射的NSG（NOD-SCID IL-2Rγnull）小鼠,8周后处死小鼠,流式细胞术检测人CD45＋细胞比例.结果 CD47的表达在初诊正常核型AML患者中高于健康志愿者,CD47在AML各法、美、英协作组（FAB）亚型中均有表达,相对表达量比较差异无统计学意义（F=0.545,P〉0.05）.137例患者中CD34＋CD38-CD47hi 37例,其中17例（46%）FLT3-ITD阴性,20例（54%）为FLT3-ITD阳性;CD34＋CD38-CD47lo的患者100例,其中63例（63%）FLT3-ITD阴性,37例（37%）FLT3-ITD阳性,CD34＋CD38-CD47hi与CD34＋CD38-CD47lo患者相比较,FLT3-ITD阳性率差异无统计学意义（χ2=3.79,P〉0.05）.将FACS分选的CD34＋CD38-CD47lo和CD34＋CD38-CD47hi接种于含有琼脂糖的甲基纤维素板12 d后,仅CD34＋CD38-CD47lo细胞可以形成CFU.NSG小鼠移植实验显示CD34＋CD38-CD47lo细胞可重建造血,CD34＋CD38-CD47hi植入失败.结论CD34＋CD38-CD47hi富集LSC,可作为AML患者化疗后微小残留病的监测指标.

Objective To investigate the CD47 expression in de novo acute myelogenous leukemia （AML） patients with normal karyotype and its clinical significance. Methods One hundred thirty-seven cases of de novo AML with normal karyotype and 3 healthy volunteers were selected. Relative CD47 expressions in normal bone marrow hematopoietic stem cells （HSC） and multipotent progenitor （MPP） from healthy volunteers, as well as bone marrow mononuclear cells （MNC） and leukemia stem cells （LSC, Lin-CD34＋CD38-CD90-） from AML patients were determined by flow cytometry. CD47 expression on the Lin-CD34＋CD38-LSC-enriched fraction of specimen was determined by flow cytometry. The FMS-like tyrosine kinase 3 internal tandem duplication （FLT3-ITD） was detected by using the Genome Analyzer platform. CD34＋CD38-CD47hi and CD34＋CD38-CD47lo expressing cells were identified and purified using FACS. Two groups of cells were inoculated with MethoCult H4445 medium on agarose-containing methylcellulose plates. After 12 days, MPP colony forming units （CFU） were counted, and 1＆#215;105 CD34＋ CD38- CD47lo and CD34＋ CD38-CD47hi cells were transplanted into NSG （NOD-SCID IL-2R γ null） mice irradiated by 280 cGy, and mice were sacrificed after 8 weeks. The ratio of human CD45＋cells was detected by flow cytometry. Results The expression of CD47 in AML patients was higher than that in the healthy control. CD47 was expressed in all FAB （French-American-British） subtypes of AML. No significant difference in CD47 expression among different FAB subtypes was found （F=0.545, P〉0.05）. Among the 37 patients with CD34＋CD38-CD47hi, 17 （46 %） were FLT3-ITD negative, and 20 （54 %） were FLT3-ITD positive. Among the 100 patients with CD34＋CD38-CD47hi, 63 （63%） cases were FLT3-ITD negative, 37 （37%） cases were FLT3-ITD positive. The rate of FLT3-ITD positive in patients with CD34＋ CD38- CD47lo had no statistical difference compared with patients with CD34＋CD38-CD47hi （χ2= 3.79, P〉 3.79）. The CD34＋CD38-CD47lo or CD34＋CD38-CD47hi which was selected by FACS, was inoculated with the methylcellulose plate containing agarose for 12 days, and CD34＋CD38-CD47lo cells could form CFU. The NSG mouse transplantation experiment showed that CD34＋CD38-CD47lo cells could be reconstructed hematopoiesis, and CD34＋CD38-CD47hi implantation failed. Conclusion CD34＋CD38-CD47hi could enrich LSC, which may be a potential marker to detect minimal residual disease.