[8;21) 急性髓系白血病中CD34白血病干细胞的体外鉴定及特性]

[In Vitro Identification and Characteristics of CD34 Leukemia Stem Cell in t(8;21) Acute Myeloid Leukemia].

作者信息

Zhang Yan-Huan, Yang Lu, Wang Ya-Zhe, Kong Yuan, Zhu Hong-Hu, Qin Ya-Zhen

机构信息

Peking University People's Hospital, Peking University Institute of Hematology, Beijing 100044, China.

Peking University People's Hospital, Peking University Institute of Hematology, Beijing 100044, China. E-mail:

出版信息

Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2017 Oct;25(5):1289-1294. doi: 10.7534/j.issn.1009-2137.2017.05.003.

DOI:10.7534/j.issn.1009-2137.2017.05.003

PMID:29070097

Abstract

OBJECTIVE

To preliminarily identify the existence of CD34 leukemia stem cell (LSC) in t(8;21) acute myeloid leukemia (AML) by in vitro test.

METHODS

Bone marrow samples collected from newly diagnosed t(8;21) AML patients were tested. LinCD34 CD38(abbreviation, CD34CD38), LinCD34CD38 (abbreviation, CD34CD38) and LinCD34CD38CD45SSC(abbreviation, CD34"LSC") cell fractions were gated by flow cytometry after staining with fluorescent antibodies. Cells in G phase were identified through Hoechst 33342 and pyronin Y staining. Aldefluor reagent was used to test aldehyde dehydrogenase (ALDH) activity. The above-mentioned 3 cell fractions were sorted, and mRNA levels of AML1-ETO and WT1 were measured by real-time quantitative PCR.

RESULTS

The 3 tested samples displayed the same tendency in ratio of the cells in G phase: CD34"LSC">CD34 CD38>CD34CD38. The paired t-test of 53 patients showed that frequency of ALDH cells of both CD34CD38 and CD34"LSC" cell fractions was significantly higher than that of CD34CD38 (P<0.01), furthermore, the ALDH cell frequency was significantly higher in CD34"LSC" than that in CD34 CD38 (P<0.01). AML1-ETO mRNA levels of cells sorted from 3 patients were similar among the 3 cell fractions within each patient, whereas WT1 mRNA levels were significantly higher in CD34"LSC" than that in other 2 cell fractions.

CONCLUSION

CD34 LSC may exist in t(8;21) AML, and may be more primitive than CD34 LSC. These results promote the necessity to perform in vivo xenogeneic transplantation mice.

摘要

目的

通过体外试验初步鉴定t(8;21)急性髓系白血病（AML）中CD34白血病干细胞（LSC）的存在。

方法

对新诊断的t(8;21) AML患者采集的骨髓样本进行检测。用荧光抗体染色后，通过流式细胞术对Lin⁻CD34⁺CD38⁻（简称，CD34⁺CD38⁻）、Lin⁻CD34⁺CD38⁺（简称，CD34⁺CD38⁺）和Lin⁻CD34⁺CD38⁻CD45⁻SSC⁻（简称，CD34⁺“LSC”）细胞亚群进行设门。通过Hoechst 33342和派洛宁Y染色鉴定G期细胞。使用醛脱氢酶（ALDH）试剂检测醛脱氢酶活性。对上述3个细胞亚群进行分选，通过实时定量PCR检测AML1-ETO和WT1的mRNA水平。

结果

3个检测样本在G期细胞比例上呈现相同趋势：CD34⁺“LSC”>CD34⁺CD38⁻>CD34⁺CD38⁺。对53例患者进行配对t检验显示，CD34⁺CD38⁻和CD34⁺“LSC”细胞亚群中ALDH细胞频率均显著高于CD34⁺CD38⁺（P<0.01），此外，CD34⁺“LSC”中ALDH细胞频率显著高于CD34⁺CD38⁻（P<0.01）。3例患者分选的细胞中，AML1-ETO mRNA水平在各患者的3个细胞亚群中相似，而WT1 mRNA水平在CD34⁺“LSC”中显著高于其他2个细胞亚群。