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摘要:
目的 研究Hes1对急性髓系白血病(AML)患者骨髓CD34+CD38-细胞的作用及其机制。 方法 收集初治AML患者及正常供者骨髓样本后，通过密度梯度离心法获取单个核细胞，流式细胞术检测CD34+CD38-细胞比例及其细胞周期。通过免疫磁珠法分选CD34+CD38-细胞后，体外集落形成实验(CFC)检测其增殖能力，并通过Realtime PCR检测其Hes1的表达量。构建Hes1过表达逆转录病毒载体，感染正常供者骨髓CD34+细胞后，流式细胞术分析其细胞周期的改变，CFC检测其增殖的改变。 结果 AML患者骨髓CD34+CD38-细胞比例明显低于正常对照，流式细胞术结果显示患者来源CD34+CD38-细胞大多数进入静止期，CFC结果显示患者CD34+CD38-细胞体外扩增能力下降。Realtime PCR结果发现患者CD34+CD38-细胞中Hes1表达上调。提高正常供者CD34+细胞中Hes1的表达后，细胞增殖减少，进入静止期。 结论 在AML中CD34+CD38-细胞比例下降，进入静止期，与Hes1的表达上调有关。 -
关键词:
- AML白血病 /
- CD34+CD38-细胞 /
- Hes1 /
- 细胞周期 /
- 增殖
Abstract:Objective To determine the effect of Hes1 on bone marrow CD34+ cells in acute myeloid leukemia (AML). Methods Bone marrow mononuclear cells were isolated by using Ficoll. Then, the proportion and cell cycle of CD34+ cells were analyzed by using fluorescence-activated cell sorting (FACS). CD34+ cells were cultured in vitro for colony-forming cells (CFC). The expression of Hes1 in CD34+ cells was evaluated by using real-time polymerase chain reaction. After upregulating the expression of Hes1 in CD34+ cells, the cell cycle was analyzed through FACS, and the colony formation of CD34+Hes1+ cells was analyzed by CFC. Results The ratio of CD34+ cells in the bone marrow was lower in the AML group than in the control group. In addition, more CD34+ cells underwent quiescence in the AML group than in the control group. In vitro assay showed that the colony formation of CD34+ cells was lower in the AML group than in the control group. The expression of Hes1 was higher in the CD34+ cells from the AML patients than that in the CD34+ cells from normal donors. After Hes1 transduction, more CD34+ cells underwent quiescence and showed weak proliferation. Conclusion The proportion of CD34+ cells in the bone marrow was lower in AML patients than in normal donors. A large proportion of CD34+ cells underwent quiescence, which was related to Hes1, in AML patients. -
Key words:
- T-ALL /
- CD34+CD38-cell /
- Hes1 /
- cell cycle /
- proliferation
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图 1 患者来源CD34+CD38-细胞向各系分化形成的集落数少于对照
Figure 1. CFC test results showed that the clone numbers of CD34+ cells were lower in the AML group than in the control group
BFU-E: burst-forming unit-erythroid; CFU-G: colony-forming unit-granulocyte; CFU-M: colony-forming unit-megakaryocyte; CFU-GM: colony-forming unit-granulocyte macrophage; CFU-GEMM: colony-forming unit-granulocyte, erythroid, macrophage, and monocyte
图 2 骨髓CD34+CD38-细胞周期分析
Figure 2. Cell cycle analysis of bone marrow CD34+CD38- cells
A. Proportion of S phase cells detected by the bromodeoxyuridine method. B. Proportion of G0/G1 phase cells detected by the Hoechst/PY method
图 3 Realtime PCR和Western检测Hes1的表达量
Figure 3. Expression of Hes1 was tested through real-time polymerase chain reaction and Western blot
A and B. Relative expression of Hes1 mRNA in T cell acute lymphoblastic leukemia (T-ALL) patients and normal donors compared with the GAPDH gene. C. Relative expression of Hes1 protein in T-ALL patients and normal donors
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