Objective: To investigate the changes and mechanism of angiopoietin1 (Ang1) in murine bone marrow during G-CSF induced mobilization of hematopoietic stem/progenitor cell.
Methods: The proportion of Lin-Sca1⁺cKit⁺ (LSK) cells in peripheral blood of C57BL/6 mice before and after G-CSF mobilization was detected by flow cytometry. Expression changes of Ang1 and osteocalcin (OCN) during HSC mobilization were determined by immunohistochemistry, enzyme linked immunosorbent assay (ELISA) and real-time fluorescence quantitative PCR. The number of osteoblasts in the bone marrow was counted under the microscope.
Results: After treated with G-CSF, the proportion of LSK cells in peripheral blood significantly increased from the controls (0.04 ± 0.01)% to (0.61 ± 0.05)% at day 5 (P<0.05). Before G-CSF mobilization, the endosteum cells expressed higher level of OCN and Ang1 than that of bone marrow nucleated cells. The mRNA expression level of OCN was significantly reduced from 28.64 ± 8.61 in the controls to 12.55 ± 7.06 on day 3 and 4.75 ± 1.62 on day 5, and the expression level of Ang1 also declined from 2.84 ± 0.95 in the controls to 0.93 ± 0.30 on day 3 and to 0.92 ± 0.22 on day 5 after G-CSF mobilization. The number of endosteum osteoblasts was significantly decreased after mobilization (P<0.05). The Ang1 expression was decreased in the BM after mobilization. The serum OCN was significantly reduced from (24.11 ± 3.17) ng/ml in the controls to (9.96 ± 2.16) ng/ml on day 3 and (8.43 ± 2.62) ng/ml on day 5, and the Ang1 also declined from (2.24 ± 0.52) ng/ml in the controls to (1.21±0.38) ng/ml on day 3 and (0.90±0.24) ng/ml on day 5.
Conclusion: In G-CSFinduced HSPC mobilization, the bone marrow osteoblasts retraction causes reduction of Ang1, and the reduction of Ang1 may contribute to HSPC mobilization.
目的: 探讨G-CSF诱导的造血干／祖细胞（HSPC）动员过程中血管生成素1(Ang1)在小鼠骨髓的表达变化及导致其变化的机制。
方法: 采用流式细胞术检测小鼠动员前及动员后外周血Lin−Sca1+cKit+(LSK)细胞比例变化，并应用ELISA、免疫组化、RQ-PCR等方法检测G-CSF动员不同时间点小鼠外周血和骨髓标本，观察Ang1、骨钙素（OCN）基因及蛋白水平变化，显微镜下计数骨髓标本成骨细胞数量。
结果: G-CSF动员5 d小鼠外周血LSK细胞比例为（0.61±0.05)%，相对于对照组的(0.04±0.01)%显著增高(P<0.05)；稳态小鼠OCN及Ang1 mRNA在骨内膜细胞表达高于骨髓有核细胞；动员后骨内膜细胞OCN及Ang1 mRNA相对表达量对照组（28.64±8.61及2.84±0.95)、G-CSF动员3 d组（12.55±7.06及0.93±0.30）及动员5 d组（4.75±1.62及0.92±0.22）均下降；动员后骨内膜成骨细胞数量显著减少，骨髓组织Ang1蛋白表达下降；对照组、动员3 d及5 d组外周血OCN及Ang1浓度分别为（24.11±3.17）及（2.24±0.52)ng/ml、(9.96±2.16）及（1.21±0.38)ng/ml和（8.43±2.62）及(0.90±0.24) ng/ml，动员3 d及5 d组均明显低于对照组，差异均有统计学意义(P值均<0.05)。
结论: 在G-CSF介导的HSPC动员过程中，骨髓微环境中成骨细胞数量下降、功能减低，进而导致骨髓Ang1表达下降，导致骨髓对HSPC的趋化和黏附能力下降，有利于HSPC动员的发生。