[Cheek blood sampling of mice].

OBJECTIVE: To explore the method of cheek blood sampling in mice in continuous blood test. METHODS: Cheek blood sampling was carried out in 10 9-week-old mice once every 2-3 days. The volumes of blood samples were recorded. Reactions of the mice were observed. RESULTS: Cheek blood sampling collected 0.3-0.6 ml of blood every time, and did not affect the normal activities of the mice. The blood sampling could be repeated every 2-3 days. CONCLUSIONS: Cheek blood sampling has several advantages including being capable to draw adequate blood, simple and easy to operate, well tolerated, and ethical in treating laboratory animals.

[Changes of angiopoietin 1 expression in G-CSF induced hematopoietic stem progenitor cells mobilization].

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.

Granulocyte Colony-Stimulating Factor Induces Osteoblast Inhibition by B Lymphocytes and Osteoclast Activation by T Lymphocytes during Hematopoietic Stem/Progenitor Cell Mobilization.

In the bone marrow (BM), hematopoietic stem and progenitor cells (HSPCs) reside in specialized niches near osteoblast cells at the endosteum. HSPCs that egress to peripheral blood are widely used for transplant, and mobilization is most commonly performed with recombinant human granulocyte colony-stimulating factor (G-CSF). However, the cellular targets of G-CSF that initiate the mobilization cascade and bone remodeling are not completely understood. Here, we examined whether T and B lymphocytes modulate the bone niche and influence HSPC mobilization. We used T and B defective mice to show that G-CSF-induced mobilization of HSPCs correlated with B lymphocytes but poorly with T lymphocytes. In addition, we found that defective B lymphocytes prevent G-CSF-mediated osteoblast disruption, and further study showed BM osteoblasts were reduced coincident with mobilization, induced by elevated expression of dickkopf1 of BM B lymphocytes. BM T cells were also involved in G-CSF-induced osteoclast activation by regulating the Receptor Activator of Nuclear Factor-κ B Ligand/Osteoprotegerin (RANKL/OPG) axis. These data provide evidence that BM B and T lymphocytes play a role in G-CSF-induced HSPC mobilization by regulating bone remodeling.