Résumé
The mobilization efficiency of granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) to bone marrow mononuclear cells (MNCs) in mice was observed,and the changes of CXCL12/CXCR4 signal were detected in order to find out the mobilization mechanism of stem cells.Kunming mice were randomly divided into two groups.The mice in treatment group were subjected to subcutaneous injection of G-CSF at a dose of 100 μg/kg and SCF at a dose of 25 μg/kg every day for 5 days,and those in control group were given isodose physiological saline.The MNCs were separated,counted and cultured,and the colony-forming unit-fibroblast (CFU-F) was evaluated.CD34+CXCR4+ MNCs were sorted by flow cytometry.The expression of CXCL12 protein in bone marrow extracellular fluid was detected by ELISA,and that of CXCL12 mRNA in bone marrow was measured by RT-PCR.The results showed that the counts of MNCs in peripheral blood and bone marrow were increased after administration of G-CSF/SCF (P＜0.01).The factors had a dramatic effect on the expansion capability of CFU-F (P＜0.05).Flow cytometric of bone marrow MNCs surface markers revealed that CD34+CXCR4+ cells accounted for 44.6%±8.7% of the total CD34+ MNCs.Moreover,G-CSF/SCF treatment induced a decrease in bone marrowCXCL12 mRNA that closely mirrored the fall in CXCL12 protein.In this study,it is evidenced that G-CSF/SCF can effectively induce MNCs mobilization by disrupting the balance of CXCL12/CXCR4 signaling pathway in the bone marrow and down-regulating the interaction of CXCL12/CXCR4.
Résumé
The present study was aimed at finding an effective method to isolate and purify the subtype of type A spermatogonial stem cells (SSCs) in juvenile rats. Testes from 9-days-old rats were used to isolate germ cells by using two-step enzymatic digestion. The expression of c-kit in the testes of the rats was immunohistochemically detected. After isolation, cell suspension was enriched further by discontinuous density gradient centrifugation. Then type A1-A4 spermatogonia was isolated from the purified spermatogonia with c-kit as the marker by using fluorescence-activated cell sorting(FACS). Electron microscopy was used to observe their ultrastructure. Finally, highly purified and viable subtype of SSCs was obtained. Cells separation with discontinuous density gradient centrifugation significantly increased the concentration of c-kit positive cells [(18.65±1.69)% after the centrifugation versus (3.16±0.84)% before the centrifugation, P＜0.01]. Furthermore, the recovery and viability were also high [(65.9±1.24)% and (85.6±1.14)%]. It is concluded that FACS with c-kit as the marker in combination with discontinuous density gradient centrifugation can well enrich type A1-A4spermatogonia from the testes of 9-days-old rats.