痕量小鼠骨髓造血干细胞移植体系的建立

Establishment of Trace Amount Bone Marrow Hematopoietic Stem Cells of Adult Mouse Transplantation System

目的:建立痕量小鼠骨髓造血干细胞(hematopoietic stem cells,HSCs)移植体系。方法:利用ESLAM(CD45^+EPCR^+CD150^+CD48-)组合流式分选成体GFP/CD45.2小鼠骨髓细胞与保护细胞(CD45.2小鼠),共同移植给致死剂量照射的受体小鼠(CD45.2小鼠),分析移植后不同时间点,受体小鼠外周血嵌合以及其重建效率和谱系分化情况,随后处死受体小鼠,获取骨髓、胸腺、脾脏和外周血等组织的细胞,检测各组织嵌合情况。结果:移植后受体小鼠外周血结果显示第4周之后重建效率为100%,GFP^+细胞在各系细胞(髓系、红系、B细胞、T细胞、巨核)中不同时间点的比例不同,且各系重建水平的变化趋势不同;6个月之后各组织(外周血、骨髓、脾脏、胸腺)嵌合情况显示外周血红系和巨核细胞中有较高比例GFP^+细胞,且在髓系和淋系细胞中无GFP^+细胞,而骨髓的髓系细胞含也有较高比例GFP^+细胞,脾脏中的B细胞也有较高比例的GFP^+细胞,胸腺中的T细胞也含有较高比例GFP^+细胞。结论:ESLAM组合能够较高效率的富集具有长期重建能力的HSCs;这群细胞移植后6个月外周血结果提示可能具有红系和巨核分化倾向。

Objective： To establish a transplantation system for a trace amount of adult mouse bone marrow hematopoietic stem cells（ HSCs）. Methods： The HSCs from bone marrow cells of GFP adult mouse were seperated by using marker combination ESLAM（ CD45 ＋ CD201 ＋ CD150 ＋ CD48-）. The goal cell population acquired by flow cytometry sorting were injected into lethally irradiated recipient mouse plus protective cells. And the peripheral blood cells from recipient mice tail veins at different time points were acquired to observe the reconstruction level and lineage differentiation. Then the receptor mouse was killed and the cells from thymus,spleen,marrow,and peripheral blood were acquired to measure tissue chimerism. Results： The results of peripheral blood of recipient mice after transplantation showed that the reconstructive efficiency was 100% after 4 th week. The proportion of GFP ＋ cells at different time points in various lines（ myeloid,erythroid,B,T and megakaryocytes） were different and the change tendency of reconstruction were different. From different tissues（ peripheral blood,bone marrow,spleen,thymus） after6 months,the intramedullary spleen and thymus chimerism can be seen and showed a high proportion of GFP ＋ cells in the peripheral blood erythroid and megakaryocytes and there were no GFP ＋ cells in the myeloid and lymphatic cells,but the myeloid cells in the bone marrow cells of the recipient mice contained a higher proportion of GFP ＋ cells. The B cells in the spleen had a higher proportion of GFP ＋ cells,and the T cells in the thymus contained a higher proportion of GFP ＋cells. Conclusion： The ESLAM combination can be highly efficient one for the enrichment of HSCs with long-term reconstructive ability,and the results of peripheral blood 6 months after the transplantation suggests that it may have the inclination to erythroid and megakaryocyte differentiation.