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目的:观察粒细胞集落刺激因子(G-CSF)动员的人外周血中CD34~+造血干细胞(HSC)在免疫缺陷NPG~(TM)(NOD.Cg-Prkdcscid II2rgtm1vst/vst)小鼠模型上造血重建的水平。方法:用免疫磁珠分选G-CSF动员人外周血CD34~+的造血干细胞,经骨髓腔移植到亚致死剂量照射的NPG小鼠。移植后2、4周观察小鼠血象恢复情况;移植后4、6、8、10、12周用流式细胞仪动态监测小鼠外周血人源CD45~+、CD19~+细胞表达;12周后处死各组小鼠,检测骨髓、肝脏、脾脏中细胞表面CD45~+、CD19~+细胞表达;用PCR方法检测小鼠骨髓细胞人Alu基因的有无。结果:免疫磁珠分选人的CD34~+造血干细胞纯度可达96.3%;NPG小鼠经过照射后骨髓腔内有核细胞及巨核细胞数量均明显减少或消失,达到了清髓的效果;移植组小鼠4周外周血各系血细胞恢复到移植照射前水平;所有小鼠全部存活,移植组小鼠在4、6、8、10、12周均检测到人源CD45~+、CD19~+细胞;应用PCR方法检测移植组小鼠人Alu基因均为阳性。结论:经骨髓腔途径移植G-CSF动员的人外周血CD34~+干细胞至NPG小鼠,可以建立人鼠嵌合模型。
OBJECTIVE: To observe the effect of granulocyte colony-stimulating factor (G-CSF) mobilizing human peripheral blood CD34 ~ + hematopoietic stem cells (HSC) on hematopoiesis in a mouse model of immunodeficiency NPG TM (NOD.Cg-Prkdcscid II2rgtm1vst / vst) Reconstruction level. Methods: G-CSF was used to mobilize CD34 + hematopoietic stem cells from human peripheral blood by immunomagnetic beads and then transplanted into sub-lethally irradiated NPG mice via the bone marrow cavity. At 4, 6, 8, 10, and 12 weeks after transplantation, the peripheral blood of mice were dynamically monitored for the expression of CD45 ~ + and CD19 ~ + cells by flow cytometry at 4, 6, The mice in each group were sacrificed and the expression of CD45 ~ + and CD19 ~ + cells in the bone marrow, liver and spleen were detected. The presence or absence of human Alu gene was detected by PCR. Results: The purity of CD34 ~ + hematopoietic stem cells was up to 96.3% by immunomagnetic bead sorting. The number of nucleated cells and megakaryocytes in NPG mice significantly decreased or disappeared after irradiation, and the effect of myeloablation was achieved. The blood cells of peripheral blood of each group were recovered to the pre-irradiation level before transplantation. All the mice survived. The levels of CD45 +, CD19 + Cells; application of PCR method to detect mouse Alu gene transplanted mice were positive. CONCLUSION: Chimeric human-mouse models can be established by transplanting G-CSF mobilized human peripheral blood CD34 + stem cells into NPG mice via the bone marrow cavity.