D183．49壳聚糖对脐血造血干细胞移植小鼠免疫重建的研究

Immune reconstruction of D183.49 chitosan in the cord blood hematopoietic stem cell-transplanting mice

目的:探讨D183.49壳聚糖对脐血造血干细胞的体外扩增作用及其对脐血造血干细胞移植小鼠的免疫重建并初步探索对脐血造血干细胞移植有调控作用的药物筛选方法。方法:分离脐血中单个核细胞体外扩增培养,测定细胞增殖率及CD34+细胞数。60Coγ射线亚致死量照射小鼠,尾静脉注射扩增培养后的单核细胞,每日腹腔注射D183.49壳聚糖,28天。观察记录生存状况;测定血象及CD3、CD4、CD8、CD19水平;脾脏病理切片观察。结果:①体外研究:D183.49壳聚糖10μg组体外增殖效率最显著,48小时达峰值;流式细胞仪测定CD34+细胞扩增8倍,P<0.01。②体内研究:移植药物小鼠血象恢复明显短于其他实验组;CD3、CD4、CD8、CD19水平基本恢复正常,P<0.05。小鼠脾脏病理切片显示亦恢复正常。结论:D183.49壳聚糖对脐血造血干细胞有明显扩增作用,其能够促进脐血造血干细胞移植小鼠免疫造血重建。

Objective： To investigate immune reconstruction of D183.49 chitosan in the mice transplanted with hematopoietic stem cells of cord bloed.Methods： MNCs in cord blood were separated and expanded in vitro by cultures. CD34^＋ cells were counted and cell proliferation rates were measured. The mice were irradiated with sublethal dosage of 60Co gamma ray. The cultured MNCs were injected to the radiated mice via tail vein, then the mice were daily injected wih 10 mg in 0.2 ml volume of D183.49 chitosan for 28 days. Mortality of the mice and graftrejection were observed, and RBC, WBC, PLT and CD3, CD4, CD8 and CD19 positive cells in blood were measured by both routine examination and FCM. Pathological sections of the mouse spleens were routinely examined. Results：OIn vitro study： 10μg of D183.49 chitosan resulted in most efficient proliferation of CD34^＋ cells,and the proliferative rates reached peak in 48 hrs. CD34 ^＋ cells increased 8 folds by FCM measurement（ P 〈 0.01）.②In vivo study： Bloed routine examination showed that Red blood cells,White blood cells and Platelets of the group of transplanting plus（10 mg/0.2 ml） of chitosan were equal to those of the normal group. The times needed for reconstruction was distingnishedly shorter than in the other experimental groups （ P 〈 0.05）. CD3, CD4, CD8 and CD19 positive cells in blood of the group of transplanting plus chitosan were the same as in normal group（ P 〈 0.05）.Pathological examination of mouse spleens showed that the morphological structure,cell density, and splenic mesenchyme in the group of transplanting plus chitosan were similar to those of the normal mice. Conclusion： D183.49 chitosan can evidently amplify cord blood hematopoietic stem cell and promote immuno-hemopoietic reconstruction in the mice with sublethal dosages gamma irradiation.