NOD/SCID小鼠脐血单个核细胞骨髓腔内移植的实验研究

目的观察骨髓腔内移植(iBM)人脐血单个核细胞(MNC)对小鼠造血重建和免疫功能恢复的作用。方法NOD/SCID小鼠经137Cs全身照射后,在4h内无菌条件下局部麻醉后从尾静脉或骨髓腔内输注分离脐血MNC。受体小鼠随机分为5组:①对照组:骨髓腔内输注培养液;②阳性对照组(iTV):尾静脉输注脐血MNC3×107/只;③实验Ⅰ组(iBM1):骨髓腔内输注脐血MNC3×106/只;④实验Ⅱ组(iBM2):骨髓腔内输注脐血MNC1×107/只;⑤实验Ⅲ组(iBM3):骨髓腔内输注脐血MNC3×107/只。对照组4只小鼠,实验Ⅱ组7只小鼠,其余每组5只。24h后观察iBM22只小鼠未移植侧胫骨骨髓腔脐血细胞的迁移分布,动态观察移植后小鼠的存活和造血重建情况,7～8周后处死各组小鼠,检测骨髓细胞表面CD分子表达、碳青花(DilCM)染料示踪研究和脐血βactin的DNA标记。结果①照射后骨髓腔内输注脐血MNC,24h后在未输注脐血MNC的一侧胫骨骨髓细胞膜有DilCM标记;②7～8周后小鼠存活14只,其中对照组存活1只,iTV组、iBM1组各存活2只,iBM2组存活4只,iBM3组存活5只;③外周血常规检查结果显示iBM组白细胞的恢复速度比iTV组和对照组快而且稳定;④移植后存活小鼠骨髓细胞表面CD45标记、DilCM染料示踪研究和βactin均显示人源的标记。结论经iBM途径移植脐血MNC至NOD/SCID小鼠骨髓可以重

Intra-bone marrow cavity transplantation of human umbilical cord blood mononuclear cells into NOD/SCID mice

OBJECTIVE: To evaluate the hematopoietic reconstitution of implanted NOD/SCID mice, after intra-bone marrow cavity injection (iBM) of human umbilical cord blood (CB) mononuclear cells (MNCs). METHODS: 24 female NOD/SCID mice were divided into different MNCs dosage iBM groups (3 x 10(6), 1 x 10(7), 3 x 10(7) cells), tail vein intravenous injection (iTV) group (3 x 10(7) cells) and control group (iBM of medium only). CB MNCs sorted by Ficoll-Hypaque were transplanted into left tibia bone marrow cavity of 6- to 8-week-old NOD/SCID mice, which were anesthetized and sublethally irradiated (270 cGy (137)Cs-gamma irradiation). The distribution of injected CB MNCs in noninjected right tibia of the same implanted mice was observed 24 hours after iBM. The establishment of hematopoiesis and the survival of mice were observed. BM cell surface CD marker expressions, dye Dil-CM tracing and human beta-actin from implanted mice were assessed 8 weeks after iBM or iTV. RESULTS: Dil-CM marker could be detected on BM cells from noninjected right tibia 24 hours after iBM. Fourteen engrafted mice survived at the end of our study. Among them two, four and five were of iBM-1, iBM-2 and iBM-3 groups respectively, and one of control group and two of iTV group. White blood cell reconstitution was better in iBM mice than in iTV and control mice. There were human markers including CD45, Dil-CM and beta-actin DNA in the marrow cells from the human CB MNC engrafted mice. CONCLUSION: The preliminary results showed that hematopoiesis reconstitution by iBM was significantly better than iTV.