摘要
目的探讨人脐血CD34+造血干细胞在非肥胖糖尿病/重症联合免疫缺陷(no obese diabetic/severecombined Immunodeficiency,NOD/SCID)小鼠模型上造血重建的作用。方法利用密度梯度离心法,从新鲜脐血中分离出单个核细胞,利用免疫磁珠分选法筛选CD34+造血干细胞,经尾静脉输注入经亚致死剂量照射后的NOD/SCID小鼠体内,移植后3、7、10、14 d分别用断尾法取小鼠外周血,血常规计数外周血动态变化情况;移植后4、6、8、10周,取其外周血,运用PCR法检测外周血中人特异性Alu基因的表达情况。结果免疫磁珠分选得到的CD34+细胞浓度达91.2%,照射后小鼠骨髓腔内有核细胞和巨细胞数量明显减少或消失,达到清髓目的。移植后第3天移植组小鼠外周血各系细胞均明显低于正常组(P<0.01),移植后第7天,移植组小鼠外周血象开始恢复,明显高于阴性对照组[白细胞:(3.90±0.53)×109/L vs(1.30±0.18)×109/L,血红蛋:(139.8±5.0)g/L vs(79.8±11.0)g/L,白血小板:(253.0±17.5)×109/L vs(52.0±6.9)×109/L,(P<0.01)],移植后第10天,移植组小鼠外周血象恢复到辐照前水平,与正常组无差别。移植4周后,PCR方法在小鼠外周血中可检测到人特异Alu基因序列,未移植组小鼠照射后2周内全部死亡。结论经照射后的NOD/SCID小鼠通过人脐血CD34+细胞植入可建立起人鼠嵌合模型。NOD/SCID小鼠经照射后,不破坏骨髓造血微环境及造血基质,可用于异基因细胞移植模型。人脐血CD34+细胞移植入NOD/SCID小鼠,其造血系统能有效重建。
Objective To investigate the hematopoietic reconstitution by human umbilical cord blood CD34＋ cells in no obese diabetic/severe combined immunodeficiency （NOD/SCID） mice. Methods Mono- nuclear cells （MNCs） were isolated from human fresh cord blood by gradient centrifugation, and then CD34 ＋ hematopoietic stem cells were selected by magnetic activated cell sorting. The selected cells were injected into the tail of the NOD/SCID mice after irradiation of sublethal doses. After the sample of peripheral blood were collected by cutting mouse tail at 3, 7, 10, and 14 d after transplantation, dynamic monitoring was performed on the mouse blood . The mice alive were sacrificed and the blood samples were harvested at 4, 6, 8 and 10 weeks after transplantation. The mRNA expression of human-specific gene Alu in the peripheral blood cells were analyzed by PCR. Results The yield proportion of CD34 ＋ cells accounted for 91.2%. Irradiation of sublethal doses obviously reduced the number of karyocytes and giant ceils and even demolished the cells in medullary space. At the third day after transplantation, the peripheral blood cells in each department were significantly lower than those in the normal group （P 〈0.01 ）. From the seventh day after transplantation, peripheral blood in transplantation mice began to recover, significantly higher than negative control group [ white cells： （3.90 ± 0. 53） 109/L vs （ 1.30 ±0. 18） 109/L, hemoglobin ： （ 139.8 ± 5. 0） g/L vs （79.8 ± 11.0） g/L, platelets ： （253.0 ±17.5） 109/L vs （52.0 ± 6.9） 109/L,P 〈 0.01 ]. At 10 d after transplantation, peripheral blood of transplanted mice was back to the level before irradiation, with no significant difference with that of the normal group （P 〉 0. 05 ）. In 4 weeks after cell transplantation, there were more and more human-derived hematopoi-etic cells, human specific Alu gene was detected in the peripheral blood in mice. All mice died who received no transplantion in 2 weeks after irradiation. Conclusion Human-mouse chimeric immune model is built in irradiated NOD/SCID mice transplanted by cord blood CD34＋ cells. Irradiation can not destroy the bone marrow microenvironment and hemopoietic stromal in NOD/SCID mice, and the mice can be used for allogeneic cell transplantation model. The hematopoietic system, humoral immunity is effective reconstructed in NOD/SCID mice after human umbilical cord blood CD34 ＋ cells transplantion.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2011年第11期1136-1140,共5页
Journal of Third Military Medical University
基金
贵州省科技厅(黔科合计2008-1003)
遵义市"15851"人才工程项目(市15851人才办2008-9)
贵州省教育厅培育项目(黔科教2009-113)~~
关键词
人脐血
NOD/SCID小鼠
CD34+干细胞
造血重建
human umbilical cord blood
no obese diabetic/severe combined immunodeficiencymice
CD34 ＋ stem cells
hematopoietic reconstitution
