卵黄囊移植人脐血CD34^＋细胞构建人源化血液系统小鼠模型

背景：与较为成功的羊等大型动物宫内移植方法相比，利用小鼠等小型动物宫内移植异种造血干细胞仍面临困境，在外周血内检测到的异种血液细胞重建率仍在1％以下。目的：观察小鼠卵黄囊移植人脐血来源CD34^＋造血干细胞后的异种造血重建效率，并与腹腔移植的效果进行比较。设计、时间及地点：细胞学体内移植实验，于2007—10在北京大学干细胞与再生生物学实验室完成。材料：健康新生儿脐血取自北京海淀区妇幼保健院。ICR小鼠由北京大学医学部实验动物中心提供，孕8．5d鼠8只，孕12．5d鼠9只。藻红蛋白标记的抗人CD45单克隆抗体与流式细胞仪均为BD公司产品。方法：采用Ficoll法梯度离心获得人脐血单个核细胞，经磁珠分选系统纯化获得CD34^＋细胞。将孕8．5d鼠麻醉后，打开腹腔并暴露子宫，显微镜下抽取5～10μL脐血CD34^＋细胞（5×10^4个），缓慢注入胎鼠的卵黄囊中。相同操作条件下将等量脐血CD34^＋细胞注射到孕12．5d胎鼠的腹腔中。快速抽针，将孕鼠子宫归位并缝合腹腔，继续妊娠，等待分娩。待小鼠出生后3个月，经尾静脉取血，在避光状态下加入藻红蛋白标记的抗人CD45单克隆荧光抗体，流式细胞仪检测血细胞表面标记CD45的表达，通过其阳性率判断人血液细胞在小鼠体内的重建效果。主要观察指标：不同移植方式对小鼠造血重建的影响。结果：CD34^＋细胞通过卵黄囊移植方式共获得健康出生小鼠52只，3个月后86．5％（45／52）小鼠CD45呈阳性表达，嵌合率高达4．34％。通过腹腔移植方式共获得健康出生小鼠48只，3个月后81.3％（39／48）小鼠CD45呈阳性表达，明显低于卵黄囊移植方式（t=2．363，P〈0．05）。结论：早期卵黄囊移植可以获得人造血干细胞在小鼠体内的长期重建，效果优于腹腔移植，同时也证明了通过胎鼠卵黄?

Construction of humanized blood mouse models using human cord blood CD34^＋ cells by yolk sac transplantation

BACKGROUND： Compared with utero transplantation in large animals such as goat, the utero heterogeneous hematopoietic stem cell transplantation in small animals such as mouse is facing difficulties. Rebuilding rate of heterogeneous hematopoietic stem cells in peripheral blood was below 1%. OBJECTIVE： To observe rebuilding rate of heterogeneous hemopoiesis following human cord blood hematopoietic stem cell transplantation in mouse yolk sac, and to compare with outcomes of abdominal transplantation. DESIGN, TIME AND SETTING： The cytology in vivo experiment was performed at the Laboratory of Stem Cells and Regenerative Biology, Beijing University in October 2007. MATERIALS： Cord blood from healthy newborns was collected at the Maternal and Child Health Hospital of Beijing Haidian District. ICR mice were obtained from Animal Experimental Center of Medical Department of Peking University, including 8 mice at gestational day 8.5, and 9 mice at gestational day 12.5. Phycoerythrin labeled anti-human CD45 monoclonal antibody and flow cytometer were purchased from BD. METHODS： Human cord blood mononuclear cells were harvested by Ficoll method. CD34^＋ cells were purified and collected using magnetic bead sorting. Mice at gestational day 8.5 were anesthestized prior to abdominal cavity was opened to expose uterus. Under a microscope, 5 10 μL cord blood CD34^＋ cells （5×10^4） were harvested, and slowly injected into the yolk sac of fetus. Under the same operation condition, the same volume of cord blood CD34^＋ cells were injected into the abdominal cavity of mice at gestational day 12.5. Needles were rapidly taken. Pregnant mouse uterus was homing, and then the abdominal cavity was sutured for continued pregnancy, waiting for parturition. At 3 months after birthing, blood was obtained via the caudal vein. In the dark, phycoerythrin labeled anti-human CD45 monoclonal fluorescent antibody was added. Flow cytometry was used to measure CD45 expression. Rebuilding effect of human blood cells in mice in vivo was