【作者】 苏丽萍；

【导师】 缪竞诚； 张澜生；

【作者基本信息】 苏州大学 ， 病原生物学， 2001， 硕士

【摘要】 脐血作为一种新的造血干/祖细胞来源，可以成功地替代骨髓和动员外周血用于临床上骨髓衰竭病人的造血重建。这已经被越来越多的实验研究所证明。这是因为脐血中含有丰富的造血干/祖细胞：CD34~+CD38~-细胞、集落形成细胞（CFC）、长期培养启动细胞（LTC-IC）的含量均高于骨髓和动员外周血。另外脐血移植后移植物抗宿主疾病（GVHD）的发生率和严重程度也比骨髓和动员外周血细胞移植低。但是由于单份脐带血中所含有的造血细胞数量较少，限制了脐血移植的应用，目前脐血移植主要用于儿童和低体重成人患者。为了扩大脐血移植的应用范围，首先必须对脐血造血干/祖细胞进行体外扩增，这也是目前移植领域中倍受关注并急需解决的课题之一。 近年来陆续发现了几种新的造血生长因子如FL、TPO、SCF等，并已证明它们在早期造血调控中发挥着重要作用。因此我们参考国内外新的方法并加以改进，在原有实验结果的基础上，采用细胞因子组合FL+TPO+SCF+IL-6来扩增脐血造血细胞，通过无基质细胞接触的液体悬浮培养、细胞表型动态测定、体外集落形成试验等方法探讨了这一因子组合对脐血CD34~+细胞的体外扩增效应，发现与经典的细胞因子组合（SCF+IL-3+IL-6+GM-CSF+EPO）相比，该扩增方案能更有效扩增CD34~+细胞，在产生了相当数量的较成熟细胞的同时，能在较长时间（至少4周）内仍保持一定比例的造血干细胞成分。 为了进一步了解扩增后细胞的移植能力和造血潜能，我们以严重联合免疫缺陷（SCID）小鼠作为脐血移植的动物模型，将扩增后的脐血造血细胞通过尾静脉注入受亚致死量辐照的SCID小鼠体内，无菌饲养4周后取其骨髓细胞，通过人CD45~+细胞的流式细胞仪检测和Alu 人脐血造血干／祖细胞体外扩增和移植 SC小鼠的实验研究 中文摘要 基因的PCR检测，在小鼠体内均发现了人源细胞，表明了扩增后的 脐血造血细胞可以长期植人SCID小鼠体内，从而改善或重建其造血 功能。更多还原

【Abstract】 Umbilical cord blood (UCB) is an alternative source to bone marrow(BM) and mobilized peripheral blood(MPB) of hernatopoietic stemlprogenitor cells. Many studies have demonstrated that UCB has been used successfully for hematopoietic reconstitution in pediatric patients and low weight adults who are in bone marrow failure. It has been shown that UCB contains abundant hematopoietic stem/progenitor cells, the content of CD34~CD38 and the numbers of colony forming cells and long-term culture-initialing cells are much greater in UCB than in BM and MPB from adults. Another characteristic of UCB transplantation is the lower severity and accident of acute graft-versus-host(GVHD) than BM and MPB. However, due to the relative volumes of placental blood and the limited number of progentor cells collected routinely, cell dose is presently a potential restriction for the widespread use of UCB for transplantation in adults. The study of ex vivo expansion of UCB hematopoietic stem/progenitor cells is an interesting projection in basic and clinical reseach of the transplantation field. Recently, several cytokines such as FL .~ TPO SCF which play essential role in early stage hematopoiesis are identified in succession. Based on the previous experimental results, we study the expansion effects of the new cytokine combination (FL+TPO+SCF 盜L-6) on purified CD34~ cells in strom-free liquid culture system, the results show that the new cytokine combination can more effectively expand CD34~ cells compared with the classical cytokine combination (SCF+IL-6盜L-3 +GM-C SF+EPO), and maintain some proportional hematopoietic stem cells in longer time(no ILL t less than 4 weeks) while generating considerable mature cells. In order to study the engraftment capability and hernatopoietic potential of the expanded cells, we establish the SCID mice model of UCB transplantation. The sublethally irradiated SCID mice are injected with expanded cord blood cells by tail vein. Four weeks after inocuration, BM cells of the sacrificed animals are obtained from both femurs and tibias and assessed for the presence of human cells by FACS and PCR. The results show that the expanded cells can engraft the BM of sublethally irradiated SCID mice and reconstitute hematopoiesis.更多还原