Basic Research of hematopoietic stem cell transplantation utilizing CXCR4 gene transfer

目的：研究表明基质衍生因子-1(SDF-1)及其受体CXCR4在造血干细胞移植后的归巢和植入中起重要作用。最近，CXCR4的C末端截短被确定为Whim综合征的致病突变，Whim综合征是一种以尖锐湿疣、低丙种球蛋白血症、感染和骨髓异常为特征的免疫缺陷疾病。我们利用逆转录病毒载体系统检测了截短型CXCR4(Whim突变体)的强制表达是否能增强小鼠HSC对SDF-1的反应性。方法：我们利用pGCsam骨架构建了表达野生型CXCR4或Whim突变体的逆转录病毒载体，然后产生带有水泡性口炎病毒糖蛋白(VSV-G)的高浓度逆转录病毒颗粒。我们还转导了富含HSC的骨髓细胞，在体外和体内实验系统中检测了表达Whim突变体的小鼠HSC对SDF-1的反应性。结果：K562和小鼠c-Kit阳性/谱系标记阴性的BM细胞都成功地被转导到了所有的逆行方向：建立可靠的体内归巢实验为进一步的实验奠定了基础。

Objective : Studies have suggested that the stromal derived factor-1(SDF-1) and its receptor CXCR4 play an important role in HSC (hematopoietic stem cell) homing and engraftment upon transplantation. Recently, C-terminus truncation of CXCR4 has been identified as causative mutations in WHIM syndrome, an immune deficiency disorder characterized by Warts, Hypogammaglobulinemia, Infections and Myelokathexis. We have tested if forced expression of truncated CXCR4 (WHIM mutants) could confer enhanced responsiveness to SDF-1 on murine HSC using a retroviral vector system.Methods : We constructed retroviral vectors that express wild type murine CXCR4 or WHIM mutants using the pGCsam-backbone, then generated highly concentrated retroviral particles pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G).Erythroleukemia K562 cells were transduced with the above retroviral vectors to test functionality of the mutant CXCR4 molecules. We also transduced HSC-rich marrow populations to test responsiveness to SDF-1 of murine HSCs expressing WHIM mutants in both in vitro and in vivo experimental systems.Results : Both K562 and murine c-Kit positive/lineage-marker negative BM cells were successfully transduced with all the retrovi Future directions : Establishment of the reliable in vivo homing assay must be achieved for future experiments.