胎儿骨髓源Flk1+间充质干细胞的归巢机制

背景：间充质干细胞移植后是否成功，依赖于经静脉输注后能否定居于靶器官并长期存活，这个过程被称为归巢。然而，调节和控制间充质干细胞归巢的分子机制目前尚不十分清楚。 目的：探讨胎儿骨髓源Flk1+间充质干细胞向骨髓归巢的机制，观察基质细胞衍生生长因子1及其受体CXCR4对干细胞归巢效率的影响，摸索提高其归巢和长期植入效率的方法。 设计、时间及地点：细胞学体内实验，于2005-09/2006-07在中国医学科学院、中国协和医科大学组织工程中心完成。 材料：Flk1+骨髓间充质干细胞来源于流产胎儿，由天津医科大学第二附属医院妇产科提供。6~8周龄NOD/SCID小鼠购自中国医学科学院实验动物研究所。 方法：采用流式细胞仪、实时定量PCR等方法检测特定细胞因子刺激前后Flk1+间充质干细胞CXCR4表达和体外迁移能力的变化。NOD/SCID小鼠预先经全身亚致死量照射后，从尾静脉输入经或未经细胞因子刺激的Flk1+间充质干细胞，对照组注射等体积生理盐水。移植后24 h，应用流式细胞仪分析骨髓中供体细胞的含量，或在移植后定期从小鼠的尾静脉取血，分析外周血中白细胞、红细胞及血小板的数量变化。移植后6个月，实时定量PCR法检测小鼠骨髓中的人特异性DNA含量，了解人源细胞的植入情况。 结果：Flk1+间充质干细胞的胞浆中有CXCR4表达，在适当细胞因子刺激下，能够在短时间内被诱导至细胞表面表达。通过24 h短时间细胞因子刺激，不仅能够上调细胞表面及内部的CXCR4，而且可以增加细胞在体外沿基质细胞衍生生长因子1浓度梯度迁移的活性，促进细胞植入经亚致死量照射的NOD/SCID小鼠体内后向骨髓的归巢及长期存活，同时也加快了受体小鼠的造血恢复；用CXCR4中和抗体处理的Flk1+间充质干细胞，则明显减少其移植后在受体小鼠中的归巢和植入。 结论：基质细胞衍生生长因子1及其受体CXCR4在Flk1+间充质干细胞迁移和归巢中发挥着重要作用，增加细胞表面CXCR4的表达，有助于促进Flk1+间充质干细胞向骨髓归巢和加快受体造血恢复。

Homing mechanism of fetal bone marrow-derived Flk1+ mesenchymal stem cells

BACKGROUND: Whether the transplantation of mesenchymal stem cells (MSCs) is successful or not depends on homing. However, the molecular mechanism of regulating and controlling MSC homing is still unclear. OBJECTIVE: To explore the homing mechanism of fetal bone marrow Flk1+ MSCs into bone marrow, to observe effects of stromal cells-derived growth factor-1 and its receptor CXCR4 on homing efficiency of stem cells, and to investigate the method of elevating the homing and long-term implantation efficiency. DESIGN, TIME AND SETTING: The cytology in vivo study was performed at the Center of Tissue Engineering, Peking Union Medical College, Chinese Academy of Medical Sciences from September 2005 to July 2006. MATERIALS: Flk1+ MSCs were isolated from human fetal bone marrow obtained from aborted fetus, which was provided by the Department of Gynaecology and Obstetrics, Second Affiliated Hospital, Tianjin Medical University. NOD/SCID mice aged 6 to 8 weeks old were purchased from the Animal Experimental Institute, Chinese Academy of Medical Sciences. METHODS: The expression of CXCR4 in Flk1+ MSCs stimulated with a cytokine cocktail and the migration capacity to stromal cell-derived factor-1 in vitro were investigated. NOD/SCID mice were sublethally irradiated prior to transplantation with Flk1+ MSCs stimulated with cytokines or not. Control mice received the same volume of saline. At 24 hours after transplantation, the presence of donor cells were analyzed by flow cytometry. Blood was obtained from the mouse caudal vein after transplantation to analyze the changes in number of leukocytes, erythrocytes and thrombocyte in peripheral blood. Human specific DNA content was measured in mouse bone marrow using real-time PCR at 6 months after transplantation to understand the implantation of human-derived cells. RESULTS: Flk1+ MSCs harbored intracellular CXCR4 which could be rapidly induced to cell surface within a few hours. Short-term (24 hours) stimulation with the cocktail cytokines resulted in up-regulation of both cell surface and intracellular CXCR4, increasing in vitro migration capacity to stromal cell-derived factor-1 and homing to the bone marrow of irradiated NOD/SCID mice. Moreover, transplantation of cytokine-treated Flk1+ MSCs resulted in faster hematological recovery and higher levels of donor chimerism in bone marrow than non-treated cells. Neutralization of CXCR4 significantly reduced homing and engraftment of Flk1+ MSCs in murine bone marrow. CONCLUSION: Stromal cell-derived factor-1/CXCR4 axis plays an important role in the regulation of motility of Flk1+ MSCs. Increasing CXCR4 expression might be a potential strategy to improve homing of Flk1+MSCs in bone marrow and accelerate hematopoiesis recovery.