与AEC共培养对AMSC在NOD/SCID小鼠体内归巢及造血支持作用的影响

Although allogeneic hematopoietic stem cell (HSC) transplantation is an effective therapeutic modality for a variety of malignant and nonmalignant disorders, it remains complicated by treatment-related mortality because of infection and bleeding, regimen-related toxicity, engraftment failure, and acute and chronic graft-versus-host disease. Cotransplantation of mesenchymal stem cells (MSC) and HSC after myeloablative therapy could facilitate engraftment and accelerate hematopoietic recovery. And further opportunities have been identified to improve the clinical benefits after MSC transplantation via their homing/migratory capacity. Previously we have demonstrated the importance of the SDF-1/CXCR4 axis in the regulation of motility of Flk1+ MSC derived from human fetal BM. Increasing CXCR4 functional expression might be a potential strategy to improve engraftment of Flk1+ MSC in BM and accelerate hematopoiesis recovery.The human amniotic membrane is a highly abundant and readily available tissue. Amniotic MSC (AMSC) have been proposed as good candidates to be used in cell therapy or regenerative medicine to treat damaged or diseased tissues. And we have shown for the first time that the capacity of hematopoitic support of AMSC was similar to that of BMSC. Interestingly AMSC could even express the hematopoietic cytokines that were not expressed in BMSC, such as IL-3. And the co-culture with amniotic epithelial cells (AEC) resulted in up-regulation of cell surface CXCR4 of AMSC. In this study we will investigate the expression of CXCR4 in AMSC co-cultured with amniotic epithelial cells and explored their homing ability after intravenous infusion into sublethally irradiated NOD/SCID mice. The peripheral blood will be analyzed and human cells in recipients' BM will be quantified six months after transplantation. This study is designed to evaluate the effects of co-culture of AMSC with AEC on their homing efficiency and hematopoietic supportive capacity in vivo. And the role of SDF-1/CXCR4 axis will also be analyzed. Some valuable informations may be provided to good advantage for modulating AMSC in the clinical setting, particularly for enhancing the effectiveness of transplantation.

加快造血恢复对提高造血干细胞移植(HSCT)成功率、减少感染等移植并发症、改善患者生存质量具有重要意义。前期，我们发现增加骨髓间充质干细胞（BMSC）表面CXCR4的表达，可以有效促进MSC向骨髓归巢和加快受体造血恢复。但BMSC来源、数量、扩增和分化能力有限，使研究和应用受到限制。羊膜MSC(AMSC)来源丰富、增殖迅速、分离纯化方法简单，前期我们首次证实了AMSC体外具有与BMSC相似的支持造血作用，甚至表达比BMSC更丰富的造血细胞因子，并发现通过与羊膜上皮细胞（AEC）共培养可以有效增加AMSC表面CXCR4的表达，并保持良好细胞活性。本研究旨在观察AEC共培养对AMSC归巢能力的影响，探索应用AMSC加速造血恢复的可能性，阐明SDF-1/CXCR4轴在AMSC归巢、植入和促进造血过程中的作用。为AMSC临床应用奠定基础，并希望为临床提高MSC移植疗效和HSCT成功率提供新策略。

间充质干细胞（MSC）移植后能促进受体造血恢复，其向骨髓的归巢是关键环节。羊膜MSC(AMSC)来源丰富、增殖迅速、分离纯化方法简单，可能成为一种较骨髓更加理想的MSC新来源。我们的研究发现，羊膜上皮细胞（AEC）条件培养基处理是提高AMSC表面CXCR4表达的一种简便且安全有效的方法，而SDF-1/CXCR4在调节AMSC移植入NOD/SCID小鼠体内后的归巢和植入过程中发挥重要作用。首先，利用Millicell悬挂式培养皿构建AEC和AMSC的间接共培养体系，并制备AEC条件培养基，分别观察其对AMSC生物学特性的影响，结果证实与AEC共培养及AEC条件培养基处理均能在保持良好AMSC细胞活性的基础上，上调细胞表面CXCR4的表达，并增强AMSC在其体外沿SDF-1浓度梯度迁移的能力，但AEC条件培养基处理的方法较共培养更加简便、易行、经济。其次，AEC条件培养基处理AMSC 48h后采用PKH26染色标记，通过尾静脉输入经亚致死量照射的NOD/SCID小鼠体内，经流式分析归巢至受体小鼠骨髓的AMSC数量，结果证实AEC条件培养基处理后的AMSC短期归巢能力增加，且这一归巢过程依赖于CXCR4表达。进一步，研究AMSC经尾静脉移植后促进放射损伤的NOD/SCID小鼠造血恢复的情况，结果显示，AMSC移植能够明显促进受体的造血恢复，而AEC条件培养基处理的AMSC移植后受体造血恢复速度更快。长期植入实验证实，只有经AEC条件培养基处理的AMSC才能在受体骨髓中长期植入，植入的干细胞可分化成为纤维母细胞和内皮细胞等骨髓基质细胞，且CXCR4中和抗体孵育过的AMSC其长期植入能力下降。这提示AMSC移植后需要归巢到骨髓才能长期植入并进一步分化。综上，本研究证实了SDF-1/CXCR4在调节AMSC移植后体内的迁移中发挥重要作用，增加CXCR4的表达能够促进AMSC向骨髓归巢和长期植入，并加速受体的造血恢复。这为更好地将AMSC应用于临床提供了可能的理论依据，为有效地提高移植及多种损伤性疾病疗效开辟了新途径。