人脐带间充质干细胞对脐血干细胞体外扩增的影响及机制研究

Umbilical cord blood transplantation (UCBT) has become an established therapy for patient without matched related or unrelated donors. And the success of transplantation is obviously reduced owing to a limited number of stem cells in a typical UCB unit and delayed engraftment. Mesenchymal stem cells (MSC) are important cells component of bone marrow microenvironment. The recent studies have showed that MSC supported homing, self-renewal and differentiation of HSC in vivo and in vitro. Our previous studies have showed that MSC possessed hematosis-support，and Notch was involved in Endothelium/HSC interactions and in support of umbilical cord blood hematosis.However,if the same mechanism is involved in MSC/HSC is unclear. Therefore, the little cell numbers of HSCs will be resolved using MSC as feeder cells to expansion of cord blood , and the mechanism will be clarified by Notch. In this work, we design three different culture conditions to activate MSC and find best MSC/HSC coculture system; we will transplant the expansion HSC to hematopoietic failure NOD/SCID mouse to test the Hematopoietic recovery. At last we will transfect or interfere Notch ligand to MSC and compare the hematosis-support to interpret the role of Notch signaling pathway in MSC/HSC interaction. This work will offer theoretical basis for the clinical application of cord blood stem cells.

脐血干细胞HSC是骨髓配型不成功患者理想的替代性细胞，但细胞数量有限及造血恢复延迟降低了其临床移植的成功率。间充质干细胞MSC为骨髓微环境中细胞成分的前体细胞，调节造血干细胞的更新和分化。我们先前的研究表明脐带MSC有很强的体外造血支持作用，同时我们发现内皮细胞通过Notch促进脐血体外扩增，但MSC对HSC的调节是否同样通过Notch通路并不清楚。故利用MSC体外扩增脐血将解决细胞数量少的限制，并且以Notch为研究点有望阐明MSC对HSC的调节机制。本课题拟在明确脐带间充质干细胞对脐血干细胞体外扩增的基础上，进一步活化MSC而优化培养体系，建立高效稳定的扩增体系；并将扩增后脐血细胞移植到造血衰竭的NOD/SCID小鼠体内，体内验证扩增后细胞的造血重建能力；同时将MSC转染或干扰Notch配体，对比造血支持的差异，阐明Notch信号在其中的机制作用，为脐血干细胞的临床应用奠定理论基础。

脐血干细胞HSC是骨髓配型不成功患者理想的替代性细胞，但细胞数量有限及移植后造血恢复延迟降低了其临床移植的成功率。体外扩增HSC是解决这一难题的主要方法，但是扩增体系难以获得长期造血重建的HSCs。因此，寻找高效稳定的脐血扩增方法，是目前脐血干细胞移植的难点和重点。本项目中我们将IL-1beta活化后间充质干细胞（MSCs）为滋养细胞体外扩增脐血干细胞，我们发现：（1）IL-1beta 10ng/ml可以促进间充质干细胞的生物学功能；（2）与未活化MSCs相比，IL-1beta活化后MSC的培养上清及MSC均可以促进脐血造血干细胞的增殖和髓系分化；（3）采用PCR及ELISA检测发现，IL-1beta活化后MSC分泌更多的造血支持因子及趋化因子（G-CSF、GM-CSF、IL-6及IL-8）；（4）二代测序分析IL-1beta活化前后MSC的mRNA表达差异，并对差异基因进行pathway预测发现其主要与NF-kB信号通路相关，WB进一步进行验证，并采用NF-kB信号通路抑制剂PDTC及IKK-NED peptide进一步验证NF-kB信号通路在IL-1beta-MSCs在造血中的作用。总之我们的研究结果表明：采用IL-1beta预处理MSCs能够明显促进脐血造血干细胞的体外扩增的效率并保持造血干细胞的干性。