人骨骼肌肉源性血管外膜干细胞的生物学特性及其对造血干/祖细胞支持作用的研究

Mesenchymal stem/stromal cells (MSCs) as precursor cells of bone marrow stromal cells, in addition to the formation of the bone marrow microenvironment, secrete a variety of blood-related factors, promote hematopoietic recovery and reconstruction. However, Mesenchymal stem/stromal cells are extremely rare within human bone marrow, which limit their experimental and clinical applications. Our previous studies showed that CD146+ perivascular stem cells (PSCs) are possible originator of mesenchymal stem/stromal cells. As the precursor of mesenchymal stem/stromal cells, whether perivascular stem cells have effects on supporting hematopoietic stem/progenitor cells (HSPCs) is rarely reported. Although perivascular stem cells are widely distributed throughout the organism, skeletal muscle tissue is ideal source of PSCs for investigation as it is largely dispensable and readily accessible with minimal morbidity. Based on our previous studies, in current study, we intend to do the separation, purification and identification of human skeletal muscle-derived PSCs. In order to define their role in hematopoiesis, we will test the ability of CD146+PSCs to sustain human HSPCs in coculture. Furthermore, whether it can support the ex vivo maintenance of human umbilical cord blood CD34+ HSPCs is going to be investigated. The results of present study will help us to find a new the rich source of stromal cells to promote hematopoietic stem/progenitor cells.

间充质干细胞（MSCs）作为骨髓基质细胞的前体细胞，分泌多种造血相关因子，促进造血恢复和重建，但在人体骨髓中MSCs的含量极其稀少，使其在研究及临床应用中受到限制。我们前期的研究表明，MSCs起源于毛细血管和微血管壁上的血管外膜干细胞(PSCs)，然而作为MSCs前体细胞的PSCs，是否有促进造血干/祖细胞(HSPCs)扩增和可能的造血调控机制还有待于研究。PSCs广泛分布于机体组织，而骨骼肌肉是人体最大的组织器官取材容易且损伤小。课题拟在前期工作的基础上，1.通过应用多参数流式细胞仪分选、纯化出人骨骼肌源性CD146+PSCs并进鉴定；2.建立体外CD146+PSCs与HSPCs共培养扩增体系，研究PSCs能否长期持续地分泌细胞因子支持造血；3. 研究CD146+ PSCs是否可以支持人脐血CD34+HSPCs在体内长期造血重建，为HSPCs的扩增提供一个崭新而丰富的支持细胞的来源。

背景：间充质干细胞（MSCs）作为骨髓基质细胞的前体细胞，分泌多种造血相关因子，促进造血恢复和重建，但在人体骨髓中MSCs的含量极其稀少，使其在研究及临床应用中受到限制。研究表明，MSCs起源于毛细血管和微血管壁上的血管外膜干细胞(PSCs)，然而作为MSCs前体细胞的PSCs，是否有促进造血干/祖细胞(HSPCs)扩增和可能的造血调控机制还有待于研究。PSCs广泛分布于机体组织，而骨骼肌肉是人体最大的组织器官取材容易且损伤小，可应用多参数流式细胞术分选、纯化出人骨骼肌源性CD146+PSCs并进鉴定，并建立CD146+PSCs与HSPCs体外培养体系，研究CD146+PSCs能否支持造血，进一步研究CD146+ PSCs是否可以支持人脐血CD34+细胞在体内造血重建，为HSPCs的扩增提供一个崭新而丰富的支持细胞的来源。.研究内容：1.利用多参数流式细胞术分离、纯化人骨骼肌源性CD146+PSCs，并进行体外培养及生物学特性的研究；2.人脐血CD34+细胞的提取与纯化；3. 建立以CD146+PSCs为滋养层的脐血CD34+细胞体外培养体系,模拟体内造血微环境,以了解CD146+PSCs能否长期维持脐血CD34+细胞的扩增。4.采用免疫磁珠法从CD146+PSCs与脐血CD34+细胞共培养后的细胞中分选出CD45+细胞并植入经亚致死剂量照射后的NCG小鼠体内，观察造血重建的作用。.重要结果：①利用多参数流式细胞术从人骨骼肌组织中分选表型为CD146+CD56-CD34-CD144-CD45-的CD146+ PSCs，纯度为（91.5±1.85）%；②CD146+ PSCs贴壁生长，细胞形态呈纤维样，且CD146+ PSCs表达MSCs表面抗原CD44、CD73、CD90、CD105；且可分化为成骨细胞、脂肪细胞、成软骨细胞。③将人脐血 CD34+分别与CD146+ PSCs（实验组）、人BM-MSCs(阳性对照组）共培养，并设置UCB CD34+细胞单独培养体系（空白对照组），共培养1周、2周及4周后，以CD146+ hMD-PCs为滋养层培养体系与BM-MSCs为滋养层培养体系的细胞数、集落形成能力及免疫表型（CD45+、CD34+CD33-、CD14+、CD10+/CD19+）均无统计学差异（P>0.05,n=6）；但共培养1周、2周后，CD146+ hMD-