研究Notch/Jagged信号通路在脂肪源干细胞促进造血干/祖细胞增殖分化中的作用

Adipose derived stem cells(ADSCs) has similar biological characteristics which bone marrow mesenchymal stem cells(BMSCs) has, and could also promote the proliferation and differentiation of hematopoietic stem and progenitor cells(HSPCs). But its mechanism has not been illuminated. Our previous study found that when mouse Lin-Sca-1+C-Kit+ cell co-cultured with ADSCs in vitro,the Notch/Jagged indicated an increased expression , however we don't know the promotion of HPSCs proliferation and differentiation by ADSCs whether through Notch signaling pathway or not? The aim of this project is that: in vitro,to detect the expression of target gene Hes-1 of Notch signaling pathway in mouse ADSCs with HSPCs co-culture system , and then to use gamma secretase inhibitors blocking the pathway，at the same time to use Notch knockout mice ,observing whether it block the promotion of HSPCs proliferation and differentiation ;in vivo, to observe the fluorescent expression in mouse allograft transplantation mode who injected with ADSCs and HSPCs which has been marked fluorescent,then detect the content of its Jagged. Finally,we hope to judge whether or not ADSCs promote the proliferation and differentiation of hematopoietic stem and progenitor cell through Notch signaling pathway? Providing theoretical basis for clinical treatment of hematologic malignancies by ADSCs.

脂肪源干细胞(ADSCs)具有与骨髓间充质干细胞(BMSCs)相近的生物学特性，也能促进造血干/祖细胞（HSPCs）的增殖与分化，但其作用机制尚不明确。我们前期的研究发现小鼠的ADSCs与Lin-Sca-1+C-Kit+细胞共培养后，Notch/Jagged表达增高，但其是否通过此信号通路促进HPSCs增殖与分化机制尚不清楚，故本项目拟在体外利用小鼠ADSCs与HSPCs共培养体系检测Notch信号通路靶基因Hes-1是否表达？并用γ分泌酶抑制剂（GSI）阻断此通路，同时采用Notch基因敲除小鼠，看是否影响ADSCs促进HSPCs的增殖与分化；再利用小鼠异体移植模型注入ADSCs与荧光标记的HSPCs并进行定位，然后检测其Jagged在体内的表达情况，最终判断ADSCs是否通过Notch信号通路影响造血干/祖细胞增殖与分化，从而为ADSCs临床治疗恶性血液病提供理论基础。

研究内容：本研究成功地分离培养了C57/BL小鼠的脂肪源间充质干细胞，及LSK细胞。通过脂肪源干细胞与小鼠LSK细胞建立共培养体系，研究脂肪干细胞对LSK细胞体外的支持，通过对LSK细胞进行细胞计数及甲基纤维素培养，我们证实了ADSCs能够在体外短期及长期支持LSK细胞增殖，同时保留形成造血集落的能力，同时通过RT-PCR的技术，明确了Notch信号通路在这一过程中的作用。同时通过体内的归巢实验，我们同时发现ADSCs能够促进LSK的植入，同时能够迁移到较易发生损伤的器官，有助于造血干细胞的归巢和受损组织的修复。而在体内移植实验中，我们发现ADSCs能够促进造血干细胞的植入，同时具有减轻炎症反应的作用。重要研究成果：本研究发现ADSCs能够通过Notch信号通路在体内及体外支持造血干细胞的增殖，同时具有能够修复放射损伤组织，减轻炎症反应等作用。为脂肪源间充质干细胞在造血干细胞移植植入不良、移植物抗宿主疾病，放化疗后组织损伤的修复等后期临床应用，提供了翔实可靠的依据。