造血干细胞和白血病干细胞对相同CD44抗体不同反应性的相关机制研究

CD44 is a ubiquitously expressed transmembrane glycoprotein which mediates adhesive cell-cell and cell-extracellular matrix interactions through binding to its main ligand - hyaluronan. Although there are many molecular studies on CD44 available, it is rarely reported that CD44 binding with its specific antibody can directly alter the fate of leukemic stem cell ( LSC ). LSC is the only cell capable of initiating and maintaining the leukemic clonal hierarchy. Moreover, most LSC, like its normal counterpart - resides in G0, escaping from the traditional chemotherapy targeting cycling cell, and it is also responsible for the relapse of acute myeloid leukemia (AML) patient. Our previous results confirmed that AML LSCs require interaction with a supportive microenvironments (niche) to maintain their stem cell properties and CD44 eradicates LSC in nonobese diabetic/severe combined immunodeficient mice through interrupting their interaction with niche. More interestingly, the percentage or total number of normal hematopoietic stem cells (HSC) in NOD / SCID mice is inhibited to a much lesser extent than that observed with LSC. Our preliminary expriments showed that the adhesion rate of CD44 antibody -treated AML CD34+CD38- on non-specialized stromal cells(SC) and niche-specialized osteoblast(OB) is significantly higher than that of IgG1-treated cells. CD44 ligatin maybe selectively induce the AML differentiation by promoting more AML CD34+CD38- cells adhere to non-specialized stromal cells, resulting in distant from osteoblast that is required for self-renewal. It is reasonable to make a hypothesis that there is a dynamic balance between SC and OB in bone marrow niche, which meantain the stem cell suvival and self-renewal in vivo. Ligating CD44 by specific antibody selectively inhibits the AML stem cell homing, and alters their interaction with different niche components. In this study, co-culture system of three kinds of cells (HSC/LSC and BM stromal cells and osteoblasts ) will be used to explore the mechnisms of different reactivity of HSC and LSC to the same CD44 antibody. Then beta-glycerophosphoric acid and dexamethasone will be also uesd to mannipulate the SC and OB balance by inducing OB and adipose cell differentiation. Recent work has shown that interaction between CXCR4 on leukemic cells and SDF-1 at the niche is necessary for proper homing and proliferation in vivo of leukemic cells. The relationship of CD44 and CXCR4 / SDF1 axis will be also investigated using a series of in vivo and in vitro experiments. If the expected results can be obtained by fund support, new therapies eliminating AML LSCs may be devoloped by targeting their specific niche, while the normal HSCs won't be harmed.

CD44是一种广泛表达的跨膜糖蛋白，目前有关该分子的研究很多,但它与其特异性抗体结合能否直接改变白血病干细胞（LSC）的命运则鲜有报道。前期结果证实：LSC需要与骨髓微环境（干细胞龛）相互作用才能保持其干细胞特性，CD44抗体通过干扰LSC与龛之间的作用而杀灭了在非肥胖型重度联合免疫缺陷(NOD/SCID)小鼠模型体内植活的LSC，更为重要的是，正常造血干细胞（HSC）在NOD/SCID小鼠体内的植入则可逃逸CD44的作用而不受影响。本课题将进一步探讨HSC和LSC对相同CD44抗体具有不同反应性的机制，利用三种细胞的共培养体系（HSC/LSC+BM基质细胞+成骨细胞）证实CD44抗体对LSC的靶向性清除作用，并在体内外探讨CD44与CXCR4/SDF1信息轴的关系。如能获得资助，取得预期结果，可为寻找抑制白血病复发,靶向性清除LSC同时维持HSC自我更新功能的药物提供理论和实验依据。

CD44是骨髓基质中广泛存在的一种膜蛋白，它在多种生理和病理过程中起重要作用,如造血、免疫反应(淋巴细胞活化和归巢)、发育、创伤愈合、炎症和肿瘤等，但它对白血病干细胞（LSC）的作用则鲜见报道。本课题组研究结果证实CD44抗体通过干扰LSC与骨髓微环境（干细胞龛）之间的作用而杀灭了在非肥胖型重度联合免疫缺陷(NOD/SCID)小鼠模型体内植活的LSC，而正常造血干细胞（HSC）在NOD/SCID小鼠体内的植入则可逃逸CD44的作用而不受影响。该课题应用两种细胞及三种细胞的体外共培养体系进一步研究显示：HSC和LSC与骨髓干细胞龛不同细胞成分间的粘附作用确实存在一定差异，LSC与成骨细胞（OB）间的粘附明显低于基质细胞（SC）,而HSC与两种细胞间的粘附则相当，但均低于LSC与OB/SC间的作用。CD44抗体可明显降低LSC和OB间的粘附率，但并不影响HSC和OB间的作用，HSC和LSC与SC间的粘附也未发生明显变化。同时结果还证实CD44不仅作用于LSC,还可明显抑制OB的增殖和迁移，这提示HSC/LSC与BM 龛中的基质细胞和成骨细胞间确实存在一个动态平衡，这个平衡维系着 HSC/LSC 的存活和自我更新功能，CD44能够同时作用于LSC和微环境中的细胞成分而特异性干扰LSC在微环境中的平衡，减少LSC贴近OB细胞的数量，从而降低LSC的自我更新，靶向性杀伤LSC。正常HSC基本不受CD44抗体的影响。CXCR4/SDF1信息轴在细胞的粘附迁移中起重要作用，本研究结果显示CD44抗体的短期孵育并未改变LSC/HSC表面CXCR4的表达，提示CD44的作用可能并不是通过该信息轴起作用。总之，本研究结果表明HSC和LSC由于在微环境中与不同细胞组分间作用不同，导致对相同CD44抗体可产生不同的反应，CD44抗体能够特异性干扰LSC与OB间的作用，而这种作用是通过对LSC和OB两方面的作用来完成的，体内实验将提供进一步的证据。