靶向p18的小分子化合物对人造血干细胞扩增作用研究

Stem cells are capable of self-renewal and multi-lineage differentiation, thereby offering a major cellular source for regenerative and transplant medicine. However, the full therapeutic potential of human hematopoietic stem cell (HSC) has not yet been achieved. It is necessary to determine which combination of growth factors is optimal for net HSC expansion in vitro and what methods can effectively enhance the intrinsic self-renewing properties of HSC in vivo. The ultimate success of HSC expansion in vitro and efficient repopulation of the hematopoietic/ immune system in vivo will require potent, yet specific, biological or chemical agents. These agents can provide greater insights into the downstream signaling pathways of HSC self-renewal in response to microenvironmental cues. We have focused on the role of CKIs in stem cell regulation and found that targeting specific CKIs may yield different outcomes. p18 represents a potent and specific target for enhancing the self-renewal property of HSC. Single cell analysis indicated that deleting p18 gene favored self-renewing division of HSC in vitro. Based on the structure of p18 protein and in-silico screening, we further identified novel small molecule inhibitors that can specifically block the activity of p18 protein .Our selected lead compounds were able to expand functional HSCs in a short-term culture. Thus, these putative small molecule inhibitors for p18 protein are valuable for further dissecting the signaling pathways of stem cell self-renewal and may help develop more effective chemical agents for therapeutic expansion of HSC. In this project, the regulation of p18 on human HSCs will be explored. We will design and screen new compounds targeting p18 for human HSCs expansions. Development of more effective p18 inhibitors or its combinations with other HSC expansion agents may offer effective agents for HSC expansion in the future.

造血干细胞（HSCs）具有广泛的应用前景，但在外周血、骨髓、脐带血中，造血干细胞的比例较低，限制了其临床应用。在前期工作中，我们以细胞周期抑制蛋白p18为靶点，利用计算机虚拟筛选的方法设计合成了一系列小分子化合物，这类化合物可通过特异性抑制p18与CyclinD及CDK6的相互作用，在体外有效扩增小鼠HSCs，有可能成为激活HSCs自我更新和刺激HSCs扩增的候选药物。在本项目中，我们拟利用已建立的单细胞技术平台及HSCs相关的药物筛选及安全性评估的方法，深入研究p18对人HSCs调控的作用机制，筛选对人HSCs具有扩增作用的新的化合物，并与SR1，UM171等已知化合物联用，为提出体外安全有效扩增人HSCs的方案提供理论基础及实验依据。

脐带血移植（UCBT）的应用受到单个UCB单位中造血干细胞和祖细胞数量有限的限制。小分子物质不仅在数量上而且在质量上能促进造血干细胞体外自我更新能力的增强，有利于造血干细胞移植和基因治疗的临床应用。干细胞的细胞周期状态决定了细胞的命运倾向。最近的证据表明，细胞周期蛋白依赖性激酶抑制剂p18INK4C（p18）是造血干细胞自我更新和长期造血重建的关键调节因子。在这里我们报告p18基因敲除可以增强人长期HSCs体外自我更新。然后，我们确定了一种优化的p18 005A小分子抑制剂，以诱导HSC的体外扩增，该HSC能够在免疫受损小鼠中重建人类造血至少4个月，005A处理的细胞在二次移植中仍能存活。机制研究表明，005A可能延缓细胞分裂，激活Notch信号通路和转录因子HoxB4的表达，从而促进长期移植HSC和祖细胞库的自我更新。综上所述，这些观察支持p18在人类HSC维持中的作用，以及p18抑制剂005A在促进HSC治疗的临床应用中的作用。

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