利用唐氏综合征患者诱导多能干细胞（iPSCs）研究其易发白血病的机制

Down syndrome (DS) is the most frequent single cause of human birth defects and intellectual disability (ID). Its etiology has been known for over 50 years, DS is caused by trisomy of chromosome 21 (Ts21). In addition, there are more evidences in clinical show that patients of DS are also have high incidence of leukemia, but its underlying mechanisms have yet to be discovered. Elucidation of these mechanisms has been hindered by the difficulties in isolating and expanding enough hematopoietic stem cells (HSCs) from the patients. To overcome this limitation,we developed Ts21-induced pluripotent stem cells (iPSCs) from mononuclear cells using Episomal vectors. Under certain conditions, these Ts-21 specific iPSCs could be further induced into any cell types, including HSCs. Initial characterization of the iPSCs generated from DS and control patients confirmed these cell lines to be fully reprogrammed and carry the original mutation. Next, we are trying to induce the patient-specific iPSCs differentiate into HSCs under serum free and feeder free condition in vitro. By using flow cytometry, qRT-PCR and CFU assays, we can make well comparisons between HSCs derived from TS-21 iPSCs and control iPSCs, and expolre the pathological feature and mechanisms. We have observed that myeloid differentiation potential of HSCs derived from DS patients is significantly higher than normal patinets. By performing LncRNA-seq analysis, we are trying to identify key gene and key lncRNA regulatory networks in DS with hematopoietic disorder. On the basis of that, we can further search for the potential drug targets that effective against leukemia of DS patients. This approach would provide a powerful cell resource for clinical research and a useful model for the study of the mechanisms of DS and drug screening.

唐氏综合征（DS）源于21号染色体三倍体化，是一种常见的出生缺陷疾病。患者除了表现出中枢神经系统发育异常导致的认知障碍，亦有高发白血病的风险。然而导致其高发白血病的机制尚未被阐明，难点之一在于患者的HSCs难以获取以及在体外扩增。为了探究DS患者高发白血病的机理，我们使用Episomal电转法，将病人的外周血单核细胞重编程为hiPSCs，在无基质无血清条件下诱导其定向分化成HSCs，建立DS患者造血分化的体外模型。之后通过流式分析、qRT-PCR和CFU等手段，多层面地将DS患者来源的HSCs与正常人的相比较，获得病理特征。我们发现患者来源的HSCs在体外向髓系分化的潜能显著高于正常人，同时伴有淋系的过早衰老。在此基础上，我们将通过LncRNA-seq分析进一步探寻与DS白血病相关的关键基因及关键lncRNA，为DS伴白血病的诊断和治疗提供新的靶点，亦将为开展精准医疗药物研发提供有效工具。

DS患者来源的iPSC诱导分化的HSC存在造血分化异常，具体表现为髓系细胞过度增殖和红系的异常分化，多携带GATA1/3突变。通过数据库分析结合临床标本验证，我们筛选出在AML中与GATA1具有高度相关性，且在急性红白血病（AEL）中特异性高表达的基因KEL，其作用尚未见报道。AEL是急性髓细胞白血病(AML)中一种少见亚型，预后较差，尚未发现相关的分子标志物作为诊断标准。环状RNA（circRNAs）在多种疾病发生发展中起着重要作用。数据库显示KEL在AEL病人中显著高表达，其作用尚不清楚。研究中我们发现：KEL和circ-KEL在AEL血液样本和细胞系中均高表达；KEL过表达细胞中circ-KEL亦有升高，红系分化能力整体提升，进一步敲降circ-KEL，红系分化能力得到回复；circ-KEL可吸附miR-671，解除miR-671对KEL mRNA的抑制从而促进红系分化。二者在红系分化中通过circ-KEL/ miR-671/KEL通路发挥协同促进作用。同时，蛋白芯片分析发现KEL过表达的AEL细胞有多条增殖及耐药相关通路被显著活化，经验证PKC/RSK2/CREB通路的激活可能是KEL促进AEL细胞增殖的分子机制。本研究将为探寻AEL的诊断分子标志物和潜在的治疗靶点提供新思路。. 另一方面，围绕非编码RNA在白血病发生发展中的作用进一步开展相关研究，着重研究了circRNA在慢性淋巴细胞白血病（CLL）中的的分子机制和临床意义，主要发现：（1）血浆中circ-RPL15可以作为CLL的致癌驱动因子，初步证明了circ-RPL15在CLL疾病进程和临床诊断中的价值，揭示了circ-RPL15/miR-146b-3p/ RAF1通路调控在CLL细胞增殖中的作用；（2）线粒体circRNA mc-COX2在CLL患者的血浆外泌体中高表达，保护CLL细胞免于凋亡并促进细胞增殖，可能与CLL疾病进展有关。这些发现有望为靶向治疗，以及预测和克服化疗耐药提供理论依据，同时也为白血病个体化治疗提供新思路。