白血病细胞来源微粒在allo-HSCT后供者细胞白血病发生中的作用及机制研究

Allogeneic hematopoietic stem cell transplantation (HSCT) remains the main treatment modality for curing hematologic malignancies. Post-transplantation relapse of leukemia, the most common cause of treatment failure, is frequently observed. Most relapses refer to primary disease progression, and in some cases, leukemia is derived from the donor cells; thus, it is termed donor cell leukemia (DCL). Although it is a rare complication, insight into the mechanism of DCL will provide more information on the prognosis allo-HSCT, and more importantly, yield further clues into leukemogenesis of normal cells, as leukemia in the general population is usually sporadic and unpredictable. For the occurrence of DCL, most researchers have expanded on this idea recently, speculating that some non-viral leukemogenic materials, might be released from residual leukemia cells and fuse with incoming donor cells, resulting in transformation. Microparticles (MPs) are released by eukaryotic cells, especially tumor cells, by outer cell membrane budding. The biological functions of MPs are due to their complexity of bioactive cargo, including proteins, RNA, microRNA (miRNA), and DNA derived from parent cells. We demonstrated that leukemia MPs could initiate malignant transformation of normal hematopoietic transplants. Consequently, the aim of this study was to investigate whether MPs participate in the occurrence of DCL via intercellular transferring of onco-materials. Further work (in vitro, mice models and clinical analysis) will be performed to prove that MPs participate in the occurrence of DCL. Besides,we will figure out the key recipient cell of leukemogenesis and the mechanisms that lead to the leukemia transformation; as well as compounds in MPs that could alter the transformation. Findings of this subject will sheed much-needed light on an unconventional and poorly understood mechanism of intercellular communication and the manner in which it may have significant consequences in the tumorigenesis of DCL and other secondary tumors. Aberrant transformation of normal cells that have been exposed to MPs shed by cancer cells also should be considered, which reveals new insights into the cellular basis of DCL and the potential to translate this knowledge into innovative approaches for diagnostics and personalized therapy. It also presented a unique opportunity to prospectively study the changes undergone by normal grafts, leading to their malignant transformation, serving as a convenient and operable model for investigating leukemogenesis, especially for DCL.

移植后残留白血病细胞恶性转化供者造血细胞是供者细胞白血病(DCL)发生的关键机制。微粒(MP)是一种在细胞间传递信息的高效载体，我们新近发表文章证明移植后患者骨髓中仍存在白血病细胞来源MP，且MP能够体外诱导正常移植物成瘤。因此我们推测：异基因移植后残留的白血病细胞通过MP恶性转化正常供者细胞诱发DCL。本课题拟在前期研究基础上，通过体内外实验及临床研究进一步证明白血病MP参与移植后DCL的发生；明确MP作用的确切靶细胞，证明MP通过投递miRNA分子群诱导正常细胞成瘤，并从miRNA-靶基因-转录因子共调控环角度初步阐明正常细胞恶变的主要分子事件。本项目的完成，将从MP这一全新视角初步阐明白血病移植后DCL的发生机制；MP可望发展为一种早期预测移植后疾病进展及DCL发生的优秀预警指标；更重要的是，本课题为研究白血病发生提供了一种极佳的模型选择，并初步完成了对正常细胞白血病化机制的探讨。

大部分细胞都可以释放微泡（MP）作为细胞间交流的介质。MP是多种信号分子的复合体，包裹母细胞的蛋白、脂质和核酸，因此微泡可以作为监测疾病进展或复发的潜在标记。我们前期已证明慢性髓细胞白血病 （CML）K562细胞来源的MP可以恶性转化正常的造血移植物，使其成为急性白血病样细胞。我们发现在早期恶性转化时微泡中抗肿瘤miRNA增加，一些防御性通路激活。转化晚期阶段，细胞周期、DNA修复以及能量代谢通路相关的癌性miRNA和基因上调。调控网络分析发现大量转录因子和miRNA与通路紊乱、恶性转化相关。高表达miR-146b-5p增加受体细胞内活性氧水平和基因组不稳定性。另外，恶性转化过程中造血细胞的全基因组甲基化水平在第三天最高，甲基转移酶（DNMT）水平也相应增加; RNA酶处理后，K562微泡丧失这一恶性转化能力并且DNMT的表达也降低。所以微泡中的RNA对恶性转化进程以及基因组DNA甲基化的改变至关重要。后续实验发现K562-MP中miR-106a/b增高加速恶性转化 （8.33±0.94 vs. 13.29±1.28天；P<0.01）；lincPOU3F3表达降低延缓恶性转化进程（8.33±0.94 vs. 17.83±0.29 天；P<0.05）。lincPOU3F3可以直接增加DNMT3a/b的表达，miR-106a/b可以通过RB间接增加DNMT3a/b的表达。临床研究方面我们发现CML停药后复发与否和白血病干细胞（LSC）数目不相关。停药后LSC鼠移植模型可以提供新的方法来分析LSC功能和预测复发。白血病来源的微泡可以为停药后微小残留病和早期复发监测提供重要信息。CD4+CD134+T细胞的比例可能是评估异基因造血干细胞移植后患者免疫抑制状态的潜在指标。