基于微泡评判残留白血病干细胞功能状态指导CML患者TKI停药的实验研究

Tyrosine kinase inhibitor（TKI） therapy has markedly improved the treatment response and overall survival in patients with chronic myeloid leukemia,most of whom may be able to achieve complete molecular remission(CMR).For these patients,wether TKI therapy can be discontinued remains controversial. Previous trials show that TKI cessation in patients in sustaining CMR results in distinct outcome:while nearly 60 percent of patients relapsed within a year,the other 40 percent of patients remained in CMR till the end of the observation.These distinct results indicates that for some patients,persistent TKI therapy is needed and for the others TKI cessation is safe.Residual Leukemia stem cells(LSCs) that are insensitive to TKI therapy are belived to be the cause of relapse after TKI discontinuation. However, persistence of LSCs dosen't necessarily lead to relapse, since LSCs could be detected by more sensitive techniques even in those who had maintained CMR after TKI cessation. Thus, the functional status of LSCs may be critial to determine relpase or not after TKI cessation and its evaluation may guide the choice of further treatment for patients in CMR. Despite the great value of evaluating the functional status of residual LSCs, residual LSCs can hardly be seperated and analysed.In order to solve this problem, novel strategies to evaluate LSC function status are needed. Microvesicles seems to be the key.Different kinds of cells are able to release large amount of microvesicles(MV) containing bioinformational moleculars from parental cells. We previously showed that BCR-ABL transcripts could be detected in MVs derived from periphery blood(PB) of Chronic myeloid leukemia patients by RT-PCR, and the level of BCR-ABL transcripts was associated with the BCR-ABL level in PB cells and treatment reponse. Moreover, BCR-ABL level could be detected in MVs from PB of patients in CMR，Thus providing evidence that MVs could be used to predict functional status of their parental cells.So in our study,we intend to investigate whether the level of β-catenin，which plays a key role in the maintenance of self-renewal ability of stem cells，and β-gal，a classical marker of cell senescence，in MVs，could predict the tumor initiating ability of residual LSC，and validate the sensitivity and specificity of this method.And Then, assess the ability of MV content to predict relapse by using mouse model and by clinical observation, and finally，establish a kind of new risk stratification system to guide safe cessation of TKI.

达到完全分子学缓解（CMR）的慢性髓性白血病(CML)患者能否停用酪氨酸激酶抑制剂(tyrosine kinase inhibitor，TKI)治疗是目前关注焦点。残留白血病干细胞（LSC）功能状态是决定患者停药后是否复发的关键因素，但临床极难准确评判。我们前期研究发现CMR患者外周血存在残留LSC来源微泡（MV），基于MV含有大量遗传信息并能反映母体细胞特征，我们设想通过MV评判残留LSC功能状态，为预测TKI 停药后疾病复发提供新型指标。本课题拟通过蛋白质组学技术筛选LSC来源微泡特异性表面标记；研究基于MV中β-catenin/β-gal水平评判LSC功能状态，并衡量其灵敏度与特异性；通过建立人CML残留动物模型模拟TKI停药,观察MV及基于MV检测的上述指标与停药后疾病复发的相关性，以期建立一种指导CMR患者安全停用TKI的新型靶标及其"安全线"阈值。

达到完全分子学缓解（CMR）的慢性髓性白血病(CML)患者能否停用酪氨酸激酶抑制剂治疗是目前关注焦点。残留白血病干细胞（LSC）功能状态是决定患者停药后是否复发的关键因素，但临床极难准确评判。我们前期研究发现CMR患者外周血存在残留LSC来源微泡（MVs），基于MVs含有大量遗传信息并能反映母体细胞特征，我们设想通过MV评判残留LSC功能状态，为预测TKI 停药后疾病复发提供新型指标。本研究通过体外分离临床初治CML患者骨髓(BM)/外周血(PB)中干祖细胞(CML-CD34+细胞)及其MV，证实了MV具有选择性富集母细胞表面标记及miRNAs的功能。结果发现：(1)检测了8例慢性期(CP)，4例急变期(BP)的CML-CD34+细胞为试验组与4例供者采集物残余样本为正常对照组作对比，证实了CML-BP来源CD34+细胞与正常采集物来源CD34+细胞相比，CD123表达显著增高，CML-CP来源CD34+细胞与正常对照组相比，CD123表达有增加的趋势，而CD34+细胞来源的MVs表面CD123的表达量与细胞呈相同的趋势；(2)对CML-CD34+细胞及其来源MVs进行miRNA表达谱芯片检测，筛选出了15个在MVs中选择性富集的miRNAs，对于这些选择性富集的miRNAs我们进一步进行了靶基因预测、GO和pathway分析，证实了这些miRNAs组分在多种生物学进程中都发挥重要调控作用，包括肿瘤发生、细胞粘附及肿瘤信号转导通路等。(3)我们以qRT-PCR法对芯片检测中某些CML-CD34+细胞来源MVs选择性富集的miRNAs进行验证，同时比较其与母细胞中相应miRNAs的表达差异，结果显示MVs内miR-1290、miR-638、miR-438-5p、miR-627表达显著上调。我们的研究首次从白血病干细胞的层面验证细胞分泌的微泡具有选择性富集母细胞的表面标记CD34、CD123的功能，可能为白血病干祖细胞的特征研究提供新的思路，也为干祖细胞来源MVs的分选提供了实验基础；此外，我们的研究首次报道了CML-CD34+细胞及其MVs内miRNA表达谱的差异，结果表明MVs中上调的miRNA可能与CML-CD34+细胞发育、肿瘤发生及重要信号通路密切相关，为我们进一步研究CML-CD34+细胞来源MVs对母细胞的功能状态的调节提供了重要依据。