【作者】 杨杨；

【导师】 蔡真； 孙晓平；

【作者基本信息】 浙江大学 ， 内科学， 2011， 博士

【摘要】 急性淋巴细胞性白血病(acute lymphoblastic leukemia, ALL)是一种进行性恶性血液病,好发于儿童及成年人。近年来,随着对疾病认识的深入及治疗方案的不断改进,ALL的预后明显改善。然而,此病在大多数病人仍易复发及产生耐药,整体存活率低。越来越多的证据表明ALL的复发和耐药与骨髓微环境,如骨髓基质细胞(BMSC)对ALL细胞的保护作用有关。但白血病细胞及微环境相互作用的机制还不清楚。因此研究骨髓微环境对ALL细胞的保护机制,对于提高疗效乃至治愈ALL有重要作用。本研究中我们检测了BMSC对阿糖胞苷(Ara-C)诱导的ALL细胞凋亡的保护作用,并利用各种分子生物技术,体内体外实验研究其可能的分子机制。首先我们将Reh, RS4;11, SEMK2 3种ALL细胞株与人和鼠的BMSC细胞共培养,模拟骨髓微环境,并用Ara-c诱导ALL细胞凋亡。流式细胞术检测发现BMSC能显著减少ALL细胞的凋亡。BMSC同样可抑制ALL病人的原代白血病细胞的自发性凋亡及Ara-C诱导的凋亡。Western blot检测结果也证实了BMSC可逆转Ara-C诱导的ALL细胞Caspase3, PARP, Bcl-2蛋白被剪切,同时伴有p21,Cyclin A, CDK2表达的减少。此外,BMSC还能加快ALL的细胞周期进程,促进其增殖。我们发现与BMSC共培养可导致Akt的磷酸化,而用P13k抑制剂LY29400抑制Akt的磷酸化可增加ALL细胞的凋亡。但是利用CXCR4的拮抗剂AMD3100并不能抑制BMSC的保护作用。为了进一步探索参与保护机制的分子,我们利用基因芯片,蛋白质芯片等,检测了在Ara-C作用下单独培养和共培养Reh细胞基因和蛋白表达的变化。结果表明许多的信号通路参与该过程,包括凋亡通路,细胞周期等,与上述结果相符。半定量RT-PCR验证了显著差异表达的基因。我们从中发现共培养组的Lef-1, C-myc及Cyclin D1表达增加,Western blot也同样发现了β-catenin的表达增加,提示BMSC可通过活化ALL细胞的Wnt通路起到保护作用。进一步研究发现,小分子抑制剂XAV939能够有效抑制β-catenin的表达,从而抑制共培养系统中ALL细胞的Wnt通路的活化,并最终逆转BMSC对ALL细胞的保护作用,增加Ara-C诱导的细胞凋亡。此抑制剂不但作用于ALL细胞,而且作用于保护ALL细胞的BMSC。预计这种双重作用使它可能成为将来ALL治疗中与化疗联合用药的优先选择。最后我们联合Ara-C及XAV939治疗Rehluc/SCID小鼠,通过持续定期活体IVIS荧光成像系统对小鼠体内白血病的发生发展进行实时跟踪观察,发现XAV939联合Ara-C治疗,可减缓白血病的进展,延长小鼠的生存时间。综上所述,BMSC可通过多个信号途径保护Ara-C作用下的ALL细胞,如PI3k/Akt通路,Wnt通路。靶向这些通路,为打破微环境对ALL细胞的支持保护作用并最终消灭白血病提供新的策略和治疗。更多还原

【Abstract】 Acute lymphoblastic leukemia (ALL) is one of the fastest-growing hematological malignancies affecting patients with all ages, particularly children. Significant advances have been made in recent years in our understanding of the disease and new therapies have been developed, which have led to a greatly improved outcome. Nevertheless, in a significant number of patients with ALL, the disease relapses and becomes resistant to treatment, causing death of the patients. Increasing evidence suggests that relapse of the disease and resistance to treatment are largely attributed to the protection of the leukemic cells by various components in the microenvironment. such as bone marrow stromal cells (BMSC). However, the cross-talk between leukemic cells and their microenvironment remains poorly understood. Therefore, better understanding the mechanisms underlying the protection of ALL cells by the microenvironment is of ultimate importance in developing new therapies targeting such protection and eventually eradicating all the leukemic cells to cure the disease. In this study, we evaluated the effects of BMSC on apoptosis of ALL cells induced by Ara-C and further investigated the underlying molecular mechanisms in vitro and in vivo by different molecular biologe techniques.We co-cultured human leukemia cell lines Reh, SEMK2 and RS4.11 with human and murine BMSC to mimic in vivo bone marrow microenvironment and used Ara-C to induce apoptosis. Flow cytometry analysis showed that BMSC could significantly inhibit apoptosis of ALL cells. BMSC could also provide protection of primary ALL cells from both spontaneous and Ara-C induced apoptosis. The reduced apoptosis in the co-culture was confirmed by Western blot which showed that BMSC could protect ALL cells from Caspase-3 and PARP cleavage, associated with decreased p21, Cyclin A and Cdk2 expression. In addition, BMSC could increase proliferation of ALL cells by accelerating cell cycle. We found that co-culture with BMSC resulted in phosphorylation of AKT in ALL cells and PI3K inhibitor LY294002 specifically inhibited MSC-induced activation of AKT and promoted ALL cell apoptosis. CXCR4 inhibitor AMD3100, however did not increase ALL cell apoptosis. To identify candidate molecules potentially involved in the protection of ALL cells by MSC, we performed gene expression microarray and reverse phase protein array analyses on ALL cells exposed to Ara-C in presence or absence of BMSC. Our data indicated that several signaling pathways were involved in this process, including apoptosis signaling and cell cycle checkpoint control, which was consistent with the apoptosis data described above. A group of top differentially expressed genes identified in the microarray studies were confirmed by RT-PCR.By analyzing the microarray data, we found that expression of several important members of the Wnt pathway, such as Lef-1, C-myc, and Cyclin Dl was increased in ALL cells when co-cultured with BMSC. Though (3-catenin expression was not significantly changed at mRNA level on microarray, elevated expression ofβ-catenin at protein level was confirmed by Western blot. These results suggested that Wnt pathway could play a role in BMSC-mediated protection. Subsequently, we found that small molecule inhibitor XAV939 could inhibit expression ofβ-catenin and activation of Wnt pathway, which reversed protection of BMSC and increased Ara-C induced apoptosis. Our data also suggested that the inhibitor acted on both ALL cells and BMSC, making it a potential highly effective molecule in ALL treatment in combination with chemotherapy. We tested this possibility in NOD/SCID mice. We transplanted NOD/SCID mice with Rehluc ALL cells and treated them with Ara-C with or without XAV939. Growth and development of leukemia was monitored by IVIS imaging systems. We found that combined treatment significantly slowed down leukemia development and resulted in better survival of the recipient mice.Collectively, our results demonstrated that BMSC can protect ALL cells from Ara-C induced apoptosis by multiple signaling pathways, such as those involving PI3K/AKT and Wnt signaling. Hence, targeting these pathways may become potential novel therapeutic strategies to disrupt the support of the microenvironment to ALL cells and to eventuallv eradicate leukemic cells.更多还原