实验血液学研究(赵维莅)
发布日期:2010-04-03 浏览次数:470

2009年课题组继续致力于恶性淋巴瘤的疾病进展分子标志物和靶向治疗的研究。
1. 分子标志物的大规模筛选。运用高通量拷贝数变异(CNV)芯片对25例弥漫性大B细胞淋巴瘤(DLBCL)和11例T细胞淋巴瘤(T-NHL)患者病变组织进行检测。DLBCL患者中发现小片段扩增区域51处、缺失区域16处。结合基因表达谱结果,选择在CNV检测中位于扩增/缺失片段,在基因表达谱中异常表达的12个候选基因进行定量PCR验证。T-NHL患者中,发现小片段扩增区域8处,缺失4处,但未发现集中的特异区段。考虑表观遗传学调控,特别是甲基化修饰可能和T细胞恶性增殖密切相关,进一步运用甲基化芯片对6例T-NHL进行检测。发现与反应性增生相比42个基因在所有6例患者中均存在高甲基化。我们将对上述基因中和细胞信号传导通路相关的12个基因先进行定量PCR验证。同时,运用MassARRAY技术对73例淋巴瘤及500例正常对照进行DNA修复基因的筛选。发现XRCC1R280H和MGMT L84F与淋巴瘤的发生率密切相关。
2. 靶向治疗的探索。发现蛋白酶体抑制剂和组蛋白去乙酰化酶抑制剂联合应用对T-NHL的治疗作用。体外试验显示蛋白酶体抑制剂硼替佐米和组蛋白去乙酰化酶抑制剂SAHA具有协同作用,加速肿瘤细胞凋亡。伴随明显的线粒体膜电位降低、caspase家族蛋白异常激活。同时,对多个细胞信号传导通路产生影响,包括抑制细胞保护性信号传导通路NF-B、Raf-1/MEK/ERK和AKT,和激活应激相关信号传导通路JNK和p38MAPK而发挥细胞杀伤作用,体内试验显示联合应用明显抑制小鼠移植瘤的生长,肿瘤细胞原位凋亡明显增加。为临床上采用硼替佐米-SAHA联合方案治疗T-白血病/淋巴瘤提供理论依据,上述结果发表于《LEUKEMIA》杂志上,并受主编之邀撰写专家论坛。此外,我们进一步研究疏花毛萼香茶菜中提取得到的二萜类化合物毛萼乙素(Eriocalyxin B,EriB)的抗淋巴瘤活性和作用机制。结果显示,EriB对多种B和T-NHL细胞均具有生长抑制作用,并在早期即激活caspase家族,显著诱导细胞凋亡。在caspase活化的过程中伴随着Bid裂解及Bcl-2/Bax 和Bcl-xL/Bax下调。毛萼乙素诱导淋巴瘤细胞凋亡的机制涉及多途径、多靶点,不仅抑制NF-B的表达,而且下调AKT活性,致其下游一系列底物,如p-Bad、IKKα/β和Raf-1失活,P21上调,最终导致细胞周期阻滞,细胞凋亡发生。毛萼乙素还能以依赖细胞内活性氧的方式激活p-ERK表达而诱导细胞凋亡。体内试验证实毛萼乙素可显著抑制小鼠淋巴瘤生长,促进肿瘤细胞原位凋亡,是一种非常有前景的治疗淋巴瘤的候选靶向药物。

Ⅷ. Study on experimental hematology (Wei-Li Zhao)
In 2009, our group still focused on the biomarkers related to disease progression and targeted therapy in malignant lymphoma. 
1. Large-scale screening of biomarkers. High resolution copy number variation (CNV) was detected in 25 cases of diffuse large B-cell lymphoma (DLBCL) and 11 cases of T-cell non-Hodgkin’s lymphoma (T-NHL). A total of 51 amplification regions and 16 deletion regions were found in DLBCL. Comparing with gene expression profile, 12 candidate genes were selected for further detection by quantitative PCR. In T-NHL, although 8 amplification regions and 4 deletion regions were observed, they were scattered in different chromosomes. Considering that epigenetic regulation, especially gene methylation, plays an important role on T-cell malignancies, methylation chips were further performed on 6 cases of T-NHL. Forty-two candidate genes with abnormal methylation in all 6 cases and 12 of them were closely related to cell signaling pathways and selected for further verification. Meanwhile, using MassARRAY technique, 73 cases of NHL and 500 healthy controls were screened for DNA repair genes. The results showed that in XRCC1 R280H and MGMT L84F were associated with an increased risk of NHL. 
2. Target therapy. Interactions between inhibitors of the proteasome and histone deacetylases have been examined in human T-NHL cells both in vitro and in vivo. Co-exposure of cells to bortezomib and suberoylanilide hydroxamic acid (SAHA) synergistically induces T-lymphoma cells to undergo apoptosis, consistent with a significant increase in mitochondrial injury and caspase activation. These events are accompanied by inhibition of cyto-protective signaling pathways, including the NF-B, Raf-1/ MEK/ERK and AKT pathways, and activation of stress-related cascades, including the stress-activated kinases JNK and p38MAPK. Moreover, bortezomib in conjunction with SAHA efficiently induces apoptosis of primary T-lymphoma cells and inhibits tumor growth in a murine xenograft model established with subcutaneous injection of Jurkat cells. Taken together, these findings confirm the synergistic anti-tumor effect of the proteasome and histone deacetylase inhibitors, and provide an insight into the future clinical applications of bortezomib-SAHA combining regimen in treating T-cell malignancies. The results have been accepted by , with spotlight review invited. Meanwhile, we further investigated the effect of Eriocalyxin B (EriB) and its mechanism of action in human lymphoma both in vitro and in vivo. The results indicated that EriB significantly inhibited proliferation in a series of B and T lymphoma cell lines. EriB-induced lymphoma cell apoptosis was observed in association with caspase activation. Anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xL were downregulated, with pro-apoptotic member Bax stable or upregulated, resulting in reduced Bcl-2/Bax and Bcl-xL/Bax ratios. Multiple signal transduction pathways were involved in lymphoma cell apoptosis in response to EriB, including the inhibition of NF-B and AKT pathways, and the activation of ERK pathway. AKT inactivation was related to increased expression of cyclin dependent kinase inhibitor P21, decreased expression of anti-apoptotic phosphorylated form of Bad and NF-B activator IKK/. ERK activation corresponded to reactive oxygen species production and could be blocked by antioxidant dithiothreitol. In murine xenograft lymphoma models, EriB remarkably inhibited tumor growth and induced in situ tumor cell apoptosis. Together, these findings emphasized the value of EriB as a promising candidate targeting apoptosis cascade in lymphoma treatment.

年度发表论文:
国家重点实验室第一作者单位文章:
1. Zhao WL. Targeted therapy in T-cell malignancies: dysregulation of the cellular signaling pathways. Leukemia 2010;24:13-21.
2. Zhang QL, Wang L, Zhang YW, Jiang XX, Yang F, Wu WL, Janin A, Chen Z, Shen ZX, Chen SJ, Zhao WL. The proteasome inhibitor bortezomib interacts synergistically with the histone deacetylase inhibitor suberoylanilide hydroxamic acid to induce T-leukemia/lymphoma cells apoptosis. Leukemia 2009; 23: 1507-1514.
合作文章:
1. Soltani-Arabshahi R, Leboeuf C, Rivet J, Pisonero H, Zhao WL, Bachelez H, Ameisen JC, Janin A. Bcl-xL Gene Expression Correlated with Lower Apoptotic Cell Numbers and Shorter Progression-Free Survival in PCFCL. J Invest Dermatol. 2009;129:1703-9.