Dll4-Notch1通路调控血管新生在急性T淋巴细胞白血病中作用的研究（斑马鱼模型）

Many studies have confirmed that angiogenesis, the formation of new blood vessels from existing vasculature, plays an essential role in growth, invasion, and metastasis of acute lekeukmia. Dll4-Notch1 and VEGF signaling pathway play as the two key roles in tumor angiogenesis. Activating mutations in the Notch1 gene have been found in about 60% of patients with T-cell acute lymphoblastic leukemia(T-ALL), as the independent prognostic factor, promoting cell proliferation, inhibiting differentiation and apoptosis, collaborating cancer gene effects, but there is no report about the effect in T-ALL angiogenesis. Development of the vascular system has been well characterized in zebrafish embryos. Many zebrafish blood vessels form by angiogenic sprouting and appear to require the same proteins that are necessary for blood-vessel growth in mammals. And we have established stable transgenic line in which green fluorescent protein (GFP) expression is directed to endothelial cells by the use of endothelial cell-specific promoter- - fli1. In this study, we plan to construct the fusion rag2 promoter (specifically target to inmature T lymphocytes) and ICN (human truncated notch1 gene active clips), tag with RFP, microinject into the Tg(fli1:EGFP) zebrafish embryo, establish double color tracer and double transgenic (Notch1 and fli1) zebrafish, verify and select the T-ALL model by TCR-α positive. Taking the advantage of the transparent characteristic of zebrafish, we can dynamically observe green vessels network and leukemia cell with red fluorograph real time by laser confocal microscope imaging technology. Combining with transcription group sequencing technology, qRT-PCR, Western Blot, whole-mount in situ hybridization, immunohistochemistry, and flow cytometry to detect kinds of transcription factors in Dll4-Notch1 and VEGF signaling pathway, comparing with wild type, we may get the crucial differentially expressed genes (DEGs)，then select the possible target genes through "reciprocal best hit". Next, we plan to use RNA interference technology to prove the interaction between Dll4-Notch1 and VEGF signaling pathway. Ultimately, basis on the above research, high throughput drug screening will be carried out on this T-ALL animal model in vivo and T-ALL cell lines, patients' leukemia cells in vitro. These experiments may illuminate and supplement the existing hypothesis in T-ALL pathogenesis, infiltration, and prognosis with the role of angiogenesis mediated by Dll4-Notch1 signaling pathway, and provide new theory basis and the more safe and effective molecular therapic target. The applicant has rich experience in leukemia drug resistance research and transgenic zebrafish technology, it is expected that the results of this research can reach the advanced level.

抗血管新生是白血病靶向治疗的新热点，Dll4-Notch1与VEGF信号通路是肿瘤血管新生最主要的调控因素。60%的急性T淋巴细胞白血病（T-ALL）患者存在Notch1信号通路异常激活，与不良预后相关，但其在T-ALL血管新生中所起的作用，目前尚无报道。本课题拟在血管内皮EGFP标记的斑马鱼模型上，显微注射Rag2驱动的人源Notch1和RFP重组质粒，建立双色示踪双转基因斑马鱼系，筛选出T-ALL模型，通过转录组测序法检测差异表达谱，对主要差异表达基因进行过表达或Morpholino Knockdown干预，采用激光共聚焦显微镜、基因芯片、qRT-PCR、Western Blot、原位杂交、免疫组化、流式细胞术等方法，遴选出Notch1参与T-ALL发病和血管新生的关键基因和可能机制，并应用该T-ALL模型高通量筛选出2-3个靶向药物，最后在 T-ALL细胞株和原代细胞中进行药效验证。

为研究急性T淋巴细胞白血病（T-ALL）中普遍高表达的Dll4-Notch1通路对T-ALL血管新生及预后影响，我们建立了过表达人源Notch的双色示踪双转基因斑马鱼系，显示血管网增生、紊乱，FCM提示Notch鱼系的肾髓细胞红系受抑，粒系增生，淋系G2/S期阻滞，凋亡增加；2月龄时外周血红系降低，出现成簇小淋巴细胞，原始造血细胞增生，类似人类T-ALL，提示Notch1参与T-ALL发病并促进血管新生。.RT-PCR证实斑马鱼淋系造血主要调控因子ikaros、rag1、lck均受抑，即淋巴细胞分化严重受阻。转录组测序检测发现VEGF通路，NF-κB通路，PI3K-AKT-mTOR通路均不同程度的激活，部分基因，如CDK4/CDK6，Myc基因等显著转录上调，提示细胞增殖和血管新生上调，而凋亡受阻。.不同浓度三氧化二砷（ATO：0，50, 100和150μM）、槲皮素（0，80, 160 和240μM）及0.1% DMSO（对照）处理14hpf转基因斑马鱼胚胎，可见ATO主要抑制其肠下静脉（SIVs）形成，并下调VEGF、 Dll4、Notch1 与 Hes1 mRNA 的表达，槲皮素可显著抑制其背部节段血管发育及血管新生芽，并下调 Notch1、Hes1 mRNA，或可成为此类T-ALL的新型候选药物。Dll4或VEGF的Morpholino-RNA，均可抑制Notch斑马鱼白血病细胞增殖和血管新生，RT-PCR证实抑制VEGF可使Notch1、D114、Hes1和Hey2的RNA表达下降；抑制Dll4使VEGF、VEGFR1和VEGFR2基因增高。可见VEGF对Notch/D114信号通路有正调节作用，而后者反馈抑制VEGF信号通路。.ATO、槲皮素处理高表达Notch的T-ALL细胞株A3与Molt4，发现ATO可显著抑制细胞增殖，并下调Notch1、Hes1、Dll4及VEGF mRNA与蛋白的表达。槲皮素抑制细胞株的增殖并诱导其凋亡，且呈时间和浓度依赖性，下调Notch1、Hes1、Dll4、VEGF等基因及蛋白的表达水平。在40例T-ALL患者原代白血病细胞，已获得类似数据结果。.该工作为T-ALL的综合治疗提供新的思路和候选药物。针对Notch1/VEGF的通路研究，为理解并克服白血病新生血管形成这一不良预后因素的存在和机制提供理论基础。