Notch配体刺激白血病细胞内Cleaved Notch1蛋白及基因的表达

背景：Notch信号系统对干细胞的自身复制与分化有调节的作用.目的：监测Notch配体刺激下白血病细胞蛋白与基因表达的变化,解析Notch信号系统在细胞内的作用.方法：应用被Notch配体包被的培养皿,培养TMD7和THP-1细胞株,进行短期细胞增殖效果的评估.并且在刺激不同时间段后,应用免疫印迹法对Notch蛋白的活性进行检测,应用定量RT-PCR法测定各种基因表达的变化.结果与结论：Notch配体刺激对TMD7细胞株的增殖有促进作用,对THP-1细胞株的增殖有抑制作用.配体刺激下,免疫印记结果显示2种不同相对分子质量的活性化Notch1蛋白片段.配体刺激后,各种基因的发现量的变化是不同的.而同一细胞株对3种Notch配体刺激的反应基本类似.可见Notch信号系统比现在所知道的更为复杂,可能存在着两种正向和负向的影响.     

Notch信号通路在肿瘤治疗中的研究进展

跨膜受体蛋白Notch信号通路的活性在决定细胞命运(包括增殖、分化以及凋亡)过程中发挥着重要作用,它的功能异常将导致未分化细胞向肿瘤细胞转化.人类许多肿瘤中均可检测到Notch信号通路活性的改变,并且其受体配体的表达水平可以作为判断肿瘤预后的指标.由于肿瘤来源及其微环境的不同,Notch信号通路具有促进或抑制肿瘤发生、发展的双重作用.以Notch信号通路作为肿瘤治疗的靶点.正逐渐引起人们的重视.     

Notch配体的原核表达与纯化及其对四氯化碳损伤后造血的影响

目的:通过原核表达并纯化靶向内皮细胞的小鼠Notch重组配体m D1R蛋白,观察其对四氯化碳损伤后造血的影响。方法:PCR克隆并构建p ET22b(+)-m D1R表达载体,转化大肠杆菌,IPTG诱导,镍珠亲合层析纯化,SDS-PAGE确认目的蛋白表达。应用流式细胞术、细胞粘附实验、免疫荧光染色及实时定量RT-PCR检测蛋白的内皮靶向性和Notch激活程度。建立四氯化碳损伤小鼠模型,应用流式细胞术观察m D1R蛋白对造血干细胞(HSC)、髓系和淋系细胞的影响,应用磁珠分选Lin~-Scal-1~+c-Kit~+细胞并分析细胞周期的变化,RT-PCR检测IL-10的表达。结果:成功克隆并构建原核表达载体,获得高纯度且有生物学活性的m D1R重组蛋白。m D1R能激活四氯化碳损伤小鼠造血干/祖细胞的Notch信号途径,内源性扩增HSC和长期HSC达2.96倍和6.18倍,促进了髓系祖细胞及髓源性抑制细胞的自体扩增,尤其有利于粒/单核细胞进入血液循环,其具体机制可能与Notch信号促进应激损伤后造血干细胞增殖和上调IL-10表达有关。结论:成功构建了连接造血干细胞和造血微环境的新型Notch配体活性蛋白,证实Notch信号途径可能通过促抗炎因子表达,实现应激损伤后自体造血动员。     

Notch信号通路在儿童肿瘤中的作用

哺乳动物Notch蛋白包括四种(Notch1～Notch4),其配体分为Jagged和Delta两个家族.Notch通路由受体、配体及许多下游靶基因组成复杂的网络结构,调控着组织细胞的发育和生理过程,且在不同组织及细胞类型中起着致癌或抑癌作用.本文主要介绍Notch信号通路的调控及其在儿童肿瘤中的作用.     

Notch通路与白血病的研究进展

Notch信号通路在细胞增殖、分化、凋亡及肿瘤发生等病理生理过程中起重要作用,由Notch受体、Notch配体和细胞内效应分子CSLDNA结合蛋白3部分组成.Notch信号通路不仅对维持机体正常的造血功能起重要作用,而且其信号异常和白血病的发生、细胞周期阻滞和疗效预后相关.     

Notch信号途径与肾细胞癌发生发展的相关性研究

目的通过免疫组织化学方法检测人肾细胞癌中Notch信号途径相关蛋白的表达水平,探究Notch信号途径分子与人肾细胞癌发生发展的可能关系。方法手术方法获得6例肾细胞癌组织标本,通过HE染色和免疫组织化学染色的方法对肾细胞组织和癌旁组织中Notch信号通路受体、配体蛋白表达水平进行检测。结果与癌旁组织相比,癌症组织中Notch信号的受体Notch-1以及配体Jagged-1的表达水平有明显的降低;而相对于癌旁组织,Notch信号途径的另一个受体Notch-2及配体Jagged-2表达水平有明显的升高。结论肾细胞癌的发生与Notch信号途径的表现出了一定的相关性,Notch信号途径可能参与了肾细胞癌的发生与发展。     

丙戊酸钠对多发性骨髓瘤RPMI 8226细胞Notch通路的影响

目的:探讨丙戊酸钠(VPA)对多发性骨髓瘤RPMI 8226细胞Notch通路中Notch1受体活性片段ICN1和靶基因Hes1表达的影响。方法:实验分为4组:空白对照组、VPA 2、4和8 mmol/L组,应用四甲基偶氮唑蓝(MTT)检测VPA对多发性骨髓瘤RPMI 8226细胞的增殖抑制作用,采用反转录聚合酶链式反应(RT-PCR)检测Notch通路中Notch1受体活性片段ICN1、靶基因Hes1 mRNA的表达,应用蛋白印记法(Western blot)检测ICN1、Hes1蛋白的表达。结果:同一时间、不同浓度VPA(0、2、4、8 mmol/L)处理RPM I 8226细胞,其细胞的生长明显受到抑制。相同浓度的VPA作用于RPMI 8226细胞不同时间(24、48、72 h)时,细胞的生长明显受到抑制。2、4、8mmol/L VPA处理48 h时与空白对照组相比,ICN1 mRNA及蛋白表达水平均明显降低(P0.05),靶基因Hes1mRNA及蛋白表达水平均明显降低,(P0.05)。结论:VPA明显抑制多发性骨髓瘤RPM I 8226细胞的增殖,并且这种抑制作用在一定范围内呈时间-浓度依赖性(r=0.945)。VPA可以下调Notch通路中Notch1受体活性片段ICN1、靶基因Hes1 mRNA及蛋白的表达水平,VPA可能通过抑制Notch信号通路实现对多发性骨髓瘤细胞的生长抑制作用。     

CXCR7在急性单核细胞白血病中的表达及对THP-1细胞功能的影响

目的:探讨基质细胞衍生因子-1α(SDF-1α)受体CXCR7在急性单核细胞白血病(AML-M5)中的表达,及其对急性单核细胞白血病细胞系THP-1增殖、凋亡和侵袭能力的影响。方法:应用流式细胞术、Western blot、RTPCR分别检测初诊AML-M5患者和正常人外周血单个核细胞(PBMNC)及THP-1细胞CXCR7蛋白和mRNA表达。CCK8法、Annexin V/PI标记法和Transwell法观察CXCR7对THP-1细胞体外增殖、凋亡和侵袭的作用。结果:在初诊AML-M5患者PBMNC幼稚细胞表面CXCR7表达高于正常对照(P0.05),在THP-1细胞表面CXCR7也有高表达;THP-1细胞和AML-M5患者PBMNC中CXCR7蛋白和mRNA水平均明显高于正常对照(P0.05);SDF-1α刺激可增高THP-1细胞增殖活性,但这种增殖活性可被CXCR7抗体抑制(P0.01);CXCR7抗体不影响THP-1细胞凋亡(P0.05);CXCR7抗体可明显抑制SDF-1α诱导的THP-1细胞侵袭能力(P0.01)。结论:CXCR7在急性单核细胞白血病患者及THP-1细胞中均有高表达,并参与细胞增殖和侵袭,阻断CXCR7表达有可能降低急性单核细胞白血病的浸润风险。     

Notch分子与其配体在32D细胞分化过程中的作用

目的 探讨Notch信号传导系统的作用机制及其对造血系统的影响。方法 将报告基因TP1瞬时转染Notch－CHO和Notch2－CHO细胞后，分别加入转染有不同Notch配体Deltal,Delta4,Jagged1,Jagged2的CHO细胞及正常未转染的CHO细胞，使Notch分子与其配体充分结合并发挥作用，加入发光底物PGL－100，用Lumat测定发光情况。将Notch1－32D细胞加入含G－CSF培养液的CHO，Jagged2-CHO及Delta4-CHO细胞中，观察Notch1－32D细胞分化及分化抑制情况。结果 5种Notch配体与Notch1结合后均能引起荧光素酶活性增高，Jagged2-CHO,Delta4-CHO1-4,Delta4-CHO1-5尤为明显。对Notch2来讲，5种配体均可引起TP1活性的增高。在G－CSF存在下，Notch1－32D细胞可分化为成熟的粒细胞；通过分化抑制实验发现Jagged2能抑制G－CSF引起的Notch1－32D细胞分化，但Delta4不能抑制GCSF引起的Notch1－32D细胞分化。Jadded2,Delta4为Notch1分子的配体，Delta4不能抑制G－CSF引起的Notch1－32D细胞分化，但Jagged2能抑制G－CSF引起的Notch1－32D细胞分化。     

Notch1信号通路对AGEs诱导大鼠心脏肌成纤维细胞转分化的调控

目的:探讨Notch1信号通路在糖基化终末产物诱导的大鼠心脏肌成纤维细胞转分化中的表达及其可能的调控作用。方法:体外培养SD乳鼠心脏成纤维细胞,予以200μg/ml浓度AGEs刺激、Notch信号抑制剂处理。运用CCK-8检测细胞的增殖情况;Western blot检测Notch1信号通路及肌成纤维细胞相关蛋白α号通路及的表达。结果:(1)AGEs促进心脏成纤维细胞的增殖,并呈时间依赖性(P0.05),同时Notch信号抑制剂DAPT显著抑制细胞的增殖(P0.05)。(2)AGEs刺激心脏成纤维细胞α-SMA及Notch1信号通路蛋白Notch1和Hes1的表达(P0.05)。(3)运用γ内分泌酶抑制剂DAPT抑制心脏成纤维细胞Notch1信号可下调α-SMA的表达(P0.05)。结论 :Notch1信号可能参与促进AGEs诱导的大鼠心脏成纤维的增殖及向肌成纤维细胞方向转化。     

Retinoic acid. Inhibition of the clonal growth of human myeloid leukemia cells.

Vitamin A and its analogues (retinoids) affect normal and malignant hematopoietic cells. We examined the effect of retinoids on the clonal growth in vitro of myeloid leukemia cells. Retinoic acid inhibited the clonal growth of the KG-1, acute myeloblastic leukemia, and the HL-60, acute promyelocytic leukemia, human cell lines. The KG-1 cells were extremely sensitive to retinoic acid, with 50% of the colonies inhibited by 2.4-nM concentrations of the drug. A 50% growth inhibition of HL-60 was achieved by 25 nM retinoic acid. Complete inhibition of growth of both leukemia cell lines was seen with 1 microM retinoic acid. Exposure of KG-1 cells to retinoic acid for only 3-5 d was sufficient to inhibit all clonal growth. The all-trans and 13-cis forms of retinoic acid were equally effective in inhibiting proliferation. Retinal, retinyl acetate, and retinol (vitamin A) were less potent inhibitors. Clonal growth of the human K562 and mouse M-1 myeloid leukemic cell lines was not affected by 10 microM retinoic acid. Retinoic acid also inhibited the clonal growth of leukemia cells from five of seven patients with acute myeloid leukemia. Retinoic acid at concentrations of 5 nM to 0.3 microM inhibited 50% clonal growth, and 1 microM retinoic acid inhibited 64-98% of the leukemic colonies. The inhibition of clonal growth of KG-1 and HL-60 cell lines and of leukemic cells from two patients was not associated with the presence of a specific cytoplasmic retinoic acid-binding protein. Our study suggests that retinoic acid may prove to be effective in the treatment of human myeloid leukemia.     

Notch2 governs the rate of generation of mouse long- and short-term repopulating stem cells

HSCs either self-renew or differentiate to give rise to multipotent cells whose progeny provide blood cell precursors. However, surprisingly little is known about the factors that regulate this choice of self-renewal versus differentiation. One candidate is the Notch signaling pathway, with ex vivo studies suggesting that Notch regulates HSC differentiation, although a functional role for Notch in HSC self-renewal in vivo remains controversial. Here, we have shown that Notch2, and not Notch1, inhibits myeloid differentiation and enhances generation of primitive Sca-1(+)c-kit(+) progenitors following in vitro culture of enriched HSCs with purified Notch ligands. In mice, Notch2 enhanced the rate of formation of short-term repopulating multipotential progenitor cells (MPPs) as well as long-term repopulating HSCs, while delaying myeloid differentiation in BM following injury. However, consistent with previous reports, once homeostasis was achieved, neither Notch1 nor Notch2 affected repopulating cell self-renewal. These data indicate a Notch2-dependent role in assuring orderly repopulation by HSCs, MPPs, myeloid cells, and lymphoid cells during BM regeneration.     

Mind bomb 1 in the lymphopoietic niches is essential for T and marginal zone B cell development

Notch signaling regulates lineage decisions at multiple stages of lymphocyte development, and Notch activation requires the endocytosis of Notch ligands in the signal-sending cells. Four E3 ubiquitin ligases, Mind bomb (Mib) 1, Mib2, Neuralized (Neur) 1, and Neur2, regulate the Notch ligands to activate Notch signaling, but their roles in lymphocyte development have not been defined. We show that Mib1 regulates T and marginal zone B (MZB) cell development in the lymphopoietic niches. Inactivation of the Mib1 gene, but not the other E3 ligases, Mib2, Neur1, and Neur2, abrogated T and MZB cell development. Reciprocal bone marrow (BM) transplantation experiments revealed that Mib1 in the thymic and splenic niches is essential for T and MZB cell development. Interestingly, when BM cells from transgenic Notch reporter mice were transplanted into Mib1-null mice, the Notch signaling was abolished in the double-negative thymocytes. In addition, the endocytosis of Dll1 was impaired in the Mib1-null microenvironment. Moreover, the block in T cell development and the failure of Dll1 endocytosis were also observed in coculture system by Mib1 knockdown. Our study reveals that Mib1 is the essential E3 ligase in T and MZB cell development, through the regulation of Notch ligands in the thymic and splenic microenvironments.     

手足口病患儿外周血单个核细胞Notch配体的表达及其意义

目的：了解Notch信号通路在手足口病发病中的作用。方法2012年6月-2012年12月苏州大学附属儿童医院感染病房和儿科重症监护病房收住的82例手足口病患儿,设为手足口病组,根据其临床表现和实验室结果分为单纯组和脑炎组,对照组为外科腹股沟斜疝择期手术患儿40例,将其流行病学、临床理化特征、外周血免疫细胞水平和Notch配体水平进行分析比较。结果手足口病组患儿具有更低的外周血CD3＋细胞、CD3＋CD4＋细胞和CD3＋CD8＋细胞水平,更高的CD3-CD19＋细胞水平（P＜0.05）;手足口病组外周血单个核细胞Notch配体Dll1和Dll4表达显著高于对照组（P＜0.05）,而单纯组和脑炎组之间差异无统计学意义;外周血单个核细胞Dll4表达和CD3＋、CD3＋CD8＋淋巴细胞表达呈负相关（P＜0.05）,与CD3-CD19＋淋巴细胞表达呈正相关（P＜0.05）;外周血单个核细胞Dll4表达与脑脊液中WBC总数和总蛋白含量（S-TP）呈正相关（P＜0.05）。结论 Notch信号通路可能参与手足口病的发病;Notch配体Dll4与外周血CD3＋、CD3＋CD8＋和CD3-CD19＋淋巴细胞有较强的相关性,可能通过影响这些免疫细胞的数量和状态从而影响疾病的预后。     

Notch与恶性血液病研究进展

Notch信号通路由Notch受体、配体和胞内的效应分子组成。1991年,第1个人类Notch家族成员Notch1基因在具有t（7;9）（q34;q34.3）染色体易位的急性T淋巴细胞白血病中发现,由此揭示了Notch信号通路是一条影响细胞命运的保守而重要的信号通路,几乎涉及所有细胞的增殖、分化和凋亡。与此同时,Notch信号通路在恶性血液病和实体瘤的形成和发展中所起的作用也日趋清晰。在超过半数的急性T淋巴细胞白血病中发现了突变型Notch1基因,但其作为原发还是继发仍不确定。近年来的研究发现,Notch信号通路具有致癌和抑癌双重作用。现就Notch信号通路在不同类型恶性血液病发生和发展中所起的作用、急性T淋巴细胞白血病中突变型Notch1基因类型、Notch信号通路与其它相关信号通路的关系及Notch信号通路抑制剂在恶性血液病治疗中的作用等相关研究进展进行综述。     

High-level IGF1R expression is required for leukemia-initiating cell activity in T-ALL and is supported by Notch signaling

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer of immature T cells that often shows aberrant activation of Notch1 and PI3K-Akt pathways. Although mutations that activate PI3K-Akt signaling have previously been identified, the relative contribution of growth factor-dependent activation is unclear. We show here that pharmacologic inhibition or genetic deletion of insulin-like growth factor 1 receptor (IGF1R) blocks the growth and viability of T-ALL cells, whereas moderate diminution of IGF1R signaling compromises leukemia-initiating cell (LIC) activity as defined by transplantability in syngeneic/congenic secondary recipients. Furthermore, IGF1R is a Notch1 target, and Notch1 signaling is required to maintain IGF1R expression at high levels in T-ALL cells. These findings suggest effects of Notch on LIC activity may be mediated in part by enhancing the responsiveness of T-ALL cells to ambient growth factors, and provide strong rationale for use of IGF1R inhibitors to improve initial response to therapy and to achieve long-term cure of patients with T-ALL.     

MEDI0639: A Novel Therapeutic Antibody Targeting Dll4 Modulates Endothelial Cell Function and Angiogenesis In Vivo

The Notch signaling pathway has been implicated in cell fate determination and differentiation in many tissues. Accumulating evidence points toward a pivotal role in blood vessel formation, and the importance of the Delta-like ligand (Dll) 4-Notch1 ligand-receptor interaction has been shown in both physiological and tumor angiogenesis. Disruption of this interaction leads to a reduction in tumor growth as a result of an increase in nonfunctional vasculature leading to poor perfusion of the tumor. MEDI0639 is an investigational human therapeutic antibody that targets Dll4 to inhibit the interaction between Dll4 and Notch1. The antibody cross-reacts to cynomolgus monkey but not mouse species orthologues. In vitro MEDI0639 inhibits the binding of Notch1 to Dll4, interacting via a novel epitope that has not been previously described. Binding to this epitope translates into MEDI0639 reversing Notch1-mediated suppression of human umbilical vein endothelial cell growth in vitro. MEDI0639 administration resulted in stimulation of tubule formation in a three-dimensional (3D) endothelial cell outgrowth assay, a phenotype driven by disruption of the Dll4-Notch signaling axis. In contrast, in a two-dimensional endothelial cell-fibroblast coculture model, MEDI0639 is a potent inhibitor of tubule formation. In vivo, MEDI0639 shows activity in a human endothelial cell angiogenesis assay promoting human vessel formation and reducing the number of vessels with smooth muscle actin-positive mural cells coverage. Collectively, the data show that MEDI0639 is a potent modulator of Dll4-Notch signaling pathway. Mol Cancer Ther; 11(8); 1650-60. ?2012 AACR.