天花粉蛋白及其肽段诱导的免疫抑制依赖APC表面Notch配体的表达

新近发现,Notch信号途径参与调节外周成熟T细胞及其亚群的分化和功能发挥。本研究应用天花粉蛋白及其衍生肽处理骨髓来源的小鼠树突状细胞(DC),检测Notch配体家族分子的表达及DC对CD8+T细胞分泌细胞因子的影响。结果表明,天花粉蛋白或其衍生肽PB处理DC可使Notch配体Jagged1、Delta1分子表达明显增加,并改变CD8+T细胞细胞因子分泌格局,明显抑制Th1相关细胞因子IFN-γ的分泌,而Th2相关细胞因子IL-4和IL-10分泌明显增加。Notch信号的阻断剂可以部分逆转Tk及肽段的抑制作用。表明天花粉蛋白及其衍生肽可诱导一群具有抑制能力的CD8+T细胞,该作用依赖于DC表面Notch配体的表达。     

下调Notch3对小鼠T淋巴细胞体外活化增殖及NF-κB的影响

研究siRNA下调Notch3对小鼠T淋巴细胞体外增殖的影响,并初步探讨其免疫调节机制。从小鼠脾脏分离制备T淋巴细胞悬液;将化学合成的靶向Notch3基因的siRNA在Lipofectamine 2000介导下转染小鼠淋巴细胞;通过Westernblot检测各组细胞中Notch3蛋白水平的变化;以不同浓度的Notch3 siRNA作用于该小鼠T淋巴细胞模型,流式细胞术检测CD3~+T细胞早期活化标志CD69分子的表达;MTT检测Notch3-siRNA对小鼠淋巴细胞增殖的抑制作用;EMSA检测NF-κB活性。在淋巴细胞体外培养试验中,转染Notch3 siRNA可以明显降低小鼠淋巴细胞内Notch3的表达,下调Notch3可以显著抑制刀豆蛋白A(ConA)诱导的T细胞CD69的表达;同时下调Notch3对小鼠淋巴细胞的增殖有抑制作用;而且发现下调Notch3能明显抑制ConA诱导的NF-κB活化。实验结果提示,下调Notch3信号可通过抑制NF-κB活化对小鼠T淋巴细胞活化与增殖发挥抑制作用。     

Notch信号通路与T淋巴细胞发育

Notch信号通路在器官、细胞发育及肿瘤发生等病理生理过程中起重要作用。近年研究表明，Notch信号通路尤其是Noteh1对T淋巴细胞发育的各个阶段均有作用。Notch1能促进进入胸腺的淋巴样前体细胞向T淋巴细胞分化，抑制其向B淋巴细胞分化，并使T淋巴细胞表达TCRαβ，促进前T细胞向αβT细胞发育。此外，Notch1信号的活化可促进CD4^ CD8^ 双阳性胸腺细胞向CD8^ 单阳性细胞分化。本文综述了Notch信号通路的组成、活化，及其在T淋巴细胞发育中的作用。     

Notch信号通路与T淋巴细胞发育

Notch信号通路在器官、细胞发育及肿瘤发生等病理生理过程中起重要作用。近年研究表明 ,Notch信号通路尤其是Notch1对T淋巴细胞发育的各个阶段均有作用。Notch1能促进进入胸腺的淋巴样前体细胞向T淋巴细胞分化 ,抑制其向B淋巴细胞分化 ,并使T淋巴细胞表达TCRαβ ,促进前T细胞向αβT细胞发育。此外 ,Notch1信号的活化可促进CD4 + CD8+ 双阳性胸腺细胞向CD8+ 单阳性细胞分化。本文综述了Notch信号通路的组成、活化 ,及其在T淋巴细胞发育中的作用。     

Notch信号介导共培养脐带间充质干细胞和造血干细胞的相互作用

目的:探讨脐带间充质干细胞(UC-MSC)体外与造血干细胞共培养后Notch信号分子的改变。方法:通过胶原酶消化方法分离UC-MSC,通过流式细胞仪检测以及成脂、成骨和成软骨诱导鉴定UC-MSC具备间充质干细胞的特性。进而,将UC-MSC与脐血CD34+造血干细胞(HSC)体外培养,实时PCR方法检测MSC及CD34+细胞表面Notch配体及受体表达以及表达是否存在变化;在共培养体系中加入Notch信号阻滞剂DAPT(γ-secretase抑制剂),比较Hes-1基因活化状态的改变。结果:体外实验显示:UC-MSC在形态学、细胞表面表型和诱导分化能力上均具备间充质干细胞的特性。UC-MSC及CD34+细胞表面存在Notch信号配体及受体的表达,共培养后Jagged 1、Notch1基因表达明显增加;共培养后CD34+细胞中的Hes-1基因表达明显增加而加入DAPT后Hes-1基因表达未检出明显改变。结论:UC-MSC支持造血中,Notch信号可能发挥重要作用。     

间充质干细胞诱导小鼠CD8α+ CD11b+ jagged2high调节性树突状细胞的方法

目的:研究小鼠骨髓间充质干细胞(BMSCs)体外诱导CD8α+CD11b+jagged2high调节性树突状细胞(DCregs)的方法。方法:应用4种细胞因子体外刺激BALB/c(H-2d)小鼠骨髓有核细胞(BMCs)3 d,流式细胞术(FCM)分选树突状细胞(DCs),与BMSCs共培养10 d,诱导DCregs产生。FCM分析共培养前后DCs的表型、细胞周期及Notch信号通路中Jagged1、Jagged2配体的表达变化。结果:小鼠BMSCs在体外成功诱导新型DCs转变为DCregs,其CD86、CD80、CD40、MHC-II表达均明显下降(P<0.05),而CD205、Jagged1、Jag-ged2表达均明显上升(P<0.05);细胞周期中(G2+S)期细胞增加。结论:MSC可诱导DC细胞向DCregs转化,该MSC-DCregs具有致免疫耐受性表型,增殖能力提高;MSC诱导免疫耐受的机制可能与上调DC的Jagged1和Jagged2基因表达,激活T细胞Notch信号通路相关。     

体外定向诱导小鼠胚胎干细胞发育为造血干/祖细胞方法的初步研究

目的:探讨体外定向诱导胚胎干细胞(ESC)发育为造血干细胞(HSC)的方法。方法:将小鼠E14胚胎干细胞在含干细胞生长因子(SCF)和血管内皮生长因子(VEGF)的甲基纤维素培养基中首先诱导发育为胚胎体(EB),再将EB置于均含SCF、VEGF、IL-3、IL-6及促红细胞生成素(EPO)的3种不同培养体系中定向分化为HSC,并观察HSC表面标志性抗原、造血集落形成及瑞氏-姬姆萨染色的结果。结果:经两阶段诱导ESC分化为HSC,发现在甲基纤维素半固体培养体系中HSC发育缓慢,分化14d后CD34+/Sca-1+细胞数最高为(31.5±4.7)%;而在骨髓基质细胞饲养层上HSC发育较快,细胞数量较多,分化第10dCD34+/Sca-1+细胞数即达到峰值,为(47.8±6.3)%;骨髓基质细胞饲养层+胎肝基质细胞上清培养体系中HSC发育同样迅速,所产生的CD34+/Sca-1+细胞数量在3个体系中最高,为(53.6±7.2)%。经瑞氏-姬姆萨染色证实上述细胞为早期造血细胞,均有形成各系造血细胞集落的能力。结论:使用骨髓基质细胞饲养层+胎肝基质细胞上清培养体系及SCF、VEGF、IL-3、IL-6及EPO等细胞因子,通过两阶段诱导分化,可从小鼠ESC获得较高比例的HSC。     

Jagged-1抑制小鼠胚胎干细胞分化为造血干/祖细胞

目的探讨Notch信号通路在小鼠胚胎干细胞(ESC)分化为造血干/祖细胞(HSC/HPC)中的作用。方法 1)体外培养小鼠拟胚体细胞(EBs),使用Jagged-1蛋白活化Notch信号通路及DAPT(γ-分泌酶抑制剂)抑制Notch信号通路。实验分为拟胚体细胞复苏组(EB组)、对照组、Jagged-1组、DAPT组和Jagged-1-DAPT组。2)流式细胞计量术检测ESC特异性表型和HSC/HPC特异性表型的表达。3)实时定量PCR检测各组Notch信号通路基因、小鼠ESC表型基因和HSC/HPC表型基因的表达。结果 1) Jagged-1组细胞胚胎干细胞数较对照组和Jagged-1-DAPT组明显增多(P0.05), 2)Jagged-1-DAPT组分化的HSC/HPC数较control组及Jagged-1组明显增多(P0.05);3)Jagged-1组Notch1、Notch2、Notch4 mRNA表达量较对照组明显升高(P0.05);4)DAPT组和Jagged-1-DAPT组中Notch1、Notch4 mRNA表达量较对照组降低(P0.05)。结论 Jagged-1激活Notch信号通路可抑制小鼠ESC向HSC/HPC的分化。     

银屑病患者骨髓CD34+细胞Notch受体的表达

Notch信号传导通路广泛参与了包括造血干细胞在内的诸多干细胞的发育分化过程,对干细胞状态的维持和终末分化的方向等起着决定性的作用.在造血细胞的发育过程中,Notch受体1和2不同程度地表达以及被不同的配体活化决定了造血十细胞向淋巴细胞系或髓细胞系分化.为进一步研究Notch信号通路是否参与了银屑病的发病过程,本研究采用半定量RT-PCR法检测了24例寻常型银屑病患者和18例正常对照骨髓CD34+细胞两种Notch受体mBNA的表达情况.     

Delta 1与Jagged 1在诱导胸腺细胞分化中的不同作用（文献综述）

Delta 1与Jagged 1是脊椎动物Notch的两个配体，它们与相同或不同的Notch受体结合激活Notch信号通路，决定细胞分化的命运，参与调控许多组织的生长发育。单独敲除Delta 1或Jagged 1小鼠呈现不同的表型，提示这两个配体具有非重叠作用。Delta 1与Jagged 1在造血细胞、骨髓间质细胞、淋巴细胞、抗原提呈细胞（antigen presenting cells，APC）等表面均有表达，诱导淋巴细胞的分化。近来发现，Delta 1诱导胸腺细胞其向T细胞分化，而Jagged 1则诱导胸腺细胞向NK细胞分化。     

A B220+ CD117+ CD19- hematopoietic progenitor with potent lymphoid and myeloid developmental potential

In this report, we identify in the bone marrow (BM) of normal mice a subpopulation of B220+ CD117+ CD19- NK1.1- cells with potent lymphoid and myeloid developmental potential. These cells represent 0.1-0.2% of nucleated BM cells. By limiting dilution analysis in the presence of the appropriate combination of stromal cells and cytokines, 1 in 5-10 sorted cells formed B cells, 1 in 10-15 formed T cells and 1 in 5-10 generated macrophages. When cultured on a mixture of OP9 stroma and OP9 stromal cells expressing the Notch ligand Delta-like-1, single cells generated both T and B cells. Following intravenous infusion, freshly sorted cells transiently reconstituted both the T and B cell progenitor compartments, generating cohorts of mature T and B lymphocytes. The relationship between B220+ CD117+ CD19- NK1.1- cells of wild-type mice and other multi-lineage BM progenitors is discussed.     

Transient Notch signaling induces NK cell potential in Pax5-deficient pro-B cells

Unlike early B/T cell development, NK cell lineage commitment is not well understood, with a major limitation being the lack of a robust culture system to assay NK cell progenitors. Here we have exploited the multi-lineage potential of Pax5(-/-) pro-B cells to establish an effective system to direct differentiation of progenitors into the NK cell lineage. Cultivation of Pax5(-/-) pro-B cells on OP9 cells expressing the Notch ligand Delta-Like1 (OP9-DL1) in the presence of IL-7 efficiently induced T and NK cell potential. For NK cells, Notch was only transiently required, as prolonged signaling decreased NK and increased T cell development. Pure NK cell populations could be obtained by the culture of these Notch signal-experienced cells onto OP9 stroma and IL-15. A similar transient exposure to Notch was also compatible with the differentiation of NK cells from hematopoietic progenitors, while sustained Notch signaling impaired NK cell generation. Pax5(-/-) pro-B cell-derived NK cells were cytotoxic, secreted cytokines and expressed all the expected NK cell-specific surface markers examined except the Ly49 family, a phenotype similar to fetal NK cells. These data indicate that Notch signaling induces T/NK cell differentiation in Pax5(-/-) pro-B cells that is strikingly similar to early thymopoiesis.     

Both Notch1 and its ligands in B cells promote antibody production

Notch1 signaling regulates B and T lymphocyte development and also in vitro promotes antibody secretion upon B cell activation. However, it is still unclear about the role of Notch1 in antibody production upon in vitro and in vivo mixture lymphocytes activation. We first showed that Notch1 expressed in LPS-activated CD19^hi B cells and CD19^cre mediated Notch1 knock-down in LPS-activated B cells. Furthermore, we demonstrated that Notch1 knock-down in B cells reduced antibody production in LPS-stimulated B cells but did not affect antibody production in LPS-stimulated splenocytes and in experimental allergic encephalomyelitis (EAE) mice. Importantly, Notch1 ligands Dll1 and Jag1 expressed in B cells and pre-coated Notch1 protein promotes Notch1-knocked down B cells to produce antibody in LPS-stimulated B cells suggesting that Notch1 in other cells may promote antibody production by binding its ligands Dll1 and Jag1 in B cells. Together, our results suggest that both Notch1 and its ligands in B cells play an important role in antibody production.     

In vitro differentiation of adult bone marrow progenitors into antigen-specific CD4 helper T cells using engineered stromal cells expressing a notch ligand and a major histocompatibility complex class II protein

A murine stromal cell line (OP9-DL1) expressing a notch ligand, Delta-like-1, has been shown to be able to drive the differentiation of both murine and human hematopoietic progenitors into T cells in vitro. Further studies showed that hematopoietic progenitors transduced by a retroviral vector to express a human CD8 T-cell receptor (TCR) followed by an OP9-DL1 monolayer coculture could generate antigen-specific cytotoxic T lymphocytes in vitro. It remains unknown if a similar method could be applied to produce CD4 helper T cells. In this report, we show that murine adult bone marrow (BM) cells transduced with an OT2 CD4 TCR and cocultured with OP9 stromal cells expressing Delta-like-1 can differentiate into antigen-specific CD4 T cells in vitro. These cells are capable of inducing the expression of T-cell activation markers and producing cytokines upon stimulation. We have also constructed a new stromal cell line (OP9-DL1-IA(b)) ectopically expressing a murine major histocompatibility complex class II protein, I-A(b), in OP9-DL1 cells. This new line could accelerate the development of TCR-transduced BM cells into CD4 T cells, resulting in cells with an improved capacity to respond to T-cell stimulation to secrete cytokines. Taken together, we demonstrate a general and potentially useful method to generate autologous antigen-specific CD4 helper T cells in vitro from easily accessible BM cells.     

Constitutive Notch signalling promotes CD4 CD8 thymocyte differentiation in the absence of the pre-TCR complex, by mimicking pre-TCR signals

Notch1 signalling is essential for the commitment of multipotent lymphocyte precursors towards the alphabeta T-cell lineage and plays an important role in regulating beta-selection in CD4(-)CD8(-) double-negative (DN) thymocytes. However, the role played by Notch in promoting the development of CD4(+)CD8(+) double-positive (DP) thymocytes is poorly characterized. Here, we demonstrate that the introduction of a constitutively active Notch1 (ICN1) construct into RAG(-/-) lymphocyte precursors resulted in the generation of DP thymocytes in in vitro T-cell culture systems. Notably, developmental rescue was dependent not only on the presence of an intact Notch1 RAM domain but also on Delta-like signals, as ICN1-induced DP development in RAG(-/-) thymocytes occurred within an intact thymus or in OP9-DL1 co-cultures, but not in OP9-control co-cultures. Interestingly, ICN1 expression in SLP-76(-/-) precursors resulted in only a minimal developmental rescue to the immature CD8(+) single-positive stage, suggesting that Notch is utilizing the same signalling pathway as the pre-TCR complex. In support of this, ICN1 introduction resulted in the activation of the ERK-MAPK-signalling cascade in RAG(-/-) thymocytes. Taken together, these studies demonstrate that constitutive Notch signalling can bypass beta-selection during early T-cell development by inducing pre-TCR-like signals within a T-cell-promoting environment.