NOTCH配体Jagged2和Delta4对32D细胞的分化抑制研究

目的探讨Notch信号传导系统中相关配体Jagged2和Delta4对髓系前体细胞32D的分化抑制作用.方法构建Delta4高表达载体pTracer.CMV.Delta4.FLAG,制备Delta4-CHO细胞;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. 结果构建成功Delta4高表达载体pTracer.CMV.Delta4.FLAG并稳定转染CHO细胞.在G-CSF存在下,Notch1-32D细胞可分化为成熟的粒细胞; Jagged2能抑制G-CSF引起的Notch1-32D细胞分化,但Delta4不能抑制G-CSF引起的Notch1-32D 细胞分化.结论分化抑制实验发现NOTCH 配体中,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,为进一步揭示Notch信号传导系统的作用机制及其对造血系统的影响提供重要理论依据.     

NOTCH配体Jagged2和Delta4对32D细胞的分化抑制研究

目的：探讨Notch信号传导系统中相关配体Jagged2和Delta4对髓系前体细胞32D的分化抑制作用．方法构建Delta4高表达载体pTracer.CMV.Delta4.FLAG，制备Delta4-CHO细胞；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：构建成功Delta4高表达载体pTracer．CMV．Delta4．FLAG并稳定转染CHO细胞．在G-CSF存在下，Notch1-32D细胞可分化为成熟的粒细胞；Jagged2能抑制G-CSF引起的Notch1-32D细胞分化，但Delta4不能抑制G-CSF引起的Notch1-32D细胞分化．结论：分化抑制实验发现NOTCH配体中，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，为进一步揭示Notch信号传导系统的作用机制及其对造血系统的影响提供重要理论依据．     

Delta1与Jagged1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。     

Delta 1与Jagged 1在诱导T细胞分化中的不同作用

Delta 1（又称delta—like 1或DⅡ 1）与Jagged 1（又称Serrate 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Delta1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T细胞、抗原提呈细胞（antigen presenting cells，APC）如树突状细胞、B细胞和巨噬细胞等表面均有表达，在诱导T细胞分化中起着重要作用。近来发现，Delta 1可以诱导外周T细胞向1型辅助性T细胞（helper T cell 1，TH 1）分化，而Jagged 1诱导其向TH 2分化；     

大鼠Hes1基因真核表达载体的构建及瞬时表达在神经前体细胞分化中的作用

目的 探讨Hes1基因高表达对神经前体细胞分化的影响. 方法从大鼠脑组织中提取总BNA,用RT-PCR方法获得Hes1基因的全长cDNA,插入pGEM-T-Easy克隆载体中进行序列测定,测序正确后将其亚克隆至表达载体pCDNA3.1,用脂质体将重组质粒转染原代培养的神经前体细胞,经G418筛选获得抗性细胞克隆,用RT-PCR方法鉴定Hes1基因在神经前体细胞基因组中的存在,实时荧光定量PCR检测Hes1基因在转染细胞中的过表达情况. 结果 经限制性内切酶酶切图谱分析和DNA序列测定,证实目的 基因已插入重组质粒,RT-PCR证明,经G418筛选得到的转基因神经前体细胞克隆的基因组DNA中存在Hes1基因;实时荧光定量PCR进一步证明,转基因神经前体细胞Hes1基因的mRNA高表达. 结论 构建了大鼠Hes1基因的真核表达载体,获得了高表达Hes1基因的神经前体细胞克隆.Hes1基因高表达可抑制神经前体细胞Ngn1的表达,并促进神经前体细胞向神经胶质细胞的分化.     

Notch配体在诱导淋巴细胞分化中的不同作用

脊椎动物Notch配体为一组单次跨膜蛋白,包括Delta1, Delta3, Delta4, Jagged1 和 Jagged2,它们与Notch受体结合激活Notch信号通路,决定细胞分化的命运.这些Notch配体具有不同的生物学特性,在诱导造血干/祖细胞和胸腺细胞向T、B和NK细胞分化中起着不同作用,可能为免疫性疾病的治疗提供新的药物靶点.     

类风湿性关节炎患者外周血单个核细胞MALAT1表达降低、 hsa-miR155-3p表达增加并参与Notch信号通路调节

目的观察类风湿性关节炎(RA)患者长链非编码RNA(lncRNA)转移相关肺腺癌转录本1(MALAT1)、 hsa-miR155-3p表达及其与Notch信号通路关系,探讨RA可能发病机制。方法采取RA患者(RA组)、健康对照者(NC组)外周血,采用高通量基因测序筛选差异表达的lncRNA和mRNA。利用反转录PCR检测筛选的lncRNA MALAT1和hsa-miR155-3p及Notch信号通路受体配体表达。结果通过lncRNA测序分析发现,共有9158条差异表达的lncRNA。基因本体(GO)功能分类注释得出差异表达的mRNA参与免疫炎症反应、细胞转录调节等。通过通路(pathway)分析显示差异表达的mRNA与免疫炎症、应激、 Notch通路等功能基因变化有关。经顺式(Cis)和反式(Trans)预测, Jagged1/2、 Delta1/2、 Notch1/2可能与RA发生密切相关。与NC组相比, RA组MALAT1降低, hsa-miR155-3p明显升高; Notch通路配体Delta1、 Delta2、 Jagged1、 Jagged2及受体Notch1、 Notch2表达升高。相关性分析显示, hsa-miR155-3p与MALAT1呈负相关, hsa-miR155-3p与Notch通路Delta1、 Jagged1呈正相关, MALAT1与Jagged2呈负相关。结论 RA患者lncRNA MALAT1降低、 hsa-miR155-3p增加,共同调节Notch信号通路变化。     

Jagged1(CD339)诱导调节性T细胞分化、成熟及介导免疫耐受

Jagged1(新近被命名为CD339[1])是存在于哺乳动物细胞膜上Notch受体的主要配体之一,为单次跨膜糖蛋白,表达于骨髓、胎儿肝基质细胞和胸腺上皮细胞等多种组织,参与调控许多组织的生长发育,在维持正常造血前体细胞及其增殖过程中起着重要作用[2]。Jagged1在抗原提呈细胞(antigenp     

N-乙酰半胱氨酸通过海马区Notch1/hes1通路改善丙戊酸孤独症模型鼠的学习和记忆能力

目的探讨N-乙酰半胱氨酸(N-acetylcysteine,NAC)能否改善孤独症模型鼠的学习和记忆能力及其作用机制。方法利用丙戊酸(valproic acid,VPA)建立孤独症模型,通过三箱实验鉴定造模成功,Western blotting技术检测模型鼠海马区Notch1/hes1通路信号分子Notch1、Notch1胞内活性结构域(Notch intracellular domain,NICD)、Hes1、Jagged1变化,免疫荧光技术检测该通路信号下游靶基因Hes1的表达;然后用NAC干预,检测模型鼠学习和记忆能力的变化及Notch1/hes1信号通路关键信号分子的改变。结果与对照组相比,VPA组大鼠找到平台的时间增加,跨越平台的次数及在靶象限的时间均减少,同时Notch1、NICD、Hes1、Jagged1的蛋白表达水平增加;而NAC干预以后,与VPA组相比,VPA+NAC组大鼠找到平台的时间减少,跨越平台的次数及在靶象限的时间均增加,同时海马脑区NICD、Hes1的蛋白表达均下降。结论 NAC可以改善VPA孤独症模型鼠学习和记忆能力,其作用机制可能与降低Notch1/hes1信号通路的过度活化有关。     

类风湿关节炎患者外周血单个核细胞Notch及其配体表达

为研究Notch信号途径在类风湿关节炎(RA)发病机制中的作用,选取活动期RA患者,采用流式细胞术结合细胞表面及细胞内染色技术检测外周血单个核细胞Notch受体及配体表达情况,并与正常对照进行比较。结果发现,活动期RA患者外周血T细胞Notch2、Notch3及Notch4表达较正常人明显增加,Notch1分子表达均较少,二者未见差异。其中表达Notch分子的细胞以CD4+T细胞为主。二者B细胞Notch1、Notch2、Notch3及Notch4的表达均较低,之间未见明显差异。RA患者单核细胞Jagged1分子表达明显增加,而Delta1表达降低。结果提示活动期RA患者外周血单个核细胞中不同群体细胞存在Notch及其配体表达水平的改变。     

体外肿瘤细胞对血管内皮细胞影响的观察

目的 观察肿瘤细胞与血管内皮细胞的关系及其在体外直接接触培养时内皮细胞的形态变化。方法 免疫磁珠阳性选择法分离培养大鼠肺血管内皮细胞,生长至汇合状态时加入不同的肿瘤细胞(小鼠树突状细胞肉瘤细胞DCS、人胃癌细胞MGC-803、小鼠肺腺癌细胞LA795)共培养,光镜、扫描电镜、免疫组织化学、transwell法及荧光分子传递等方法观察内皮细胞的形态、功能的变化。结果培养的内皮细胞部分呈卵石样,部分呈长梭形。长至汇合状态的内皮细胞再加入肿瘤细胞后,内皮细胞形态发生明显变化,汇合细胞中间出现许多圆形管腔样结构;肿瘤细胞往往分布在新形成的腔隙内;肿瘤细胞条件培养基可支持内皮细胞的生长,促进内皮细胞的运动;luciffer yellow分子可直接从肿瘤细胞传递到内皮细胞。结论 肿瘤细胞与内皮细胞可进行通讯,肿瘤细胞可直接引起内皮细胞结构和功能表型的改变。     

支气管扩张症神经内分泌和免疫活性细胞的变化

应用免疫组化方法对３５例支气管扩张症和１０例正常肺组织中肺内分泌细胞和免疫活性细胞进行观测，并对人的支气管相关淋巴组织（ＢＡＬＴ）进行形态学和免疫组化观察。结果显示：支气管扩张症中，支气管上皮降钙素和五羟色胺阳性细胞显著增多，支气管周围ＩｇＧ、ＩｇＡ和ＩｇＭ阳性细胞以及ＵＣＨＬ，阳性Ｔ细胞显著增多，ＢＡＬＴ明显增生，且ＢＡＬＴ增生的区域肺内分泌细胞和免疫活性细胞增多尤为显著，提示支气管扩张症发病中有神经内分泌和免疫系统参与。     

T cells,dendritic cells and epithelial cells in intestinal homeostasis

The mucosal immune system of the intestinal tract is continuously exposed to both potential pathogens and beneficial commensal microorganism. A variety of mechanisms contribute to the ability of the gut to either react or remain tolerant to antigen present in the intestinal lumen. Antigens of the gut commensals are not simply ignored, but rather trigger an active immunosuppressive process, which prevents the outcome of immunopathology. The aim of this review is to provide an update on the mechanism of intestinal homeostasis, with particular focus on the complex crosstalk between T cells, dendritic cells and intestinal epithelial cells.     

Mast cells detected in cultures of neonatal rat heart cells

Although the presence of mast cells in heart muscle is well documented, their possible presence in heart cell cultures has not yet been considered. In this paper we show for the first time that cultures from neonatal rat hearts contain mast cells in proportions which may exert physiological influences on heart muscle cells. Evidence is based on cytochemistry, immunocytochemistry with a monoclonal antibody (F2) specific for connective tissue mast cells of the rat, and direct estimations of both cellular histamine and histamine released into the culture medium. Since the number of nonmuscle cells immunoreactive with antibody F2 exceeded the number of cells reacting with conventional cytochemical stains for mast cells, a substantial proportion of the former may represent less differentiated (precursor) cells.     

Mast cells detected in cultures of neonatal rat heart cells

Although the presence of mast cells in heart muscle is well documented, their possible presence in heart cell cultures has not yet been considered. In this paper we show for the first time that cultures from neonatal rat hearts contain mast cells in proportions which may exert physiological influences on heart muscle cells. Evidence is based on cytochemistry, immunocytochemistry with a monoclonal antibody (F2) specific for connective tissue mast cells of the rat, and direct estimations of both cellular histamine and histamine released into the culture medium. Since the number of nonmuscle cells immunoreactive with antibody F2 exceeded the number of cells reacting with conventional cytochemical stains for mast cells, a substantial proportion of the former may represent less differentiated (precursor) cells.     

Role of T cells and dendritic cells in glomerular immunopathology

Inappropriate T cell responses cause the four classical types of hypersensitivity immune reactions. All of these can target the kidney and cause distinct forms of glomerulonephritis. CD4(+) T cells can mediate glomerular immunopathology by cytokine secretion, by activating effector cells such as macrophages or by inducing auto-antibodies or immune-complexes. Cytotoxic CD8(+) T cell responses and failure of regulatory T cells may represent two additional types of anti-renal hypersensitivity. T cell activation is critically dependent on dendritic cells (DC), whose role in renal disease appears to be protective, but underlying mechanisms are largely unknown. In this paper, we summarized mechanistic information from rodent models on the roles of DC and T cells in glomerular immunopathology.     

Suppression of T cells by myeloid-derived suppressor cells in cancer

Myeloid-derived suppressor cells (MDSCs) are a population of immature myeloid cells defined by their immunosuppression. Elevated levels of certain soluble cytokines in tumor microenvironment, such as IL-6 and IL-10, contribute to the recruitment and accumulation of tumor-associated MDSCs. In turn, MDSCs secret IL-6 and IL-10 and form a positive self-feedback to promote self-expansion. MDSCs also release other soluble cytokines such as TGF-β and chemokines to exert their suppressive function by induction of regulatory T cells. Exhaustion of some amino acids by MDSCs with many secretory enzymes or membrane transporters as well as their metabolites leads to blockage of T cells development. The interaction of membrane molecules on MDSCs and T cells leads inactivation and apoptosis of T cells. There may be one or some dominant mechanism(s) by which MDSCs impair the immune system in different tumor microenvironment. Thus, it is important to identify the subpopulations of MDSCs and clarify the dominant mechanism(s) through which MDSCs inhibit antitumor immunity in order to establish a more individual immunotherapy by eliminating MDSCs-mediated suppression. Currently studies concentrated on therapeutic strategies targeting MDSCs have obtained promising results. However, more studies are needed to demonstrate their clinical safety and efficacy.     

Roles of lymphoid cells in the differentiation of Langerhans dendritic cells in mice

Langerhans dendritic cells are antigen presenting cells (APC) that reside within the epidermis and are capable of stimulating naive T cells. Reciprocally, lymphocytes may play a role in Langerhans cells (LC) differentiation. Our results show that the differentiation of skin LC is unaffected in the absence of lymphocytes and/or signaling through the common cytokine receptor gamma chain (gammac) required for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 signaling. Migration of LC and other dendritic cells (DC) from the skin to the draining lymph nodes (LNs) after FITC skin sensitization, is unaffected in the absence of lymphocytes or CD40. FITC+ LC/DC sorted from the LNs of lymphoid deficient or control mice stimulated naive T cells with similar efficiency. However, while the absence of lymphocytes did not appear to affect the phenotype or number of emigrating LN DC/LC, their persistence in the LN appears to depend on alphabeta T cells. Thus, DC are strikingly reduced in numbers in the peripheral LNs of T-cell deficient mice. Finally, CD8alpha expression on skin emigrants was low and dependent on the presence of CD8+ lymphocytes, while spleen CD8+ DC were present in the absence of lymphocytes. We conclude that the presence of T cells is not required for the differentiation and migration of resident skin DC but is critical for the maintenance of DC and LC migrating into the LNs.