雌激素受体ERα与PI3K/Akt细胞信号通路

雌激素直接与核内雌激素受体ERα或ERβ结合,活化靶基因的转录,这是经典的雌激素受体信号转导途径。近年来大量实验证实,雌激素受体ERα能够通过雌激素依赖或不依赖的方式激活,并与细胞质内的磷脂酰肌醇3-激酶-蛋白质丝氨酸苏氨酸激酶(PI3K/Akt)细胞信号传导通路相互作用,发挥重要的生物学效应,如参与人类雌激素相关肿瘤的发病、调节靶细胞的增殖和分化。此外,实验研究还提示,ERα和PI3K/Akt信号通路在哺乳动物植入前胚的发育过程中也起着重要调节作用。     

靶向ER-α36的vshRNA真核表达慢病毒载体的构建、鉴定及其对胃癌细胞增殖的影响

目的:针对雌激素受体(ER)-α36基因的特异性靶序列,构建并鉴定靶向ER-α36基因RNAi慢病毒载体,研究ER-α36沉默后对胃癌细胞增殖的影响。方法:筛选确定的ER-α36基因RNAi有效靶序列,合成靶序列的Oligo DNA,与慢病毒载体(GV307)连接,测序鉴定。转染293T细胞,包装产生慢病毒,感染胃癌SGC7901细胞株。荧光显微镜下观察SGC7901感染后荧光表达情况,real-time PCR和Western blotting方法检测ER-α36的表达变化。用1×10-10mol/L的17β-雌二醇处理沉默ER-α36的SGC7901细胞,用细胞计数法观察细胞增殖能力的变化及检测相关下游信号通路分子Src、ERK1/2、cyclin D1表达的变化。结果:阳性克隆PCR及测序证明成功构建慢病毒载体LV-ER-α36-RNAi,倒置显微镜下观察LV-ER-α36-RNAi慢病毒载体感染率达80%以上。Real-time PCR和Western blotting方法证实四环素(Te T)诱导下LV-ER-α36-RNAi明显抑制SGC7901细胞内ER-α36 mRNA和蛋白质的表达。与对照组相比,沉默ER-α36的SGC7901细胞增殖能力减弱,Src、ERK1/2、cyclin D1蛋白表达明显降低,Src蛋白活化能力减弱(P0.05)。结论:我们构建的Te T诱导靶向ER-α36的vshRNA慢病毒载体LVER-α36-RNAi,可明显沉默ER-α36的表达,为研究ER-α36蛋白的作用机理提供实验依据,ER-α36与胃癌细胞等肿瘤细胞的增殖有关。     

雌激素及其受体与自身免疫性疾病

自身免疫性疾病有明显的性别差异，女性的发病率显著高于男性，表明雌激素影响这类疾病的发生。雌激素及其代谢产物水平、雌激素受体基因多态性、雌激素受体介导的信号通路等方面与自身免疫性疾病密切相关，雌激素及其受体在自身免疫性疾病治疗中发挥重要作用。     

小鼠室旁核内雌激素受体与催产素、加压素表达的免疫组化研究

目的研究雌激素的核受体ER-α和ER-β以及催产素、加压素在成年雌性小鼠下丘脑室旁核内的表达。方法采用硫酸镍铵增强显色的免疫组化SP法检测ER-α、ER-β、催产素和加压素在室旁核内的表达。结果雌激素的两种核受体在室旁核内都有表达,但是以ER-β为主（P〈0.001）,其免疫阳性产物均在细胞核内,未见核外免疫阳性反应。催产素的免疫阳性产物主要在细胞核周围的胞浆即核周质内,而加压素的免疫阳性物质除了在核周质内有很强的反应外,在突起内也可见很强的免疫反应。ER-α的免疫阳性胞体主要在大细胞部内侧,而ER-β、催产素和加压素的免疫阳性胞体主要在背侧帽部或大细胞部的外侧。结论室旁核内两种雌激素受体可能都参与了对催产素和加压素的调节,但ER-β可能发挥了主要的调节作用。     

阿可拉定对子宫内膜癌HEC-1B细胞雌激素受体表达的影响

目的研究阿可拉定对人子宫内膜癌细胞株HEC-1B雌激素受体的影响及作用机制。方法体外培养HEC-1B细胞,采用四甲基偶氮唑蓝法(MTT)、逆转录聚合酶链反应法(RT-PCR),观察阿可拉定对HEC-1B细胞增殖作用以及雌激素受体ER-α66和新亚型ER-α36基因表达的影响。结果阿可拉定对HEC-1B细胞增殖具有明显的抑制作用,且抑制作用随药物浓度和给药时间的增加逐渐增强,72h阿可拉定的IC50值为5.26μmol/L。在抑制雌激素受体表达方面,阿可拉定组及阿可拉定联合顺铂组与空白组比较均可明显降低ER-α36的表达(P0.05,P0.01);阿可拉定联合顺铂组还可明显降低ER-α66的表达(P0.01)。结论阿可拉定抑制子宫内膜癌细胞增殖的作用机制可能与其抑制细胞雌激素受体新亚型ER-α36基因表达有关。     

miR-143靶向调控ER-α36影响胃癌细胞系SGC7901的侵袭

目的探讨miR-143通过靶向雌激素受体ER-α36介导人胃癌细胞系SGC7901的侵袭。方法通过慢病毒载体的构建及包装生成上调/下调miR-143的慢病毒LV-miR-143 up和LV-miR-143 down并感染人胃癌细胞系SGC7901,用Western blot检测ER-α36的蛋白表达水平和细胞的侵袭能力;生物信息学软件预测miR-143的靶基因;荧光素酶报告基因实验验证靶基因。结果 LV-miR-143 up与LV-miR-143 down慢病毒病毒颗粒分别感染人胃癌细胞系SGC7901,感染效率均在80%以上;上调miR-143,人胃癌细胞系SGC7901的ER-α36表达水平明显下降,侵袭能力明显降低(P<0.05),反之则增加。结论 miR-143通过靶向雌激素受体ER-α36负向调控胃癌细胞SGC7901的侵袭。     

雌激素受体基因敲除小鼠脾脏免疫学改变的形态学观察

目的 :形态学观察雌激素受体基因敲除小鼠脾脏免疫学指标是否发生改变。方法 :应用免疫组织化学和免疫荧光法进行检测。结果 :雌激素受体基因敲除小鼠 ,尤其是ERβ敲除小鼠 (BERKO)的脾脏出现多种异常 ,如促炎症细胞因子和诱导型一氧化氮增高 ,脾细胞增殖增强并能抵抗雌激素的抑制作用 ,IgM /IgG表达也明显增高。雌激素受体缺乏小鼠脾脏NF kB的激活可能是脾细胞过分活跃的原因。结论 :雌激素受体 ,尤其是ERβ在介导雌激素对免疫反应的调节中起重要作用 ,ERβ缺失可能增加机体对自身免疫性疾病的敏感性     

雌激素受体亚型与妇科肿瘤的研究进展

雌激素受体广泛分布于妇女生殖系统,是一类由配体激活的核转录因子,介导大部分的雌激素作用.本文综述雌激素受体亚型(ER-α和ER-β)的结构和功能、雌激素受体配体调节基因转录的机制、分布及生理作用等.主要介绍了在妇科肿瘤中二者表达及比例变化与组织细胞的功能和病理变化的关系.     

ER、PR及其相应拮抗剂与甲状腺癌的相关性研究进展

雌激素受体(ER)、孕激素受体(PR)在甲状腺癌中的异常表达说明性激素受体在甲状腺癌的发病机制中起重要作用.ER、PR通过经典的基因途径及非基因途径对甲状腺癌的发生、发展及生物学特征产生重要影响.性激素受体拮抗剂抑制甲状腺癌细胞增殖作用的研究,将为甲状腺癌的治疗提供新的选择方案.     

雌激素受体(ESR)基因多态性与疾病相关性的研究进展

雌激素受体是一种受配体激活的转录因子,由配体结合区、DNA结合区、转录激活区组成.雌激素受体对于对雌激素敏感的组织是一个重要的调节元件,如子宫内膜、乳腺、骨组织、肝脏等.雌激素的功能是刺激组织的增殖、分化,因此受体功能的变化可能有重要的临床意义.雌激素受体基因的多态性,单倍构型与它的生物学功能是相关的,研究认为ESR基因多态性与乳腺癌、骨质疏松症、HBV感染、子宫内膜异位症、冠心病等疾病存在相关性.该研究从雌激素受体的结构、功能与疾病的相关性方面作一综述.     

Direct Demonstration of Immunoregulatory T-Cell Defects in Patients with Systemic Lupus Erythematosus

The present study was undertaken to determine directly whether immunoregulatory T cells have a defective suppressor function in patients with systemic lupus erythematosus (SLE), and whether anti-T-cell antibodies are essential for immunoregulatory T-cell defects. Peripheral blood T cells and T-cell subsets were determined in 52 SLE patients. The ratio of T4 to T8 cells was distributed over a wider range in patients with SLE than in the controls. Patients with SLE were divided into three groups (low, normal and high) by the T4/T8 ratio. Lymphocytes from 12 SLE patients (7 with low and 5 with high T4/T8 ratios) were studied extensively. Their disease was inactive or in remission. Anti-T-cell antibodies were not detected, and yet the patients had immunological abnormalities characterized by the presence of antinuclear antibodies and hypergammaglobulinaemia. The SLE patients with high T4/T8 ratios had a decreased number of T8 cells, and defective suppressor-effector cells. In contrast, patients with low T4/T8 ratios had decreased T4 cells and/or increased T8 cells, and defective suppressor-inducer cells. Two patients with low T4/T8 ratios had both suppressor-effector and suppressor-inducer cell defects. These results indicate that immunoregulatory circuits in SLE patients are heterogeneous and that immunoregulatory defects exist even when the disease is inactive or in remission. Anti-T-cell antibodies were not essential for such immunoregulatory defects. Thus, immunoregulatory T-cell defects and the development of SLE may be independent conditions due to other unknown causes.     

Anti-DNA idiotype- and anti-idiotype-specific T cell responses in patients with systemic lupus erythematosus and their first-degree relatives.

We examined the proliferative responses of T cells of patients with systemic lupus erythematosus (SLE), their first-degree relatives, and healthy donors, to a human monoclonal antibody that bears a common anti-DNA idiotype, 16/6 Id, and to a murine, 16/6 Id-specific, monoclonal antibody. Both 16/6 Id+ and 16/6 Id-specific antibodies were previously shown to be involved in the induction of experimental SLE in mice. Here we show that T cells of fewer SLE patients, as compared with healthy donors, could proliferate to both antibodies. The difference between T cell responses of patients and controls to the 16/6 was found to be significant. The proliferative responses of T cells of first degree relatives of SLE patients to the anti-16/6 Id were found to be significantly lower compared with the responses detected in healthy donors and in SLE patients. The responses of T cells of SLE relatives to the 16/6 Id were found to be lower than those of healthy donors, but this difference was not significant. The present study suggests a possible involvement of T cells, and specifically of idiotype and anti-idiotype specific T cells, in SLE.     

Defects of Autologous Mixed Lymphocyte Reaction-Activated Immunoregulatory T Cells in Patients with Systemic Lupus Erythematosus

The present study was undertaken to determine the nature of the immunoregulatory T-cell defect after autologous mixed lymphocyte reaction (AMLR) activation in patients with systemic lupus erythematosus (SLE). Although AMLR was decreased in patients with SLE compared with normals, there was no difference in major proliferative cells (T4 cells and T4+JRA+ subset) in response to AMLR. Functional activity of AMLR-stimulated T4 subsets in patients with SLE and normals was examined in helper and suppressor/inducer assay, using pokeweed mitogen (PWM)-driven IgG synthesis. The T4+JRA- (helper) subset from SLE patients showed no greater activity than normals. However the T4+JRA+ (suppressor/inducer) subset from SLE patients showed decreased suppression induction compared with normals. This defect in the suppressor/inducer function was demonstrated even in patients with inactive SLR or in remission.     

转录因子FOXP3基因多态性与广西壮族系统性红斑狼疮的遗传易感性

目的 探讨转录因子FOXP3基因单核苷酸多态性与广西壮族系统性红斑狼疮(systematic lupus erythematosus,SLE)易感性之间的关系.方法 以120例SLE患者和160名健康对照者为研究对象,应用聚合酶链反应-限制性片段长度多态性和DNA测序的方法对FOXP3基因-2383 C/T、-3281 C/A单核苷酸多态性进行基因分型.结果 FOXP3基因-3281 C/A多态性在SLE组和正常人群中的分布差异无统计学意义(P＞0.05),而FOXP3基因-2383 C/T多态性在两组人群中的分布差异有统计学意义(P＜0.05),等位基因频率的相对风险分析发现,-2383 T等位基因携带者患系统性红斑狼疮的风险是-2383 C等位基因的1.715倍(OR=1.715,95%CI:1.165～2.525).联合基因型分析发现,FOXP3的-2383 T/-3281 A等位基因频率在SLE组中显著高于对照组(P＜0.05),-2383 T/-3281 A等位基因携带者显著增加了SLE的发病风险(OR=2.196,95%CI:1.165～4.142).结论 FOXP3基因-2383 C/T多态性与SLE的发病具有相关性,其中-2383 T等位基因可能是SLE的遗传易感基因.     

Quantitative and qualitative analysis of the balance between type 1 and type 2 cytokine-producing CD8(-) and CD8(+) T cells in systemic lupus erythematosus

The production of type 1 (IFN-gamma, IL-2) and type 2 (IL-4, IL-5, IL-10, IL-13) cytokines by CD8(-) and CD8(+) T cells from systemic lupus erythematosus (SLE) patients and normal subjects was investigated using an intracellular cytokine-staining technique. This flow cytometric method facilitates analysis of both surface markers and cytoplasmic cytokines, after a short term (6 h) culture with or without phorbol myristate acetate and ionomycin (PMA/I) stimulation. In SLE patients, more unstimulated T cells produced IL-10 in comparison with controls; other cytokines were not detected in unstimulated cells. The percentage of IL-10-secreting T cells did not significantly increase after PMA/I stimulation of cells from SLE patients. The mean intensity of fluorescence (MIF) of intracellular IL-4 staining was significantly higher in CD8(-) T cells of SLE patients than controls. Significantly fewer CD8(-) and CD8(+) T cells from SLE patients secreted IFN-gamma after PMA/I stimulation compared with controls. The MIF and percentage of IL-2, IL-5, and IL-13-secreting cell subsets were not significantly different between SLE patients and controls. These findings indicate that T cells of SLE patients are already stimulated to produce IL-10 in vivo, which may result in downregulation of IFN-gamma secreting CD8(-) and CD8(+) T cells observed following PMA/I stimulation. Thus, the population size of Th1 and Tc1 cells are reduced in SLE patients whereas the effector function of Th2 cells, with respect to IL-4 production, is enhanced in SLE patients. Furthermore, although the balance between Th1/Th2 and between Tc1/Tc2 is disrupted in SLE patients, it is significantly biased in favour of the Th2 subset only.     

Downregulation of CD94/NKG2A inhibitory receptor on decreased γδ T cells in patients with systemic lupus erythematosus

γδ T cells are characterized by recognizing conserved endogenous and stress-induced antigens without antigen presentation. It has been show that γδ T cells play an important role in anti-tumour/microbe responses, but their function in autoimmune diseases is yet not clear. Here, we reported the quantity and phenotype of peripheral blood γδ T cells from systemic lupus erythematosus (SLE). Both the percentages of γδ T cells in peripheral blood and among CD3(+) T cells of patients with SLE were significantly decreased, regardless of disease activity. However, activating marker CD69 and HLA-DR was upregulated, while inhibiting receptor CD94/NKG2A was downregulated in γδ T cells of patients with SLE. The expression of CD69 is negatively correlated with the quantity of γδ T cells. Moreover, the expression of CD94/NKG2A remained low even with antigen stimulation on those γδ T cells. Our results suggested that the low expression level of CD94/NKG2A upon γδ T cell activation might lead to the over-activation of γδ T cells in patients with SLE. These findings will be useful in elucidating the roles of γδ T cells in SLE pathogenesis.     

Impaired generation of polyclonal T cell-mediated cytolytic activity despite normal polyclonal T cell proliferation in systemic lupus erythematosus.

No differences in proliferation induced by the anti-CD3 MAb 454 were detected between systemic lupus erythematosus (SLE) and normal peripheral blood mononuclear cells (PBMC) or purified T cells. In contrast, overnight culture with soluble MAb 454, immobilized MAb 454, or rIL2 induced significantly less increase in cytolytic activity against Daudi targets in SLE PBMC than in normal PBMC. Cytolytic activity in SLE PBMC cultures sequentially stimulated with soluble MAb 454 and rIL2 over a 6-day period overall was also lower than normal (with approximately 50% of the individual SLE cultures generating clearly subnormal levels of cytolytic activity) and did not correlate with the daily corticosteroid dose or with the presence of nephritis. Phenotypic analysis of soluble MAb 454-stimulated SLE PBMC cultures maintained for up to 23 days in rIL2 indicated that greater than 90% (and often greater than 96%) of the recovered cells were CD3+. Cytolytic activity generated in cultures of purified T cells stimulated with soluble MAb 454 + rIL2 over a 6-day period was also subnormal in 4/8 SLE donors, suggesting that the impaired generation of cytolytic activity in SLE is caused, at least in part, by impaired T cell-mediated cytolytic activity. Taken together, these observations demonstrate that normal CD3/T cell antigen receptor (TCR)-triggered polyclonal T cell proliferation can be dissociated from abnormal CD3/TCR-triggered polyclonal T cell cytolytic activity in SLE. This may have important implications for the pathogenesis of SLE and/or for the immunocompromised state seen in SLE.     

T/B‐cell interactions are more transient in response to weak stimuli in SLE‐prone mice

Changes in immune function during the course of systemic lupus erythematosus (SLE) are well characterized. Class‐switched antinuclear antibodies are the hallmark of SLE, and T/B‐cell interactions are thus critical. However, changes in immune function contributing to disease susceptibility are unknown. Here, we have analyzed primary T and B cells from a mouse model of SLE prior to the onset of disease. To allow cognate T‐cell activation with low affinity, we have developed a lower potency peptide ligand for the OTII TCR. T‐ and B‐cell couples formed less frequently and retained their polarity less efficiently preferentially in response to low‐affinity stimulation in SLE‐prone mice. This matched decreased recruitment of actin and Vav1 and an enhanced PKCΘ recruitment to the cellular interface in T cells. The induction of the GC B‐cell marker GL7 was increased in T/B cell couples from SLE‐prone mice when the T‐cell numbers were limited. However, the overall gene expression changes were marginal. Taken together, the enhanced cell‐couple transience may allow a more efficient sampling of a large number of T/B cell couples, preferentially in response to limiting stimuli, therefore enhancing the immune reactivity in the development of SLE.