维生素D受体在1,25(OH)2D3调节人骨肉瘤细胞系HOS-8603增殖中的作用

目的:研究维生素D₃受体(VDR)在1,25(OH)₂D₃及其类似物调节人骨肉瘤细胞系HOS-8603增殖中的作用。方法:用RT-PCR和免疫组织化学技术分别在mRNA和蛋白水平上明确HOS-8603细胞中是否有VDR表达;瞬时转染VDR报告基因技术观察HOS-8603细胞中VDR的功能活性;并进一步利用稳定表达VDR反义mRNA的细胞株VDRas3细胞研究VDR被阻断后细胞增殖以及基因转录的变化。结果:HOS-8603细胞有VDR表达,此内源性VDR具有激素依赖性的转录激活活性。在内源性VDR被阻断后,1,25(OH)₂D₃及其类似物对细胞增殖的抑制作用以及对VDR的靶基因p21mRNA表达的诱导作用均明显减弱。结论:1,25(OH)₂D₃及其类似物对HOS-8603细胞增殖抑制作用是由VDR介导的。     

1,25-二羟维生素D3负性调控被动致敏人气道平滑肌细胞中的NF-κB信号通路

 目的：探讨1,25-二羟维生素D3［1,25-(OH)2D3］对被动致敏人气道平滑肌细胞(HASMCs)中核因子κB（NF-κB）信号通路的影响。方法：原代培养HASMCs并使之被动致敏,以1,25-(OH)2D3作为干预因素。EMSA法检测NF-κB的DNA结合活性;免疫细胞化学染色技术观察NF-κB p65的核易位情况;Western blotting法检测核因子κB抑制蛋白α(IκBα)及p-IκBα蛋白的表达水平;实时荧光定量PCR检测维生素D受体(VDR)、维生素D 24-羟化酶(CYP24)和IκBα mRNA的表达水平;放线菌素D处理实验检测IκBα mRNA的表达。结果：(1) 1,25-(OH)2D3显著削弱被动致敏HASMCs中NF-κB的DNA结合活性及其亚单位 p65的核易位;(2) 1,25-(OH)2D3能通过增加被动致敏HASMCs中IκBα的mRNA稳定性及减少其蛋白磷酸化水平2个途径显著上调细胞中IκBα的表达;(3) 1,25-(OH)2D3显著上调被动致敏HASMCs中VDR的mRNA表达并诱发其功能性反应。结论：1,25-(OH)2D3能通过上调被动致敏HASMCs中IκBα的表达抑制细胞NF-κB信号通路,且这一作用与VDR有关,这可能是其调控被动致敏HASMCs的重要作用机制。     

1,25—二羟基维生素D3与免疫

维生素D(Vitamin D)是调节骨盐平衡的主要内分泌激素之一,其活性形式1,25(OH)_2D_3一般通过与特异核内受体(VDR)相结合对靶基因的表达进行调节。此外,1,25(OH)_2D_3也可通过快速的非基因通路即通过与膜受体或膜识别元件相结合,白蛋白激酶C和/或依赖cAMP的蛋白激酶激活电压依赖的钙通道,从而产生生物效     

维生素D3受体mRNA在肝细胞增殖和肝癌发展中的作用

目的：探讨维生素D3受体mRNA在肝细胞增生和肝癌发展中的作用。方法：体外培养肝癌细胞株SMMC－7721和HCC－T细胞，培养时添加1000nmol/L、100nmol/L、10nmol/L 1，25-(OH)2D3作用1、3、6天后，用四唑盐比色试验（MTT）检测细胞的存活和生长；用反转录PCR（RT－PCR）检测维生素D3受体mRNA的表达。结果：1.25-(OH)2D3可以抑制维生素D3受体mRNA表达阳性的SMMC－7721细胞增生并且有剂量效应关系；对维生素D3受体mRNA表达阴性的HCC－T细胞没有抑制作用。9例肝癌组织标本维生素D3受体mRNA表达均为阳性。结论：1，25－（OH）2D3对于人肝癌细胞株SMMC－7721的增殖具有显著的抑制作用，其机械可能是通过维生素D3受体来实现的。     

多发性硬化症患者维生素D_3及其受体水平的检测

维生素D作为一种调节钙磷代谢的重要激素．直到1980年其参与免疫功能及对细胞增殖分化的影响才引起人们广泛的兴趣。绝大多数与免疫功能相关的细胞都含有维生素D，受体（VDR），如单核细胞和组织中巨噬细胞、活化的T、B淋巴细胞。维生素D，生物活性形式为1,25（OH）2D3，后者通过与高亲和力的特异性受体VDR结合而调节有关基因的表达，达到调节机体免疫系统功能的生物学效应．包括调节抗原提呈细胞的分化、淋巴细胞的增殖及细胞因子的产生。     

维生素D3及其类似物对人树突状细胞表达CSF-1及其受体的作用

目的 探讨维生素D3[1，25(OH)2D3]及其类似物抑制人树突状细胞分化，但保持细胞生存是否通过调节集落刺激因子(CSF-1)及其受体来实现。方法 在体外将人外周血单核细胞分化成树突状细胞。采用流式细胞术、RT-PCR和ELISA法分析1，25(OH)2D3及其类似物对树突状细胞表达和产生GSF-1及其受体的作用。结果 1，25(OH)2D3和他骨化醇(taealeitol)以剂量依赖的方式显著上调树突状细胞对CSF-1 mRNA的表达，并增高其蛋白分泌水平，而细胞表面CSF-1受体以及mRNA的表达均受到抑制。溶剂乙醇和24，25(OH)2D3对CSF-1及其受体均无调节作用。1，25(OH)2D3对树突状细胞表达GM-CSF受体mRNA无影响。结论 1，25(OH)2D3对树突状细胞分化的抑制与其特异性地调节CSF-l及其受体有关。     

1α,25-二羟基维生素D3抑制人结肠癌细胞系SW480中β-catenin转录活性

目的探讨1α,25-二羟基维生素D_3[1,25(OH)_2D_3]对人结肠癌细胞系SW480中β-catenin转录活性的影响及作用机制。方法 1,25(OH)_2D_3(100 nmol/L)干预和溶剂对照处理SW480细胞48 h;用双荧光素酶报告系统检测β-catenin的转录活性;Western blot检测1,25(OH)_2D_3干预后β-catenin在细胞核/质内的定位变化;用RT-qPCR和Western blot检测β-catenin和FOXM1 mRNA及蛋白水平。采用siRNA敲降SW480细胞中FOXM1后,检测1,25(OH)_2D_3干预后β-catenin转录活性的变化。结果 1)在SW480细胞中,1,25(OH)_2D_3能显著降低β-catenin的转录活性(P0.05),但不影响β-catenin mRNA和蛋白表达水平; 2)1,25(OH)_2D_3上调SW480细胞中FOXM1蛋白水平的表达(P0.05); 3)敲降SW480细胞中FOXM1降低1,25(OH)_2D_3对β-catenin转录活性的抑制作用(P0.05)。结论 1,25(OH)_2D_3通过上调FOXM1的表达,发挥抑制β-catenin转录活性的作用。     

维生素D3受体在人胎脑表达的研究

为研究维生素D3受体(VDR)在正常人胎脑的分布,进而探讨1,25-二羟维生素D3[1,25-(OH)2D3]与正常人胎脑发育的关系,本实验按合法程序收集2～7月龄正常人胎脑,用免疫组织化学方法研究VDR在正常人胎脑的时空表达情况。结果显示:阳性产物在海马结构存在于胞核,3月龄出现表达的高峰,此后逐渐下降;在额叶皮质以及中脑部位的胞核和胞浆都可存在,其表达随胎龄增长。以上结果表明VDR在2～7月龄的人胎脑额叶皮质、海马结构和中脑有表达,且各部位的表达模式不同,提示1,25-(OH)2D3通过VDR可在人胎脑不同部位发育中发挥作用。     

原发ITP患者外周血维生素D3水平与维生素D受体表达的相关性研究

目的 研究成人原发免疫性血小板减少症(ITP)患者血清中25-羟维生素D3[25-(OH)-D3]、维生素D活性指标与维生素D受体表达的相关性.方法 选择开滦总医院2017年1月至2019年12月收治的30例ITP患者作为研究对象,设为ITP组;选择同期来院体检的30例健康体检者作为对照组,抽取外周血,检测血清中25-(OH)-D3和1,25-二羟基维生素D3(1,25-(OH)2-D3)水平,并采用反转录-聚合酶链反应(RT-PCR)法检测外周血中维生素D受体(VDR)mRNA的表达,进行分组比较,采用Pearson分析法分析血清中25-(OH)-D3、1,25-(OH)2-D3水平与VDRmRNA的相关性.结果 ITP组25-(OH)-D3、1,25-(OH)2-D3水平均低于对照组[(10.36±1.86)vs(13.74±2.73)]ng/mL、[(68.25±9.80)vs(88.09±11.72)]pg/mL,差异有统计学意义(P＜0.05).对照组VDRmRNA的表达量为1.051±0.148,ITP组VDRmRNA的表达量为1.593±0.185,ITP组VDRmRNA的表达量高于对照组,差异有统计学意义(t = 12.844,P＜0.001).25-(OH)-D3、1,25-(OH)2-D3水平与 VDRmRNA 表达均呈负相关(r =-0.592,-0.486,P = 0.017,0.028).结论 成人ITP维生素D活性指标与维生素D受体表达水平与健康人群比较存在异常,且25-(OH)-D3、1,25-(OH)2-D3水平与 VDRmRNA表达均呈负相关,提示其可能参与了 ITP的发生,维生素D可能对ITP有一定的治疗治疗效果.     

1,25-二羟维生素D3的免疫调节作用新进展

维生素D3(VitD3)不是内分泌细胞合成的激素,但在体内经修饰活化后可成为一种重要的激素-1,25-二羟维生素D3[1,25(OH)2D3],它与靶细胞内的核受体结合后,能够影响基因转录,发挥多种生物学效应.1,25(OH)2D3以及维生素D受体在淋巴细胞的分化发育、细胞周期的进程以及细胞因子的产生方面存在广泛的影响.本文综述了1,25(OH)2D3的主要作用机制,以及在自身免疫性疾病中的潜在作用.     

p21wafl在1, 25(OH)2D3调节人骨肉瘤细胞系HOS-8603增殖分化中的作用

目的:探讨p21^wafl在1, 25(OH)₂D₃调节人骨肉瘤细胞系HOS-8603增殖中的作用。方法: 用定量RT-PCR法检测1, 25(OH)₂D₃对p21^waflmRNA表达的诱导作用, 并构建稳定表达p21^wafl真核表达载体的细胞系HOS-p21^wafl进一步观察该细胞的生长及分化情况。结果:1, 25(OH)₂D₃作用2h即可诱导HOS-8603细胞内源性p21^wafl的表达, 4h达高峰, 24h仍未回降。细胞过度表达p21^wafl后生长速度明显减慢, 培养6d时细胞数为未转染细胞的50%;并且组织化学染色结果表明细胞的分化标志性抗原碱性磷酸酶(AKP)的表达明显增强。结论:表明1, 25(OH)₂D₃对p21^waflmRNA诱导作用可能是激素调节细胞增殖分化的重要机制之一。     

糖皮质激素对糖皮质激素受体α和β mRNA表达的调节作用

目的：研究糖皮质激素对人类糖皮质激素受体α和β mRNA表达的调节作用。方法：采用定量逆转录-聚合酶链反应（RT-PCR）检测受体mRNA。结果：人骨肉瘤细胞系、卵巢癌细胞系及外周血白细胞中除有糖皮质激素受体α mRNA表达外，还有糖皮质激素受体β mRNA的表达；糖皮质激素对糖皮质受体α和β mRNA的表达均具有明显的降调作用，且具有剂量依赖性和时间依赖性特点。结论：糖皮质激素受体β在人类多种组织细胞中均有表达，而且其表达受糖皮质激素的调节。     

A novel inborn error in the ligand-binding domain of the vitamin D receptor causes hereditary vitamin D-resistant rickets

Mutations in the vitamin D receptor (VDR) cause hereditary vitamin D-resistant rickets (HVDRR), an autosomal recessive disease resulting in target organ resistance to 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. In this report, we describe the clinical case and molecular basis of HVDRR in an Asian boy exhibiting the typical clinical features of the disease including alopecia. Using cultured dermal fibroblasts from the patient, 1,25(OH)(2)D(3) resistance was demonstrated by a shift in the dose response required for 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) mRNA induction. Western blot showed that the cells express a normal size VDR but contained reduced levels of receptor compared to normal cells. At 24 degrees C, the affinity of the patient's VDR for [(3)H]1,25(OH)(2)D(3) was 50-fold lower than the VDR in normal fibroblasts. Sequence analysis identified a unique T to G missense mutation in exon 6 that changed phenylalanine to cysteine at amino acid 251 (F251C). The recreated F251C mutant VDR showed reduced transactivation activity using a 24-hydroxylase promoter-luciferase reporter. Maximal transactivation activity exhibited by the WT VDR was not achieved by the mutant VDR even when the cells were treated with up to 10(-6) M 1,25(OH)(2)D(3). However, the transactivation activity was partially rescued by addition of RXRalpha. In the yeast two-hybrid system and GST-pull-down assays, high concentrations of 1,25(OH)(2)D(3) were needed to promote F251C mutant VDR binding to RXRalpha, indicating defective heterodimerization. In conclusion, a novel mutation was identified in the VDR LBD that reduces VDR abundance and its affinity for 1,25(OH)(2)D(3) and interferes with RXRalpha heterodimerization resulting in the syndrome of HVDRR.     

1,25(OH)2D3 regulates c-myc mRNA levels in tonsillar T lymphocytes.

The effects of 1,25(OH)2D3 on proliferation, c-myc mRNA levels and 1,25(OH)2D3 receptor expression in activated tonsillar T lymphocytes were studied. Activation of resting T cells with phytohaemagglutinin (PHA) for 72 hr led to an increase in proliferation, c-myc mRNA levels and to induction of 1,25(OH)2D3 receptor expression. However, when activation was carried out in the presence of 1,25(OH)2D3, there was inhibition of PHA-stimulated proliferation and c-myc mRNA levels. Increased cell proliferation, c-myc mRNA expression and 1,25(OH)2D3 receptor number were also observed, albeit to a lesser extent, when T cells were stimulated by phorbol myristate acetate (PMA), anti-CD3 antibody or A23187. However, in these cases 1,25(OH)2D3 was unable to prevent increased proliferation or c-myc mRNA expression. PMA and anti-CD3 used in combination produced similar or greater changes in proliferation, c-myc mRNA levels, 1,25(OH)2D3 receptor expression and responsiveness to the hormone when compared to PHA alone. Thus the inhibition of c-myc expression in activated T lymphocytes by 1,25(OH)2D3 can be related to its anti-proliferative effects. Moreover this inhibition seems to be dependent on the level of 1,25(OH)2D3 receptor expression, which in turn appears to be related to the degree of cell activation.     

1,25-Dihydroxyvitamin D3 acts directly on the T lymphocyte vitamin D receptor to inhibit experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is an incurable autoimmune neurodegenerative disease. Environmental factors may be key to MS prevention and treatment. MS prevalence and severity decrease with increasing sunlight exposure and vitamin D(3) supplies, supporting our hypothesis that the sunlight-dependent hormone, 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2) D(3) ), inhibits autoimmune T-cell responses in MS. Moreover, 1,25-(OH)(2) D(3) inhibits and reverses experimental autoimmune encephalomyelitis (EAE), an MS model. Here, we investigated whether 1,25-(OH)(2) D(3) inhibits EAE via the vitamin D receptor (VDR) in T lymphocytes. Using bone marrow chimeric mice with a disrupted VDR only in radio-sensitive hematopoietic cells or radio-resistant non-hematopoietic cells, we found that hematopoietic cell VDR function was necessary for 1,25-(OH)(2) D(3) to inhibit EAE. Furthermore, conditional targeting experiments showed that VDR function in T cells was necessary. Neither 1,25-(OH)(2) D(3) nor T-cell-specific VDR targeting influenced CD4(+) Foxp3(+) T-cell proportions in the periphery or the CNS in these studies. These data support a model wherein 1,25-(OH)(2) D(3) acts directly on pathogenic CD4(+) T cells to inhibit EAE.     

1,25-dihydroxyvitamin D3 stimulates interleukin-2 production by a T cell lymphoma line (MLA-144) cultured in vitamin D-deficient rat serum

A lymphocyte T cell line (MLA-144), which constitutively secretes interleukin-2 (IL-2), was shown to express receptors for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). The proliferation of an IL-2-dependent cell line (HT-2) in response to supernatants from MLA-144 cells was employed as an index of IL-2 production by MLA-144 cells. IL-2 production was two fold higher from MLA-144 cells cultured in 2% vitamin D-deficient rat serum compared to 10% fetal calf serum (FCS). The addition of 1,25(OH)2D3 at 10(-15) M or 10(-11) M augmented IL-2 production by MLA-144 cells in vitamin D-deficient rat serum, but not in fetal calf serum. At 10(-7) M 1,25(OH)2D3 there was inhibition of IL-2 production by MLA-144 cells in either vitamin D-deficient serum or FCS. There was no effect of 1,25(OH)2D3 added directly to HT-2 cells. Monoclonal antibody to the IL-2 receptor competitively inhibited the proliferation of HT-2 cells in response to MLA-144 supernatants, suggesting that it was IL-2 from the MLA-144 supernatants which influenced HT-2 proliferation. Our findings demonstrate biphasic dose effects of 1,25(OH)2D3 on lymphokine secretion. The use of vitamin D-deficient rat serum allowed us to demonstrate the effects of 1,25(OH)2D3 in the physiologic and subphysiologic range.