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p21 mRNA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。     

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。     

1,25-二羟维生素D3抑制被动致敏人气道平滑肌细胞的增殖

目的: 检测1,25-二羟维生素D₃ 对被动致敏的人气道平滑肌细胞(HASMCs)增殖的调节作用,探讨其对哮喘气道重塑的可能作用。方法: 离体培养HASMCs,并将细胞分为对照组、哮喘组及1,25-(OH)₂D₃组。MTT法检测细胞增殖活力并确定1,25-(OH)₂D₃最佳作用浓度;用最佳作用浓度刺激HASMCs 48 h后以流式细胞术测定细胞周期,免疫细胞化学染色(SABC法)检测增殖细胞核抗原(PCNA)的表达。结果: 1,25-(OH)₂D₃在10^-9-10^-7 mol/L范围内能显著抑制哮喘血清被动致敏的HASMCs增殖(P<0.05),且10^-7 mol/L时抑制作用最强;流式细胞术检测显示这一最佳作用浓度的1,25-(OH)₂D₃能显著抑制被动致敏HASMCs由G₀/G₁期向S期的转化;此外,1,25-(OH)₂D₃能显著抑制被动致敏HASMCs中PCNA的表达。结论: 1,25-(OH)₂D₃能抑制哮喘血清被动致敏的HASMCs增殖,有助于哮喘气道重塑的防治。     

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。     

远志皂苷元对H2O2诱导的大鼠海马神经元损伤的影响及其机制

目的: 观察远志皂苷元(senegenin, Sen)对过氧化氢(H₂O₂)诱导的SD大鼠海马神经元损伤的影响并探讨其作用机制。方法: SD大鼠海马神经元培养至第6 d,随机分为正常组、H₂O₂组、Sen+ H₂O₂ 组和Sen组,药物干预后检测细胞存活率、丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性,并用 Hoechst 33258染色观察细胞核形态变化,荧光定量PCR(real-time PCR)检测神经元凋亡相关基因 bcl-2和bax 的表达,进一步采用Western blotting观察Bcl-2和Bax蛋白表达,酶荧光活性检测试剂盒测定caspase-3的活性改变。结果: 与正常组相比,H₂O₂组细胞存活率降低。与H₂O₂组相比,20 μmol/L Sen+ H₂O₂ 组细胞存活率升高,SOD活性升高,MDA含量下降,并且远志皂苷元能够上调bcl-2 mRNA表达、下调bax mRNA表达,降低caspase-3活性,Western blotting结果进一步表明Bcl-2蛋白表达升高,Bax蛋白表达下降,各指标均有显著差异(P<0.05)。结论: 远志皂苷元对H₂O₂处理的海马神经元有一定保护作用,其机制可能与远志皂苷元增强海马神经元抗氧化能力、调节细胞凋亡相关蛋白从而抑制凋亡有关。     

酪醇诱导肝癌细胞NQO1酶基因表达增加及细胞增殖的抑制

目的:研究酪醇诱导肝癌细胞Ⅱ相脱毒酶NAD(P)H:醌氧化还原酶-1(NQO₁)基因表达情况和对细胞增殖的影响以及两者之间的关系。方法:肝癌细胞SMMC-7721接种后24h经β-酪醇处理24h,分别测定NQO₁酶活性,mRNA表达和细胞增殖情况。NQO₁酶活性采用微孔板直接测定法,诱导结果用NQO₁酶比活性=NQO₁酶活性/细胞数;mRNA水平的变化采用定量RT-PCR;细胞增殖采用结晶紫显色法。结果:NQO₁酶活性诱导上,酪醇大于60mg/L时有明显的剂量效应关系,且每一浓度点(60mg/L,70mg/L,80mg/L,90mg/L)与空白组比较均有显著差异(P＜0.05),80mg/L的酪醇与80μmol/L的β-NF(阳性对照)诱导的酶比活性相当;mRNA表达量存在剂量依赖性增加(r=0.824,P＜0.05),且与酶比活性存在明显的相关性(r=0.951,P＜0.01);酪醇在70mg/L-100mg/L范围内,其抑制细胞增殖的能力随着浓度的增加而增加。另外,细胞增殖与酶比活性呈负相关(r=-0.410,P＜0.01)。结论:酪醇在培养肝癌细胞SMMC-7721上能使NQO₁酶活性与mRNA表达量诱导性增加,同时酪醇能抑制细胞的增殖,这种增殖抑制与酶活性诱导增加有关。     

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。     

脂氧素A4抑制内毒素诱导的人支气管上皮细胞环氧合酶2及前列腺素E2的表达

目的: 探讨脂氧素A₄对人支气管上皮细胞(HBECs)环氧合酶2(COX-2)表达的影响。方法: 应用不同浓度(0.1、1、10 mg/L)的内毒素(LPS)刺激HBECs 9 h,或者用1 mg/L LPS分别刺激HBECs不同时点(3 h、6 h、9 h)后,测定HBECs的COX-2 mRNA表达和细胞上清液前列腺素E₂(PGE₂)水平。应用不同浓度 (0、100、400 μmol/L) 的脂氧素A₄作用于经过LPS(1 mg/L)刺激培养9 h的HBECs,采用酶联免疫吸附法(ELISA)检测细胞上清液PGE₂的水平, 同时分别应用RT-PCR和Western blotting分别检测HBECs COX-2 mRNA及蛋白的表达。结果: LPS刺激培养条件下HBECs的COX-2 mRNA表达及其上清液PGE₂水平增加,并呈时间、剂量依赖性。脂氧素A₄能抑制LPS刺激培养HBECs COX-2蛋白和mRNA的表达及上清液PGE₂的水平,并呈剂量依赖性。结论: 脂氧素A₄能抑制LPS诱导的HBECs COX-2表达及上清液PGE₂的水平。     

热休克蛋白参与PI3K/Akt 介导H2O2 预处理的抗凋亡作用

目的: 探讨热休克蛋白(HSP)是否参与PI3K/Akt信号通路介导的H₂O₂预处理的抗细胞凋亡作用。方法: 利用PC12细胞建立H₂O₂预处理对抗高浓度H₂O₂诱导细胞凋亡的实验模型,分组如下:(1)空白对照组;(2) 损伤组;(3)预处理＋损伤组; (4)LY294002＋预处理＋损伤组;(5) LY294002组;(6) quercetin＋预处理＋损伤组;(7) 17-AAG＋预处理＋损伤组;(8) 溶剂对照组。应用Hoechst 33258染色观察细胞凋亡形态,碘化丙啶(PI)染色流式细胞术检测细胞凋亡率,比色法测定caspase-3的活性,免疫印迹法(Western blotting)测定HSP的表达水平。结果: 100 μmol/L H₂O₂预处理PC12细胞90 min可显著地抑制300 μmol/L H₂O₂引起的细胞凋亡,使caspase-3的活性降低,同时上调HSP70和HSP90的表达。HSP70和HSP90的抑制剂quercetin和17-AAG拮抗H₂O₂预处理的抗细胞凋亡作用。 PI3K抑制剂LY294002不仅拮抗了H₂O₂预处理抗细胞凋亡的作用,并且抑制H₂O₂预处理对HSP70和HSP90的表达上调。结论: PI3K/Akt通路、HSP70和HSP90均参与H₂O₂预处理诱导的细胞保护作用。并且HSP70和HSP90参与PI3K/Akt信号通路介导H₂O₂预处理的抗细胞凋亡作用。     

小鼠β2m正反义核酸表达载体转染NIH3T3细胞后的表达

目的:探讨小鼠β₂m正、反义核酸表达载体pcDNA3-β₂mSN和pcDNA3-β₂mAN对NIH3T3细胞表达β₂m的影响, 为降低MHCⅠ类分子表达的研究提供新方法和实验依据。方法:本研究应用分子生物学技术,构建小鼠β₂m正、反义核酸表达载体pcDNA3-β₂mSN和pcDNA3-β₂mAN,用脂质体转染NIH3T3细胞。经过RT-PCR及Western blot检测,观察正、反义核酸对细胞β₂m mRNA和蛋白表达的影响。 结果: 小鼠β₂m正、反义核酸表达载体成功导入NIH3T3细胞中,且有效表达,转染正义核酸的细胞β₂m mRNA和蛋白表达水平升高,而转染反义核酸则β₂m mRNA和蛋白表达水平降低。 结论:小鼠β₂m反义核酸表达载体pcDNA3-β₂mAN可以降低NIH3T3细胞β₂m基因的表达,本研究结果为降低MHCⅠ类分子的表达、解决同种异体排斥反应提供了新的研究方法和途径。     

Decreased immunosuppressive actions of 1α, 25-dihydroxyvitamin D3 in patients with immune thrombocytopenia

Primary immune thrombocytopenia (ITP) is an acquired autoimmune disease. 1α, 25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] and vitamin D receptor (VDR) play important immune-suppressive roles in immune system. It has been reported that serum 1,25(OH)₂D₃ were lower in ITP patients. In this study, we evaluated local 1,25(OH)₂D₃ level and VDR mRNA expression further, and determined whether 1,25(OH)₂D₃/VDR were correlated with T cell dysfunction in ITP patients. We found that 1,25(OH)₂D₃/VDR levels were decreased in active ITP patients, and 1,25(OH)₂D₃ had significant anti-inflammatory effects on ITP patients, including both anti-proliferation of peripheral blood mononuclear cells (PBMCs) and reversing the abnormal T cells polarization. 1,25(OH)₂D₃ inhibited the differentiation of T helper (Th)1 and Tc1 cells but induced the differentiation of Th2, Tc2 and T regulatory (Treg) cells in ITP patients. However, the percentage of Th17 cells were not affected obviously with 1,25(OH)₂D₃. In addition, 1,25(OH)₂D₃ also suppressed pro-inflammatory cytokines (INF-γ and IL-17A) but promoted anti-inflammatory cytokine (IL-10) secretion in ITP patients. In conclusion, decreased 1,25(OH)₂D₃/VDR might participate in the pathogenesis of ITP, and appropriate supplement of 1,25(OH)₂D₃ may be a promising treatment.     

Plasma 1,25 Dihydroxy Vitamin D<Subscript>3</Subscript> Level and Expression of Vitamin D Receptor and Cathelicidin in Pulmonary Tuberculosis

Introduction  Vitamin D₃, which exerts its effect through vitamin D receptor (VDR), is known for its potent immunomodulatory activities. Associations between low serum vitamin D₃ levels and increased risk of tuberculosis have been reported. Study Subjects and Methods  Plasma 1,25 dihydroxy vitamin D₃ levels (1,25(OH)₂ D₃) and ex vivo levels of VDR protein from peripheral blood mononuclear cells were studied in 65 pulmonary tuberculosis (PTB) patients and 60 normal healthy subjects (NHS) using enzyme-linked immunosorbent assay-based methods. Using real-time polymerase chain reaction (PCR), induction of VDR, cathelicidin, and CYP27B1 mRNA were studied in live Mycobacterium tuberculosis-stimulated macrophage cultures treated with or without 1,25 dihydroxy vitamin D₃. VDR and CYP27B1 (-1077 A/T) gene polymorphisms were studied using PCR-based methods. Results  1,25(OH)₂ D₃ were significantly increased (p = 0.0004), while ex vivo levels of VDR protein were significantly decreased in PTB patients (p = 0.017) as compared to NHS. 1,25(OH)₂ D₃ levels were not different between variant genotypes of CYP27B1. A trend towards decreased levels of VDR protein was observed among NHS with BsmI BB and TaqI tt genotypes compared to NHS with other genotypes. Relative quantification of mRNA using real-time PCR revealed increased VDR mRNA expression in live M. tuberculosis-stimulated culture in PTB patients (p < 0.01) than normal healthy subjects. Cathelicidin mRNA expression was significantly increased in vitamin D₃-treated cultures compared to unstimulated and M. tuberculosis-stimulated culture in both patients (p < 0.001) and NHS (p < 0.05). Conclusions  The present study suggests that PTB patients may have increased 1,25(OH)₂ D₃ levels, and this might lead to downregulation of VDR expression. Decreased VDR levels could result in defective VDR signaling. Moreover, addition of 1,25(OH)₂ D₃ might lead to increased expression of cathelicidin which could enhance the immunity against tuberculosis.     

1,25-Dihydroxyvitamin D3 inhibits proliferation but not the suppressive function of regulatory T cells in the absence of antigen-presenting cells

Vitamin D₃ is known to induce regulatory T (Treg) cells by rendering antigen-presenting cells tolerogenic, its direct effect on human naturally occurring Treg cells is unclear. Here, we investigated if and how 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] can directly affect the proliferation and function of human naturally occurring Treg cells in vitro. First, we demonstrated that these Treg cells express vitamin D receptors that were up-regulated following anti-CD3/CD28-bead stimulation. 1,25(OH)₂D₃ inhibited proliferation of Treg cells even when exogenous interleukin-2 was provided. Treg cells were more susceptible to the inhibitory effect of 1,25(OH)₂D₃ than conventional T cells_. 1,25(OH)₂D₃ neither affected the anergic state nor the suppressive function of Treg cells but induced a subtle increase in interleukin-10-secreting cells. The cell-division-inhibiting effect of 1,25(OH)₂D₃ on Treg cells was also demonstrated in vivo by supplementing vitamin D-deficient HIV-1-infected patients with 2000 IU cholecalciferol (vitamin D₃). Increased serum 1,25(OH)₂D₃ levels were associated with a drop in the number and percentage of Treg cells, which may be attributed to a decrease in the proliferating Foxp3⁺ Treg cell population. In conclusion, 1,25(OH)₂D₃ directly affects Treg cell growth and promotes interleukin-10 production without apparent effects on activation status and suppressive phenotype whereas in vivo, high serum 1,25(OH)₂D₃ levels are associated with reduced Treg cell proliferation and a reduced number of Treg cells.     

1,25-Dihydroxyvitamin D3 and Development of Tuberculosis in Cattle

This report describes the presence and activity of 1,25-dihydroxyvitamin D₃ (1,25-D₃) in experimental bovine tuberculosis. Animals that went on to develop tuberculous lesions exhibited a rapid transient increase in serum 1,25-D₃ within the first 2 weeks following infection with Mycobacterium bovis. 1,25-D₃-positive mononuclear cells were later identified in all tuberculous granulomas by immunohistochemical staining of postmortem lymph node tissue. These results suggest a role for 1,25-D₃ both at the onset of infection and in the development of the granuloma in these infected animals. Using a monoclonal antibody to the vitamin D receptor (VDR) as a VDR agonist, we confirmed that activation of the vitamin D pathway profoundly depresses antigen-specific, but not mitogenic, bovine peripheral blood T-cell responses (proliferation and gamma interferon production). Investigation of the mechanism of this suppression showed that the VDR antibody modified the expression of CD80 by accessory cells, such that a significant positive correlation between T-cell proliferation and accessory cell CD80 emerged.     

Vitamin D and the vitamin D receptor are critical for control of the innate immune response to colonic injury

Background

The active form of vitamin D (1,25(OH)₂D₃) has been shown to inhibit development of inflammatory bowel disease (IBD) in IL-10 KO mice. Here, the role of the vitamin D receptor (VDR) and 1,25(OH)₂D₃in acute experimental IBD was probed.

Results

VDR KO mice were extremely sensitive to dextran sodium sulfate (DSS) and there was increased mortality of the VDR KO mice at doses of DSS that only caused a mild form of colitis in wildtype (WT) mice. DSS colitis in the VDR KO mice was accompanied by high colonic expression of TNF-α, IL-1 α, IL-1β, IL-12, IFN-γ, IL-10, MIP-1α and KC. DSS concentrations as low as 0.5% were enough to induce bleeding, ulceration and weight loss in VDR KO mice. VDR KO mice failed to recover following the removal of DSS, while WT mice showed signs of recovery within 5 days of DSS removal. The early mortality of DSS treated VDR KO mice was likely due to perforation of the bowel and resulting endotoxemia. VDR KO mice were hyper-responsive to exogenously injected LPS and cultures of the peritoneal exudates of moribund DSS treated VDR KO mice were positive for bacterial growth. 1,25(OH)₂D₃in the diet or rectally decreased the severity and extent of DSS-induced inflammation in WT mice.

Conclusion

The data point to a critical role for the VDR and 1,25(OH)₂D₃in control of innate immunity and the response of the colon to chemical injury.     

The Active Metabolite of Vitamin D3 as a Potential Immunomodulator

In the past, vitamin D was known for its classical, skeletal action as a regulator of calcium and bone homoeostasis. Currently, vitamin D was found to have a role in numerous physiological processes in the human body; thus, vitamin D has pleiotropic activity. The studies carried out in the past two decades showed the role of vitamin D in the regulation of immune system functions. Basically, these effects may be mediated not only via endocrine mechanism of circulating calcitriol but also via paracrine one (based on cell–cell communication that leads to production of signal inducing the changes in nearby/adjacent cells and modulating their differentiation or behaviour) and intracrine mechanism (the action of vitamin D inside a cell) of 1,25‐dihydroxycholecalciferol (1,25(OH)₂D₃) synthetized from its precursor 25‐hydroxyvitamin D₃ (25(OH)D₃). Both vitamin D receptor (VDR) and 25‐hydroxyvitamin D₃ 1‐α‐hydroxylase (CYP27B1) are expressed in several types of immune cells (i.e. antigen presenting cells, T and B cells), and thus, they are able to synthetize the bioactive form of vitamin D that modulates both the innate and adaptive immune system. This review discusses the role of vitamin D as regulator of immune system, and our understanding of how vitamin D regulates both adaptive and innate immunity as well as inflammatory cascade on the cellular level.