1,25-二羟基维生素D3对PM2.5所致HBE细胞氧化损伤的保护作用

目的： 探讨1，25-二羟基维生素D₃[1，25-（OH）₂D₃]对细颗粒物（PM_2.5）致人支气管上皮细胞（HBE）氧化损伤的保护作用。方法： 根据处理因素不同将细胞分为4组，溶剂（乙醇）对照组、1，25-（OH）₂D₃干预组、乙醇+PM_2.5染毒组、PM_2.5染毒+1，25-（OH）₂D₃干预组。溶剂对照组细胞用0.1%乙醇处理48 h，1，25-（OH）₂D₃干预组用1×10^-9 mol/L 1，25-（OH）₂D₃处理48 h，乙醇+PM_2.5染毒组用0.1%乙醇溶剂处理24 h后更换为含乙醇的PM_2.5（200 μg/mL）染毒液继续处理24 h，PM_2.5染毒+1，25-（OH）₂D₃干预组用1×10^-9 mol/L 1，25-（OH）₂D₃预处理24 h后更换为含1，25-（OH）₂D₃的PM_2.5（200 μg/mL）染毒液继续处理24 h。染毒结束后，用CCK-8试剂盒测定细胞存活率、丙二醛（MDA）和还原型谷胱甘肽（GSH）/氧化型谷胱甘肽（GSSG）-Glo^TM试剂盒分别测定MDA浓度和GSH/GSSG比值，Western blot实验测定维生素D受体（VDR）、转录因子NF-E2相关因子2（Nrf-2）与血红素加氧酶-1（HO-1）蛋白的表达水平。结果： PM_2.5染毒处理后，HBE细胞的存活率降至80.8%，1，25-（OH）₂D₃预处理24 h后再进行PM_2.5染毒的细胞存活率降低至75.8%。与对照相比，PM_2.5染毒后，HBE细胞MDA浓度显著增加（P < 0.05），而GSH/GSSG的比值却明显降低（P < 0.01）。1，25-（OH）₂D₃处理48 h后可显著改善PM_2.5染毒细胞的抗氧化水平（P < 0.05），主要表现为MDA浓度的降低和GSH/GSSG比值的增加。蛋白分析结果发现，PM_2.5可诱导细胞Nrf-2和HO-1蛋白表达的增加。1，25-（OH）₂D₃干预48 h后可上调HBE细胞内VDR水平，并可增加PM_2.5染毒组细胞内Nrf-2和HO-1蛋白的表达水平。结论： PM_2.5可诱导HBE细胞氧化损伤，主要表现为脂质过氧化水平升高、GSH/GSSG比值下降和抗氧化蛋白Nrf-2与HO-1表达水平的增加。在PM_2.5所致细胞氧化应激效应中，1，25-（OH）₂D₃可起到一定的保护作用，这可能与VDR及Nrf-2/HO-1信号通路有关。而1，25-（OH）₂D₃所致细胞存活率的降低可能与其诱导细胞周期阻滞及促进PM_2.5所诱导损伤细胞的凋亡有关。     

1,25-二羟基维生素D3对抗多种肿瘤作用的研究进展

1,25-二羟基维生素D3是VD的活性形式,大量体内外研究证明它具有抗增殖、促凋亡、促分化等抗肿瘤作用。维生素D受体高度表达于多种人类肿瘤细胞,对于许多患者其VD状态可能与他们癌症的发生和发展密切相关。本文综述了1,25-二羟基维生素D_3对于乳腺癌、前列腺癌、直肠癌等多种癌症的抗癌作用机制以及近年来一些临床研究的鼓舞人心的结果。     

外周血1,25-二羟基维生素D3水平与胃癌患者临床特征的关系

目的 探讨外周血1,25-二羟基维生素D3[1,25(OH)2D3]水平与胃癌患者临床特征的关系.方法 选取98例胃癌患者作为观察组,100例健康体检者作为对照组,检测两组受试者血清1,25(OH)2D3、Ca2+水平,分析不同基线特征胃癌患者血清1,25(OH)2D3水平的差异,相关性分析采用Pearson相关分析....     

1,25二羟基维生素D3对体外培养的HL-60细胞的作用

[目的]研究1,25二羟基维生素D3[1,25(OH)2D3]对人HL-60细胞株生长、分化、凋亡及对维生素D受体(VDR)蛋白表达的影响.[方法]在体外用不同浓度的1,25(OH)2D3作用于对数生长期的HL-60细胞.甲基噻唑基四唑(MTT)比色法分析细胞增殖抑制作用,硝基四氮唑蓝(NBT)还原实验法分析HL-60细胞的分化指标,末端脱氧核糖核酸转移酶介导的dUTP缺口末端标记染色法(TUNEL)检测细胞晚期凋亡的变化,免疫细胞化学法检测VDR蛋白表达.[结果]不同浓度的1,25(OH)2D3作用后均可抑制HL-60细胞的生长,呈剂量和时间依赖性;可诱导HL-60细胞向成熟粒细胞分化并促其凋亡.1,25(OH)2D3作用前后HL-60细胞均表达VDR蛋白,药物作用后VDR蛋白表达上调.[结论]1,25(OH)2D3在一定浓度范围内可抑制HL-60细胞增殖,诱导细胞分化及凋亡,使VDR蛋白表达上调.     

宫颈癌患者血清1,25-(OH)2D3水平检测及临床意义

目的:检测不同宫颈病变患者血清中1,25-(OH)2D3的水平,初步探讨1,25-(OH)2D3与宫颈癌临床特征的相关性。方法:对2014年7月至2015年7月收治的116例宫颈病变患者,采用放射免疫法检测血清中1,25-(OH)2D3水平,并以40例健康体检者血清作对照。结果:宫颈癌及宫颈良性病变患者血清1,25-(OH)2D3水平均低于健康对照组,差异有统计学意义（P＜0.05）,宫颈癌与子宫良性病变组之间无显著性差异（P＞0.05）。宫颈癌组中,绝经前患者血清中1,25-(OH)2D3水平高于绝经后患者血清水平（P＜0.05）,宫颈癌组织分级越差,淋巴结越多,FIGO分期越晚,血清中1,25-(OH)2D3水平明显降低。结论:血清中1,25-(OH)2D3水平降低可能与宫颈癌进展有关。     

PM2.5对HK-2细胞氧化损伤和凋亡的影响

目的：探讨深圳和太原市PM_2.5对人肾上皮细胞（HK-2）氧化损伤和凋亡的影响。方法：用中流量采样器分别采集太原某大学校园和深圳市疾病预防控制中心楼顶空气,将吸附有PM_2.5的滤膜洗脱后制备PM_2.5混悬液。设置阴性对照组、50 μg/mL深圳PM_2.5样品组、50 μg/mL太原PM_2.5样品组和10 μmol/L Cr⁶⁺阳性对照组,分别处理HK-2细胞24 h,测定4组细胞氧化损伤指标丙二醛（MDA）、超氧化物歧化酶（SOD）、还原型谷胱甘肽（GSH）、谷胱甘肽过氧化物酶（GSH-PX）的变化,并应用流式细胞术检测细胞凋亡水平。结果：与阴性对照组相比,深圳PM_2.5样品组、太原PM_2.5样品组和阳性对照组中MDA含量分别升高8.16%、34.51%和72.23%;SOD活性分别降低7.49%、19.67%和29.55%;GSH含量分别降低10.43%、16.39%和37.43%。太原PM_2.5样品组与阴性对照组的差异均有统计学意义（P < 0.05或P < 0.01）。GSH-PX活性分别降低42.70%、61.62%和60.98%,细胞凋亡率分别升高197.25%、301.22%和399.08%,上述3组与阴性对照组间的差异均有统计学意义（P < 0.05或P < 0.01）。结论：深圳和太原市PM_2.5均可引起HK-2细胞发生氧化损伤和细胞凋亡率增加,且太原市PM_2.5的作用更为明显。     

1α,25-二羟基维生素D3通过表皮生长因子受体和细胞周期调节卵巢癌细胞的生长

目的:研究1α,25-二羟基维生素D3(1,25VD3)通过对表皮生长因子受体和细胞周期的调控调节人卵巢癌细胞的生长.方法:用1,25VD3处理人卵巢癌细胞株OVCAR3后,MTT法测定细胞生长情况,流式细胞仪分析细胞周期,Western blot检测EGFR表达.用pcDNA3-EGFRvIII质粒转染 OVCAR3 细胞,筛选出稳定过表达EGFR的克隆株EGFR-OVCAR3.用1,25VD3及EGFR抑制剂分别处理OVCAR3和EGFR-OVCAR3细胞,Western blot检测p27、Cyclin E和GADD45蛋白表达.结果:1,25VD3下调OVCAR3细胞EGFR蛋白表达,对细胞生长有明显抑制作用,使其G0/G1和G2/M期增多,S期明显减少,而对EGFR-OVCAR3细胞无明显作用.Western blot显示,1,25VD3上调OVCAR3细胞p27和GADD45蛋白表达,下调Cyclin E蛋白表达 ,而对EGFR-OVCAR3 细胞三种蛋白表达无调节作用,用EGFR抑制剂处理EGFR-OVCAR3细胞后,又能调节p27和cyclin E蛋白的表达.结论:1,25VD3可以调节EGFR蛋白表达,并进而调节下游分子,包括p27和GADD45,最后调控细胞周期,抑制细胞G1/S期转换和G2/M期转换,从而抑制卵巢癌细胞的生长.     

1α，25-二羟基维生素D3通过表皮生长因子受体和细胞周期调节卵巢癌细胞的生长

目的：研究1α,25-二羟基维生素D3（1,25VD3）通过对表皮生长因子受体和细胞周期的调控调节人卵巢癌细胞的生长。方法：用1,25VD3处理人卵巢癌细胞株OVCAR3后,MTT法测定细胞生长情况,流式细胞仪分析细胞周期,Western blot检测EGFR表达。用pcDNA3-EGFRvIII质粒转染OVCAR3细胞,筛选出稳定过表达EGFR的克隆株EGFR—OVCAR3。用1,25VD3及EGFR抑制剂分别处理OVCAR3和EGFR—OVCAR3细胞,Westernblot检测p27、CyclinE和GADD45蛋白表达。结果：1,25VD3下调OVCAR3细胞EG—FR蛋白表达,对细胞生长有明显抑制作用,使其G0／G1和G2／M期增多,S期明显减少,而对EGFR—OVCAR3细胞无明显作用。Western blot显示,1,25VD3上调OVCAR3细胞p27和GADD45蛋白表达,下调CyclinE蛋白表达,而对EGFR—OVCAR3细胞三种蛋白表达无调节作用,用EGFR抑制剂处理EGFR—OVCAR3细胞后,又能调节p27和cyclinE蛋白的表达。结论：1,25VD3可以调节EGFR蛋白表达,并进而调节下游分子,包括p27和GADD45,最后调控细胞周期,抑制细胞G1／S期转换和G2／M期转换,从而抑制卵巢癌细胞的生长。     

1α,25-二羟基维生素D3对人卵巢癌细胞株表皮生长因子受体的调节

目的:研究1α,25-二羟基维生素D3(1,25VD3)对人卵巢癌细胞株表皮生长因子受体(EGFR)表达的调节作用.方法:用1,25VD3 或其类似物EB1089处理人卵巢癌细胞株OVCAR3、2008和CAOV3,应用Western blot、Northern Blot和mRNA稳定性分析等方法检测EGFR表达的变化.结果:1,25VD3或EB1089作用后三种人卵巢癌细胞中EGFR的蛋白和mRNA表达水平均下降.用1,25VD3 和放线菌素D、RNA酶抑制剂处理OVCAR3细胞后,Northern blotting分析结果显示,1,25VD3 作用4h后没有改变EGFR mRNA的半衰期.结论:1,25VD3在mRNA转录水平下调人卵巢癌细胞株EGFR的表达.     

1α，25-二羟基维生素D3对人卵巢癌细胞株表皮生长因子受体的调节

目的：研究1α,25-二羟基维生素D3（1,25VD3）对人卵巢癌细胞株表皮生长因子受体（EGFR）表达的调节作用。方法：用1,25VD3或其类似物EB1089处理人卵巢癌细胞株OVCAK3、2008和CAOV3,应用Westernblot、NorthernBlot和mRNA稳定性分析等方法检测EGFR表达的变化。结果：1,25VD3或EB1089作用后三种人卵巢癌细胞中EGFR的蛋白和mRNA表达水平均下降。用1,25VD3和放线菌素D、RNA酶抑制剂处理OVCAR3细胞后,Northernblotting分析结果显示,1,25VD3作用4h后没有改变EGFRmRNA的半衰期。结论：1,25VD3在mRNA转录水平下调人卵巢癌细胞株EGFR的表达。     

1,25(OH)2D3 Attenuates IL-1b-Induced Epithelial-to-Mesenchymal Transition Through Inhibiting the Expression of lncTCF7

The activated form of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], regulates numerous cellular processes, including inhibition of cancer progression. IL-1 has been reported to facilitate cancer development, especially by inducing an epithelial-to-mesenchymal transition (EMT) in several malignant tumors. However, the underlying mechanism of 1,25(OH)2D3 and IL-1 in colorectal cancer (CRC) still remains largely unknown. To fill in this knowledge gap, we measured cell proliferation and invasion by CCK-8 and Transwell assays after stimulation with 1,25(OH)2D3 and IL-1 . E-cadherin and vimentin were chosen as markers of EMT measured by immunofluorescence, quantitative real-time PCR (qRT-PCR), and Western blot. The expression and function of the vitamin D receptor (VDR) was evaluated by Western blot and luciferase reporter assay. qRT-PCR and RNA-FISH were performed to detect the expression and location of lncTCF7 in vitro. The binding sites of VDR in the lncTCF7 promoter were confirmed by a chromatin immunoprecipitation assay. Based on the above experiments, we found that 1,25(OH)2D3 attenuates IL-1 - induced increased proliferation and invasion in colorectal cancer through enhancing VDR, which inhibits the expression of lncTCF7 by directly binding to its promoter region.     

血清25-羟基维生素D3及钙水平与胃癌的关系

目的 探讨血清25-(OH)D3及钙（Ca²⁺）水平与胃癌的关系。方法 选取郑州大学第一附属医院经病理确诊为慢性浅表性胃炎（对照组）患者63例,胃癌（病例组）患者118例。采用酶联免疫吸附法（ELISA）测定入选对象血清25-(OH)D3水平,离子选择性电极（ISE）电位法测定入选对象血清Ca²⁺水平。结果 病例组外周血25-(OH)D3水平显著低于对照组（t=15.07,P=0.000）,而其血清Ca水平与对照组比较,差异无统计学意义（t=1.109,P=0.269）,病例组内血清25-(OH)D3水平与Ca²⁺之间呈正相关（r=0.706,P=0.000）。外周血25-(OH)D3水平在不同细胞分化程度的胃癌组间差异有统计学意义（F=3.356,P=0.038）。胃癌经手术治疗后,术后外周血25-(OH) D3水平高于术前,差异有统计学意义（t=−8.017,P=0.000）。但胃癌淋巴结转移与无淋巴结转移组间血清25-(OH)D3水平无统计学意义（t=0.061,P=0.952）。结论 血清 25-(OH)D3水平可能与胃癌的发生、发展相关,手术治疗后其水平明显升高,可用于监测胃癌患者病情变化、预测预后。     

l‐Asparaginase‐mediated downregulation of c‐Myc promotes 1,25(OH)2D3‐induced myeloid differentiation in acute myeloid leukemia cells

Treatment of acute myeloid leukemia (AML) largely depends on chemotherapy, but current regimens have been unsatisfactory for long‐term remission. Although differentiation induction therapy utilizing 1,25(OH)₂D₃ (VD3) has shown great promise for the improvement of AML treatment efficacy, severe side effects caused by its supraphysiological dose limit its clinical application. Here we investigated the combinatorial effect of l ‐asparaginase (ASNase)‐mediated amino acid depletion and the latent alternation of VD3 activity on the induction of myeloid differentiation. ASNase treatment enhanced VD3‐driven phenotypic and functional differentiation of three‐different AML cell lines into monocyte/macrophages, along with c‐Myc downregulation. Using gene silencing with shRNA and a chemical blocker, we found that reduced c‐Myc is a critical factor for improving VD3 efficacy. c‐Myc‐dependent inhibition of mTORC1 signaling and induction of autophagy were involved in the enhanced AML cell differentiation. In addition, in a postculture of AML cells after each treatment, ASNase supports the antileukemic effect of VD3 by inhibiting cell growth and inducing apoptosis. Finally, we confirmed that the administration of ASNase significantly improved VD3 efficacy in the prolongation of survival time in mice bearing tumor xenograft. Our results are the first to demonstrate the extended application of ASNase, which is currently used for acute lymphoid leukemia, in VD3‐mediated differentiation induction therapy for AML, and suggest that this drug combination may be a promising novel strategy for curing AML.     

Downregulation of miR-302c and miR-520c by 1,25(OH)2D3 treatment enhances the susceptibility of tumour cells to natural killer cell-mediated cytotoxicity

Background:

NKG2D recognises several ligands, including polymorphic major histocompatibility complex class I chain-related chain-related proteins A and B (MICA/B) and unique long 16-binding proteins (ULBPs). These ligands are present on cancer cells and are recognised by NKG2D in a cell-structure-sensing manner, triggering natural killer (NK) cell cytotoxicity. However, the mechanisms that control the expression of NKG2D ligands in malignant cells are poorly understood. 1-α,25-Dihydroxyvitamin D3 (1,25(OH)2D3) was recently shown to enhance the susceptibility of melanoma cells to the cytotoxicity of NK cells. However, the function of 1,25(OH)2D3 in other cancers and its potential mechanisms of action remain unknown.

Methods:

The expression levels of miR-302c and miR-520c in Kasumi-1, K562, MCF7 and MDA-MB-231 cells were evaluated using quantitative real-time PCR. The targets of miR-302c and miR-520c were confirmed by luciferase reporter assay. The killing effects of NK92 cells against Kasumi-1, K562, MCF7 and MDA-MB-231 cells were examined using the CytoTox 96 Non-Radioactive Cytotoxicity Assay. The levels of cytokines IFN-γ and granzyme B, which indicate the activation of NK cells, were also measured by enzyme-linked immunosorbent assay.

Results:

Treatment with 1,25(OH)2D3 enhanced the susceptibility of both the haematological tumour cell line Kasumi-1 and solid tumour cell line MDA-MB-231 to NK92 cells. miR-302c and miR-520c expression was induced, and their levels inversely correlated with the levels of NKG2D ligands MICA/B and ULBP2 upon 1,25(OH)2D3 treatment. A luciferase reporter assay revealed that miR-302c and miR-520c directly targeted the 3′-UTRs of MICA/B and ULBP2 and negatively regulated the expression of MIA/B and ULBP2. Moreover, upregulation of miR-302c or miR-520c by transfection of their mimics remarkably reduced the viability of Kasumi-1 cells upon NK cell co-incubation. By contrast, the suppression of the activity of miR-302c or miR-520c by their respective antisense oligonucleotides improved the resistance of Kasumi-1 cells to NK cells.

Conclusion:

1,25(OH)2D3 facilitates the immuno-attack of NK cells against malignant cells partly through downregulation of miR-302c and miR-520c and hence upregulation of the NKG2D ligands MICA/B and ULBP2.     

PM2.5对斑马鱼胚胎发育早期氧化应激和DNA甲基化

目的：探讨细颗粒物（PM2.5）暴露后,斑马鱼胚胎发育过程中氧化应激和DNA甲基化基因表达的改变。方法：使用大功率大气PM2.5采样器收集PM2.5样品,超声后冷冻提取PM2.5,分别以0、5、20 μg/mL的浓度作用于斑马鱼胚胎。在暴露后2、6、24、48 h提取胚胎RNA,用荧光定量PCR法检测氧化应激相关基因超氧化物歧化酶1（sod1）和8-羟基鸟嘌呤DNA糖苷酶（ogg1）以及DNA甲基化相关基因DNA羟化酶1（tet1）、DNA甲基转移酶（dnmt1） mRNA的表达。结果：斑马鱼胚胎暴露于0、5、20 μg/mL的PM2.5后2~6 h,sod1、tet1和dnmt1的mRNA相对表达量均随着剂量升高而明显升高,呈剂量-效应关系（r依次=0.98、0.98、0.99,P均 < 0.05）;PM2.5暴露后2~6 h ogg1的mRNA相对表达量在5 μg/mL剂量组显著升高（P < 0.05）。而PM2.5暴露后24~48 h,sod1、ogg1、tet1和dnmt1的mRNA相对表达量相对2~6 h均明显下降（P < 0.05）。结论：PM2.5对斑马鱼胚胎的氧化应激相关基因sod1和ogg1以及DNA甲基化相关基因tet1和dnmt1的mRNA表达均有影响。     

米非司酮、维生素D3对乳腺癌细胞株MCF-7生长和凋亡的影响

目的:探讨米非司酮(RU486)和维生素D3(VitD3)对乳腺癌细胞株MCF-7生长和凋亡的影响,尤其是二者高浓度联合应用与单独高浓度应用之间的差异。方法:将MCF-7细胞株分别加入乙醇、VitD3和RU486。细胞培养24小时、48小时和72小时置光镜下观察细胞形态,利用体视学的方法进行计算细胞生存抑制率。72小时送流式细胞仪检查细胞分期、细胞凋亡率,经免疫组化、PI染色后行p53蛋白水平测定。结果:24小时、48小时、72小时低浓度组细胞生存抑制率与对照组乙醇相比较变化不大,而高浓度组和对照组相比较细胞生长抑制率明显升高。48小时比24小时抑制作用明显,72小时亦比48小时抑制作用明显。VitD310-7M RU48610-5M组比VitD310-7M组和RU48610-5M组72小时后的细胞生长抑制率明显升高。VitD310-7M组、RU48610-5M组、VitD310-7M RU48610-5M组较乙醇对照组相比,p53蛋白活化分子含量明显降低。结论:(1)RU486及VitD3的抗肿瘤作用有时间依赖性和剂量依赖性。(2)经析因分析后发现VitD310-7M和RU48610-5M两组具有交互作用(P<0.05),联合应用二者可以提高抗肿瘤作用。(3)RU486和VitD3可以引起乳腺癌细胞的凋亡,可能是通过一种或几种途径最终引起了p53蛋白的减少,推测其基因属突变型。