Wnt/β-catenin信号通路在1,25-二羟基维生素D3诱导人急性早幼粒细胞HL-60单核系分化中的作用

目的探讨Wnt/β-catenin信号通路在1,25-二羟基维生素D3[1,25-(OH)2-hydroxyvitamin D3,1,25-(OH)2 D3]诱导人急性早幼粒细胞HL-60向单核系分化中的作用。方法分别采用不同浓度的1,25-(OH)2 D3在不同时间诱导HL-60细胞向单核系分化,确定最佳药物浓度和诱导时间;使用最佳药物浓度0.1μmol/L 1,25-(OH)2 D3诱导HL-60细胞72 h,采用流式细胞技术、细胞化学染色技术检测并观察HL-60细胞的分化方向、分化程度及细胞增殖受抑情况;western blot检测ICAT、TCF1、c-Myc、cyclin D1、β-catenin蛋白的表达水平及pRB蛋白的磷酸化水平;采用免疫荧光法检测β-catenin在细胞内的分布情况。结果0.1μmol/L 1,25-(OH)2 D3于72 h时可有效诱导HL-60细胞向单核系分化;G 0/G 1期细胞增殖明显受抑(t=4.769,P<0.001),且细胞增殖抑制率升高(t=4.84,P<0.001);瑞氏染色结果发现,与对照组比较,成熟单核细胞比例显著升高[(61.2±13.6)%vs(0.8±0.2)%,t=7.49,P=0.001];western blot结果表明,ICAT蛋白表达上调(t=9.917,P<0.01),TCF1、c-Myc和cyclin D1蛋白表达下调(t分别为54.54,54.64和29.24,P均<0.001);此外,pRB蛋白的磷酸化水平亦下调(t=150.6,P<0.001);细胞β-catenin蛋白表达水平无显著性变化(P>0.99),但其在核内的表达水平下降(t=9.77,P=0.01);免疫荧光检测结果显示,β-catenin蛋白呈胞浆聚集状态。结论1,25-(OH)2 D3通过抑制Wnt/β-catenin信号通路,抑制HL-60细胞增殖并促进其单核系分化。     

mTOR信号通路调控1,25二羟维生素D_3抑制喉癌细胞Hep-2细胞增殖的研究

目的研究1,25二羟维生素D3抑制喉癌细胞Hep-2细胞增殖作用以及对雷帕霉素靶蛋白(mTOR)信号通路的影响。方法用不同剂量1,25二羟维生素D3(10-8 mol/L、10-7 mol/L、10-6 mol/L)分别处理Hep-2细胞24、48、72h,四甲基偶氮唑蓝法(MTT)检测Hep-2细胞的增殖情况,并计算抑制率;采用流式细胞仪分析1,25二羟维生素D3对Hep-2细胞周期分布的影响,Western blot检测1,25二羟维生素D3对mTOR信号通路的影响。结果不同浓度1,25二羟维生素D3均可抑制Hep-2细胞增殖,改变细胞周期分布,使G0/G1期Hep-2细胞比例增高。1,25二羟维生素D3干预后Hep-2细胞TSC1、TSC2蛋白表达较对照组增高(P0.01),Rheb蛋白表达明显降低;mTOR蛋白及其磷酸化水平表达与对照组比较均降低(P0.01),mTOR蛋白磷酸化表达降低尤为明显(P0.01);4EBP-1蛋白表达较对照组增高(P0.01)。结论 1,25二羟维生素D3改变喉癌细胞Hep-2细胞周期分布,影响mTOR信号通路蛋白表达,从而抑制细胞增殖。     

姜黄素对人类绒毛膜癌细胞增殖、凋亡的影响及机制

目的探讨姜黄素对人类绒毛膜癌细胞增殖、凋亡的影响及机制。方法将人类绒毛膜癌细胞JEG-3细胞株随机分为对照组和姜黄素干预组。姜黄素干预组加入100μl不同浓度的姜黄素与细胞孵育24 h;对照组只加入PRMI 1640培养液与等体积的二甲基亚砜(DMSO),MTT检测检测细胞抑制率。姜黄素干预组加入100μl姜黄素(终浓度为40μmol/L)与细胞孵育24 h后,TUNEL法检测细胞凋亡情况,Western blot检测增殖相关蛋白Ki-67和细胞周期蛋白D1(Cyclin D1)的表达,以及凋亡相关蛋白半胱氨酸蛋白酶(Caspase-3)、B淋巴细胞瘤-2(Bcl2)和Bcl2相关X蛋白(Bax)的表达。结果与对照组相比,姜黄素干预组JEG-3细胞培养24 h后吸光度显著下降(P 0. 05);姜黄素对JEG-3细胞抑制作用呈药物浓度依赖性,半抑制浓度(IC50)为(39. 33±1. 02)μmol/L;与对照组相比,JEG-3细胞的凋亡率明显升高,增殖相关蛋白Ki-67和Cyclin D1的表达显著降低(P 0. 05),凋亡相关蛋白Caspase-3和Bax的蛋白表达量显著升高(P 0. 05),Bcl-2的表达量显著降低(P 0. 05)。结论姜黄素可以下调人类绒毛膜癌细胞增殖相关蛋白的表达,上调凋亡相关蛋白的表达,调控其增殖和凋亡。     

高糖对大鼠肾小球系膜细胞LPA3（edg-7）表达影响及姜黄素的干预作用

目的探讨高糖对大鼠肾小球系膜细胞增殖及其LPA3（edg-7）表达和姜黄素的干预作用。方法体外培养大鼠肾系膜细胞,同步后采用高浓度的葡萄糖（30mmol/l）联合不同浓度姜黄素进行干预处理。应用MqT测量高糖及不同浓度姜黄素干预后肾小球系膜细胞增殖活力。应用RT-PCR检测正常培养及高糖作用后大鼠肾小球系膜细胞LPA3（edg-7）的表达及不同浓度姜黄素对其表达的影响。结果高糖促进大鼠系膜细胞增殖,上调LPA3（edg-7）的表达。姜黄素可抑制高糖刺激引起的系膜细胞增殖,下调LPA3（edg-7）表达。当姜黄素浓度大于或等于10／μmol／L时,系膜细胞增殖明显受到抑制（P〈0．05）,且表现为浓度依赖性。在基础状态下,系膜细胞表达一定量的LPA3（edg-7）,经高糖刺激后,LPA3（edg-7）基因表达上调;姜黄素可下调高糖刺激引起的LPA3（edg-7）mRNA高表达,当姜黄素浓度为20μmol／L时能明显下调高糖刺激引起的系膜细胞LPA3（edg-7）基因表达（P〈0．05）,且随其浓度增高,抑制作用有增强趋势。结论姜黄素能抑制高糖刺激大鼠肾系膜细胞增殖并可下调高糖刺激引起的LPA3（edg-7）基因表达。LPA3（edg-7）基因表达与肾小球系膜细胞增殖成正相关,下调LPA3（edg-7）表达,可能为姜黄素抑制肾小球系膜细胞增殖的作用途径之一。     

1,25-二羟基维生素D3诱导HL-60细胞向成熟单核细胞的分化

目的:观察1,25-二羟基维生素D3对白血病细胞株HL-60的分化诱导作用及其分子机制。方法:实验于2005-08/11在中南大学湘雅二医院中心实验室进行。将细胞浓度为2×108L-1HL-60细胞,分别以0,10-9,10-8,10-7,10-6mol/L的1,25-二羟基维生素D3孵育72h,对照组加入等量的无水乙醇。Western印迹技术鉴定维生素D受体在各组HL-60细胞的表达;Wright-Giemsa染色观察细胞形态学变化;流式细胞术检测细胞表面标志物CD14抗原的表达;反转录-聚合酶链反应法分析c-myc基因的表达。结果:①10-8mol/L1,25-二羟基维生素D3作用前后HL-60细胞均表达维生素D受体,且药物作用后维生素D受体的表达上调。②10-8mol/L1,25-二羟基维生素D3可明显诱导HL-60细胞向成熟单核细胞系分化,54.02%的细胞CD14表达阳性,对照组为5.23%(P<0.01)。③1,25(OH)2D3作用72h后,HL-60细胞的c-myc/β-actin比值为0.27,对照组为0.89,两者比较差异显著(P<0.01)。结论:①1,25-二羟基维生素D3可诱导HL-60细胞向成熟单核细胞分化,其受体维生素D受体参与了HL-60细胞代谢的调节。②1,25-二羟基维生素D3诱导细胞分化的同时伴随c-myc基因表达下调,表明c-myc基因表达下调是其诱导HL-60细胞分化的分子机制之一。     

熊果酸对人卵巢癌 SKOV3细胞增殖与凋亡的影响

【目的】探讨熊果酸(Ursolic acid,UA)对人卵巢癌 SKOV3细胞增殖与凋亡的影响。【方法】常规培养人卵巢癌 SKOV3细胞,应用 UA 浓度为5、10、20、40和80μmol/L 分别干预12 h、24 h 和48 h,采用 MTT 法检测细胞增殖;采用流式细胞仪检测细胞凋亡和细胞周期变化,应用 Western blot 技术检测细胞周期相关蛋白 P21、CyclinD1的表达水平。【结果】与空白对照组(0μmol/L UA)比较,40和80μmol/L UA 分别作用 SKOV3细胞12 h、24 h 和48 h 后 OD 值均显著降低(P <0.05);10、20和40μmol/L UA 作用24 h 后 SKOV3细胞早期凋亡率、晚期凋亡率和 G0/G1期比率较对照组升高,20和40μmol/L UA 作用24 h 后 SKOV3细胞周期蛋白 Cyclin D1明显降低而 P21水平显著升高(P <0.05)。【结论】体外实验中,UA 可抑制人卵巢癌 SKOV3细胞增殖和促其凋亡,其机制可能与细胞周期蛋白 Cyclin D1表达降低和 P21表达升高有关。     

活性维生素D3调控肾小球系膜细胞增殖与凋亡机制研究

作为一种类固醇激素,活性维生素D3（1,25（0H）1D3）可抑制肾小球系膜细胞的增殖,而细胞的增殖又与mTOR信号传导通路相关,本文就1,25（OH）2D3通过mTOR信号传导通路调控肾小球系膜细胞增殖与凋亡的机制作一综述。     

他克莫司对人肾小球系膜细胞细胞周期相关蛋白表达的影响

目的探讨他克莫司对体外培养的人肾小球系膜细胞细胞周期相关蛋白cyclin D1、cyclin E及p27kip1的影响及可能机制。方法设正常对照组和他克莫司处理组,他克莫司组设为三个组(浓度分别为10μmol/L、25μmol/L、50μmol/L)。四甲基偶氮唑盐法(MTT)检测他克莫司组处理12h后肾小球系膜细胞的增殖情况;流式细胞仪检测他克莫司组分别作用12、24、48h后系膜细胞的上述三种蛋白质及与其结合的CDK2和CDK4的表达情况;选择有明显抑制作用的时间点(12h),Westernblot检测上述三种蛋白质的表达情况。结果 (1)作用12h,他克莫司组均能抑制系膜细胞的增殖。(2)作用12、24、48h,与对照组相比,浓度为10μmol/L他克莫司对细胞周期蛋白cyclin D1、cyclin E及抑制因子p27kip1的表达无明显影响,而浓度为25、50μmol/L时,cyclin D1及cyclin E明显降低,p27kip1表达明显升高,呈一定的剂量效应关系;且相同浓度的他克莫司在作用三个时间点后,cyclin D1、cyclin E及p27kip1的表达无明显差异。(3)作用12h,与对照组相比,浓度为10μmol/L他克莫司对周期蛋白依赖性激酶CDK2、CDK4的表达无明显影响,而浓度为25、50μmol/L时,CDK2、CDK4的表达明显降低。结论一定浓度的他克莫司可抑制系膜细胞的增殖,其作用机制至少部分与其影响细胞周期相关蛋白的表达有关。     

选择性环氧合酶抑制剂NS-398对高糖诱导系膜细胞增殖的影响

目的:研究选择环氧合酶抑制剂NS-398对高糖诱导的系膜细胞抑制作用.方法:空白组,不加任何刺激剂;高糖组,加入30 mmol/L葡萄糖处理;干预组,不同浓度选择性环氧合酶抑制剂NS-398(5、10、20、40μmol/L)加入高糖培养的大鼠肾小球系膜细胞中,分别共同培养0、12、24、48、72 h,用[3H]-亮氨酸掺入法检测细胞蛋白质的从头合成情况,用流式细胞仪分析检测细胞G0-G1周期分布的变化,观察选择性环氧合酶抑制剂NS-398对系膜细胞增殖的影响.结果:高糖刺激的系膜细胞的[3H]-亮氨酸掺入量明显增加,用不同浓度的NS-398共同培养高糖系膜细胞,观察不同时间[3H]-亮氨酸掺入量,选择性环氧合酶抑制剂NS-398在5～40μmol/L浓度范围内,随着时间的延长、浓度的增加,可明显抑制MC[3H]-亮氨酸掺入,且呈浓度和时间依赖性.流式细胞仪检测干预组细胞G0-G1周期百分比较高糖组差异有显著性(P＜0.05或P＜0.01).结论:选择性环氧合酶抑制剂NS-398能抑制高糖诱导系膜细胞增殖,促进细胞凋亡.     

吲哚美辛诱导的胃癌细胞凋亡与Wnt/β-连接蛋白信号通路及其下游凋亡调控蛋白的关系

目的:探讨Wnt/β-连接蛋白(Catenin)信号通路及其下游凋亡调控蛋白Bcl-2和Caspase-3的表达与吲哚美辛引起的胃癌细胞凋亡的关系.方法:用终浓度为50、100、200 μmol/L的吲哚美辛干预SGC7901细胞24 h,并设不加吲哚美辛的对照组.用CCK8法检测细胞增殖活性,用TUNEL法检测涂片中胃癌细胞的凋亡率,用Western blot法检测细胞内β-Catenin、Bcl-2和Caspase-3蛋白的表达.结果:SGC7901细胞在50 μmol/L的吲哚美辛作用24 h后细胞存活率为(92.71±4.81)％,100和200 μmol/L浓度组的细胞存活率则明显降低.对照组细胞的凋亡率为0,吲哚美辛干预后的细胞的凋亡率明显升高.对照组β-Catenin和Bcl-2蛋白表达最高而Caspase-3表达最低.吲哚美辛干预后β-Catenin和Bcl-2的表达明显降低,Caspase-3表达明显增高,且表现出明显的浓度依赖性.结论:Wnt/β-Catenin信号通路在吲哚美辛引导的SGC7901细胞凋亡过程中发挥重要的作用,其下游调控蛋白Bcl-2、Caspase-3共同参与了这一过程的调控.     

Effect of 1,25-dihydroxyvitamin D3 and 13-cis-retinoic acid on in vitro hematopoiesis in the myelodysplastic syndromes

The effect of 2 X 10(-10) to 2 X 10(-7) mol/L 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) and 10(-9) to 10(-6) mol/L 13-cis-retinoic acid on in vitro differentiation and proliferation of marrow cells from patients with myelodysplastic syndrome (MDS) was assessed. Cells from 17 patients were studied by the semisolid technique, and cells from seven patients by both liquid and semisolid cultures. After incubation in liquid culture with 1,25(OH)2D3, in six of seven patients evaluated an increasing number of myeloid cells (185% to 470%) acquired the morphologic appearance of mature monocyte-macrophages, and a decrease in the number of immature myeloid cells (26% to 75%) was observed. Phagocytosis and killing of Candida albicans by monocyte-macrophages incubated with 1,25(OH)2D3 were normal and similar to those processes in untreated cells. 1,25(OH)2D3 increased the percentage of monocytes that phagocytosed C. albicans in three patients. Thirteen of 17 patients showed reduced myeloid cloning, and eight showed increased cluster formation. Cloning efficiency was significantly lower in patients with refractory anemia with excess of blasts and chronic myelomonocytic leukemia. Concentrations of 2 X 10(-9) to 2 X 10(-8) mol/L 1,25(OH)2D3 and 10(-8) to 10(-7) mol/L retinoic acid had a stimulatory effect on myeloid colony growth in five of the six patients with sideroblastic and refractory anemia, but in only two of the 11 patients with refractory anemia with excess of blasts and chronic myelomonocytic leukemia. The results indicate that the differentiation pattern of myeloid precursor cells from a subset of patients with MDS was altered by exposure to 1,25(OH)2D3.     

Effect of 1,25-dihydroxyvitamin D3 on mesangial cell proliferation

We have studied the effect of 1,25-dihydroxyvitamin D3 (1,25, (OH)2D3) on mesangial cell growth. Previous studies have shown that the monocyte-macrophage is the principal effector cell in immune-mediated nephritis; this cell infiltrates the glomerular mesangium, and its products may have important effects on the physiology of the mesangial cell. One of the substances produced by the activated macrophage is 1,25,(OH)2D3. We have investigated the effect of 1,25,(OH)2D3 on mesangial cell growth and found that this vitamin D metabolite suppresses the proliferation of mouse mesangial cells as assessed by mesangial cell tritiated thymidine uptake and by cell counts; this substance also antagonizes the mitogenic effect of epidermal growth factor on mesangial cell growth. By comparison, the vitamin D metabolite 25 hydroxyvitamin D3 has no significant suppressive effect on the proliferation of mesangial cells. It has also been possible to demonstrate that 1,25,(OH)2D3 could suppress the growth of mesangial cells that had been committed to proliferate by the prior addition of epidermal growth factor. The results of these studies are relevant to our understanding of the pathogenesis of the cellular abnormalities that occur in immune-mediated nephritis, and especially in subjects who have concurrent hypertension, because a segment of subjects with hypertension have demonstrable abnormalities in the levels of circulating 1,25,(OH)2D3.     

良性原发性甲状旁腺功能亢进症患者血清1,25-二羟基维生素D3水平变化

目的探讨良性原发性甲状旁腺功能亢进症(primary hyperparathyproidism,PHPT)患者血清1,25-二羟基维生素D3[1,25-dihydroxyvitamin D3,1,25(OH)2D3]水平变化及与甲状旁腺激素(parathyroid hormone,PTH)、血钙、血磷的关系。方法良性PHPT患者56例为观察组,同期体检健康者1118例为对照组,采用电化学发光法检测2组血清1,25(OH)2D3、PTH水平,比色法测定血钙水平,磷钼酸盐法测定血磷水平。比较2组维生素D缺乏[1,25(OH)2D3<20μg/L]、严重缺乏[1,25(OH)2D3<10μg/L]的比率及不同年龄分层患者血清1,25(OH)2D3水平变化,Pearson法分析观察组维生素D缺乏、严重缺乏患者血清1,25(OH)2D3与PTH、血钙及血磷的相关性。结果观察组维生素D缺乏比率、严重缺乏比率(94.64%、46.43%)高于对照组(62.79%、14.13%)(P<0.05);Pearson相关分析显示,观察组血清1,25(OH)2D3与PTH、血钙及血磷均无线性相关性(r=-0.226,P=0.352;r=-0.274,P=0.256;r=0.073,P=0.593)。观察组年龄18~40岁、>40~60岁、>60岁患者血清1,25(OH)2D3[(10.76±3.17)、(10.61±5.01)、(10.72±4.85)μg/L]低于对照组[18~40岁:(18.19±9.86)μg/L,>40~60岁:(17.18±9.19)μg/L,>60岁:(17.91±10.52)μg/L](P<0.05);观察组维生素D缺乏患者血清PTH[(818.86±233.49)ng/L]、血钙[(2.98±0.59)mmol/L]、血磷[(0.78±0.17)mmol/L]与维生素D严重缺乏患者[(640.09±622.69)ng/L、(2.96±0.69)mmol/L、(0.75±0.20)mmol/L]比较差异无统计学意义(P>0.05);观察组维生素D缺乏、严重缺乏患者血清1,25(OH)2D3与PTH(r=-0.360,P=0.060;r=0.071,P=0.723)、血钙(r=-0.225,P=0.250;r=-0.228,P=0.252)、及血磷(r=0.239,P=0.221;r=-0.208,P=0.297)均无线性相关。结论良性PHPT患者血清1,25(OH)2D3低于正常人群,维生素D缺乏比率较高,且血清1,25(OH)2D3与PTH、血钙及.血磷无线性相关。     

Vitamin D-dependent rickets type II. Defective induction of 25-hydroxyvitamin D3-24-hydroxylase by 1,25-dihydroxyvitamin D3 in cultured skin fibroblasts.

1,25(OH)2D3 induces 25(OH)D3-24-hydroxylase (24-OHase) in cultured skin fibroblasts from normal subjects. We evaluated 24-OHase induction by 1,25(OH)2D3 in skin fibroblasts from 10 normal subjects and from four unrelated patients with hereditary resistance to 1,25(OH)2D or vitamin D-dependent rickets type II (DD II). Fibroblasts were preincubated with varying concentrations of 1,25(OH)2D3 for 15 h and were then incubated with 0.5 microM [3H]25(OH)D3 at 37 degrees C for 30 min; lipid extracts of the cells were analyzed for [3H]24,25(OH)2D3 by high performance liquid chromatography and periodate oxidation. Apparent maximal [3H]24,25(OH)2D3 production in normal cell lines was 9 pmol/10(6) cells per 30 min and occurred after induction with 10(-8) M 1,25(OH)2D3. 24-OHase induction was detectable in normal fibroblasts at approximately 3 X 10(-10) M 1,25(OH)2D3. [3H]24,25(OH)2D3 formation after exposure to 1,25(OH)2D3 was abnormal in fibroblasts from all four patients with DD II. In fibroblasts from two patients with DD II, [3H]24,25(OH)2D3 formation was unmeasurable (below 0.2 pmol/10(6) cells per 30 min) at 1,25(OH)2D3 concentrations up to 10(-6) M. Fibroblasts from the other two patients with DD II required far higher than normal concentrations of 1,25(OH)2D3 for detectable [3H]24,25(OH)2D3 induction. In one, [3H]24,25(OH)2D3 production reached 2.9 pmol/10(6) cells per 30 min at 10(-6) M 1,25(OH)2D3 (30% normal maximum at 10(-6) M 1,25(OH)2D3). In the other, [3H]24,25(OH)2D3 production achieved normal levels, 7.3 pmol/10(6) cells per 30 min after 10(-6) M 1,25(OH)2D3. The two patients whose cells had a detectable 24-OHase induction by 1,25(OH)2D3 showed a calcemic response to high doses of calciferols in vivo. Our current observations correlate with these two patients' responsiveness to calciferols in vivo and suggest that their target organ defects can be partially or completely overcome with extremely high concentrations of 1,25(OH)2D3. The two patients whose cells showed no detectable 24-OHase induction in vitro failed to show a calcemic response to high doses of calciferols in vivo. In conclusion: (a) the measurement of 24-OHase induction by 1,25(OH)2D3 in cultured skin fibroblasts is a sensitive in vitro test for defective genes in the 1,25(OH)2D effector pathway. (b) This assay provides a useful tool for characterizing the target tissue defects in DD II and predicting response to calciferol therapy.     

Evidence that blood ionized calcium can regulate serum 1,25(OH)2D3 independently of parathyroid hormone and phosphorus in the rat.

This study asks whether arterial blood ionized calcium concentration (Ca++) can regulate the serum level of 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3] independently of serum phosphorus and parathyroid hormone (PTH). We infused either PTH (bovine 1-34, 10 U/kg body wt/h) or saline into awake and unrestrained rats for 24 h, through a chronic indwelling catheter. PTH raised total serum calcium and arterial blood ionized calcium, yet serum 1,25(OH)2D3 fell from 35 +/- 6 (mean +/- SEM, n = 10) with saline to 12 +/- 3 pg/ml (n = 11, P less than 0.005 vs. saline). To determine if the decrease in serum 1,25(OH)2D3 was due to the elevated Ca++, we infused PTH into other rats for 24 h, along with varying amounts of EGTA. Infusion of PTH + 0.67 micron/min EGTA reduced Ca++, and 1,25(OH)2D3 rose to 90 +/- 33 (P less than 0.02 vs. PTH alone). PTH + 1.00 micron/min EGTA lowered Ca++ more, and 1,25(OH)2D3 increased to 148 +/- 29 (P less than 0.01 vs. saline or PTH alone). PTH + 1.33 micron/min EGTA lowered Ca++ below values seen with saline or PTH alone, and 1,25(OH)2D3 rose to 267 +/- 46 (P less than 0.003 vs. all other groups). Thus, during PTH infusion lowering Ca++ with EGTA raised 1,25(OH)2D3 progressively. There were no differences in serum phosphorus concentration or in arterial blood pH in any group infused with PTH. The log of serum 1,25(OH)2D3 was correlated inversely with Ca++ in all four groups infused with PTH (r = -0.737, n = 31, P less than 0.001), and also when the saline group was included (r = -0.677, n = 41, P less than 0.001). The results of this study indicate that serum 1,25(OH)2D3 may be regulated by Ca++ independent of PTH and serum phosphorus levels in the rat. Since 1,25(OH)2D3 regulates gastrointestinal calcium absorption, there may be direct feedback control of 1,25(OH)2D3, by its regulated ion, Ca++.     

Influence of parathyroid hormone, calcitonin, 1,25(OH)2 cholecalciferol, calcium, and the calcium ionophore A23187 on erythrocyte morphology and blood viscosity

Parathyroid hormone and calcitonin, both endocrine modulators of calcium homeostasis, may influence blood rheology. Parathyroid hormone is known to reduce erythrocyte survival, leading to anemia. Calcitonin has been found to have some vascular effects. We have analyzed the Influence of parathyroid hormone (10(-7) to 10(-10) mol/L), calcitonin (10(-6) to 10(-12) mol/L), 1,25(OH)2 cholecalciferol (10(-7) to 10(-10) mol/L), additional calcium in plasma (+1 and 2 mmol/L), and the calcium lonophore A23187 (50 micromol/L) on erythrocyte morphology and blood viscosity at high shear rate (94 s(-1)) and low shear rate (0.1 s(-1)) in vitro. The loading of erythrocytes with calcium by the ionophore A23187 produced a marked echinocytic shape transformation, an increased blood viscosity at high shear rate caused by decreased deformability of these cells, and a decreased viscosity at low shear rate caused by decreased aggregation of echinocytes. In contrast, increasing plasma calcium concentrations, parathyroid hormone, calcitonin, and 1,25(OH)2 vitamin D3 had no effect on erythrocyte morphology and blood viscosity. We conclude that an increase in intraerythrocytic calcium leads to severe echinocytosis and altered blood viscosity. The endocrine modulators of calcium homeostasis--namely, parathyroid hormone, calcitonin, and 1,25(OH)2 vitamin D3--apparently do not influence intraerythrocytic calcium to a significant degree and have, therefore, no influence on cell morphology and blood viscosity.     

A vitamin D analogue (EB1089) inhibits parathyroid hormone-related peptide production and prevents the development of malignancy-associated hypercalcemia in vivo.

We have examined the effects of 1,25 dihydroxyvitamin D3 (1,25[OH]2D3) and a low calcemic analogue EB1089 on parathyroid hormone-related peptide (PTHRP) production and on the development of hypercalcemia in Fischer rats implanted with the Leydig cell tumor H-500. Leydig cell tumors were implanted subcutaneously into male Fischer rats, which received constant infusions intraperitoneally of either 1,25(OH)2D3 (50-200 pmol/24 h), EB1089 (50-400 pmol/24 h), or vehicle for up to 4 wk. A control group of animals received similar infusions without tumor implantation. Plasma calcium, plasma levels of immunoreactive iPTHRP, and tumor PTHRP mRNA levels were determined as well as tumor size, animal body weight, and animal survival time. Non-tumor-bearing animals receiving > 50 pmol/24 h of 1,25(OH)2D3 became hypercalcemic, whereas no significant change in plasma calcium was observed in animals receiving < or = 200 pmol/24 h of EB1089. Tumor-bearing animals receiving vehicle alone or > 50 pmol/24 h of 1,25(OH)2D3 became severely hypercalcemic within 15 d. However, animals treated with low dose 1,25(OH)2D3 and all doses of EB1089 maintained near-normal or normal levels of plasma calcium for up to 4 wk. Additionally, reduced levels of tumor PTHRP mRNA and of plasma iPTHRP were observed compared with controls in both vitamin D- and EB1089-treated rats. Infusion of 50 pmol/24 h of 1,25(OH)2D3 and 200 pmol/24 h of EB1089 significantly reduced tumor volume by the end of experiment. The analogue but not 1,25(OH)2D3 substantially prolonged survival time in tumor-bearing animals with longer survival achieved at the highest dose, 400 pmol/24 h, of EB1089. These studies demonstrate that 1,25(OH)2D3 and a low calcemic vitamin D analogue are potent inhibitors of PTHRP production in vivo. Low calcemic analogues may therefore represent important alternative therapy for malignancy-associated hypercalcemia.     

Impaired stimulation of 25-hydroxyvitamin D-24-hydroxylase in fibroblasts from a patient with vitamin D-dependent rickets, type II. A form of receptor-positive resistance to 1,25-dihydroxyvitamin D3.

We describe studies of the molecular defect in 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] action in cultured skin fibroblasts from a patient previously reported to have vitamin D-dependent rickets, type II. Binding of [3H]1,25-(OH)2D3 in fibroblast cytosol was normal with a Bmax (amount of high affinity binding) of 26 fmol/mg protein and a half-maximal saturation of 0.2 nM. Nuclear binding of [3H]1,25-(OH)2D3 following whole cell uptake was 1.5 fmol/micrograms DNA in patient fibroblasts compared with a range of 0.5-2.9 fmol/micrograms DNA in five control strains. The size of the [3H]1,25-(OH)2D3-receptor complex on sucrose density gradients, 3.8 S, was the same as in normal cells. This patient, therefore, appeared to have a receptor-positive form of resistance to 1,25-(OH)2D3. To document resistance to 1,25-(OH)2D3 in the fibroblasts we developed a method for detection of 1,25-(OH)2D3 action in normal skin fibroblasts. Following treatment of normal cell monolayers with 1,25-(OH)2D3 there was more than a 20-fold increase of 25-hydroxy-vitamin D-24-hydroxylase (24-hydroxylase) activity. Treatment of 10 control cell strains with 1,25-(OH)2D3 for 8 h increased the formation of 24,25-dihydroxy-vitamin D3 from 25-hydroxyvitamin D3 in cell sonicates from less than 0.02 to 0.11-0.27 pmol/min per mg protein. When cells from the patient with vitamin D-dependent rickets, type II were treated with 1,25-(OH)2D3 in a similar manner, maximal 24-hydroxylase activity was only 0.02 pmol/min per mg protein, less than a fifth the lower limit of normal. 24-Hydroxylase activity in fibroblasts from the parents of the patient increased normally following treatment with 1,25-(OH)2D3. We conclude that impaired induction of 24-hydroxylase in the presence of normal receptor binding is evidence for postreceptor resistance to the action of 1,25-(OH)2D3.     

1,25-(OH)_2D_3减轻糖尿病大鼠炎症因子的排泄及podocin表达缺失

目的:观察1,25-(OH)2D3对糖尿病大鼠尿液炎症因子单核细胞趋化蛋白- 1(MCP-1)、肿瘤坏死因子-α(TNF-α)、干扰素-λ(IFN-λ)及podocin表达水平的影响。方法:雄性Wistar大鼠随机分为对照组和糖尿病模型组;后者经链脲菌素诱导糖尿病模型成功后再随机分为糖尿病组和1,25-(OH)2D3组。给予1,25-(OH)2D3第6周测定大鼠尿红细胞、24小时尿蛋白、MCP-1、TNF-α、IFN-λ排泄及血糖水平。肾组织光镜电镜、肾小球podocin免疫组化及荧光染色定量分析检测podocin的表达。结果:与对照组相比,1,25-(OH)2D3组及糖尿病组尿红细胞、尿蛋白、MCP-1、TNF-α、IFN-λ、血糖明显升高(P<0.01),podocin蛋白表达明显下降(P<0.01)。与糖尿病组相比,1,25-(OH)2D3组尿红细胞、尿蛋白、MCP-1、TNF-α、IFN-λ、血糖明显降低(P<0.05或P<0.01),podocin蛋白表达明显增加(P<0.01)。结论:1,25-(OH)2D3可减轻糖尿病大鼠血尿、蛋白尿,减轻尿液炎症因子的排泄,恢复podocin的表达而有肾保护作...