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。     

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。     

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。     

肾上腺髓质素拮抗转化生长因子β1的促纤维化作用及其机制

目的:探讨肾上腺髓质素(AM)在人肾小管上皮细胞系(HK-2)对转化生长因子β₁(TGFβ₁)促纤维化作用的影响及机制。方法:细胞总胶原的合成和分泌以[³H]-脯氨酸掺入量及培养液内[³H]-羟脯氨酸放射活性来判断;ELISA法检测培养液中纤维连接蛋白(FN)含量;FN、IV型胶原和组织型金属蛋白酶抑制剂-1(TIMP-1)mRNA的表达采用RT-PCR法;信号蛋白Smad2及Smad6蛋白表达采用Westernblot法。结果:(1)AM在蛋白和mRNA水平均明显抑制TGFβ₁刺激的胶原和纤维连结蛋白的表达;(2)AM抑制TGFβ₁刺激的TIMP-1mRNA的上调;(3)AM(10^-8mol/L)本身对Smad2和Smad6的表达无明显影响,且对TGFβ₁刺激的Smad2的表达也无明显影响;但是可以明显上调被TGFβ₁抑制的Smad6的表达。结论:在HK-2细胞,AM通过上调抑制性Smad6的表达拮抗TGFβ₁的促纤维化作用。     

维甲酸对转化生长因子β1 诱导的HFL-I细胞Ⅲ型胶原、STAT3和PIAS3表达的影响

目的: 研究全反式维甲酸(ATRA)对转化生长因子β₁(TGF-β₁)诱导的人胚肺成纤维细胞(HFL-I)中Ⅲ型胶原(collagen Ⅲ)、信号转导子和转录激活子3(STAT3)和活化STAT3蛋白抑制剂(PIAS3)表达的影响。方法: 体外培养HFL-I细胞,5 μg/L TGF-β₁诱导0 h、6 h、12 h、24 h、48 h和72 h后,RT-PCR法检测collagen Ⅲ、STAT3和PIAS3 mRNA表达,诱导0 d、1 d、3 d和5 d后,Western blotting法检测STAT3和p-STAT3蛋白表达。不同浓度维甲酸干预,24 h后用RT-PCR法检测collagen Ⅲ、STAT3和PIAS3 mRNA表达,3 d后用Western blotting法检测STAT3和p-STAT3蛋白表达。结果: TGF-β₁诱导后,HFL-I细胞中collagen Ⅲ和STAT3 mRNA表达明显上调,PIAS3 mRNA表达明显下调,STAT3和p-STAT3蛋白表达明显上调(P< 0.05)。各浓度ATRA都下调TGF-β₁诱导的HFL-I细胞中collagen Ⅲ、STAT3 mRNA和STAT3、p-STAT3蛋白的表达,上调PIAS3 mRNA表达(P<0.05)。结论: ATRA可通过抑制TGF-β₁诱导的HFL-I细胞collagen Ⅲ和STAT3表达、上调PIAS3表达而起到抗肺纤维化作用。     

细胞周期调控蛋白p21WAF1在腹膜间皮细胞肥大中的作用

目的:观察p21^WAF1在高糖作用下腹膜间皮细胞肥大中的作用。方法:用RT-PCR和WesternBlot法测定在高浓度葡萄糖(含糖为1.38%、3.86%)作用24h后体外培养的大鼠腹膜皮细胞p21^WAF1水平;用流式细胞仪分析细胞周期分布。结果:高糖作用24h后大鼠腹膜间皮细胞大多出现细胞肥大和停滞于细胞周期的G1期,以含糖3.86%组明显。p21^WAF1mRNA和蛋白随着糖浓度增高表达增高(含糖组间P<0.05)。在和含糖组相同渗透压的甘露醇组和无血清常规培养基几乎无p21^WAF1mRNA和蛋白的表达。结论:p21^WAF1可能在高糖作用下间皮细胞的肥大及G1期阻滞中起重要的调节作用。     

溶血磷脂酸对上皮细胞整合素β6表达的影响

目的: 探讨溶血磷脂酸(LPA)在体外对整合素β₆(ITGB6)表达的影响,并进一步研究LPA诱导的活化转化生长因子β(TGF-β)在此过程中的作用。方法: 原代培养的正常人支气管上皮(NHBE)细胞接种于6孔板中,经LPA诱导,收集细胞,分别利用RT-PCR和流式细胞术检测ITGB6 mRNA及细胞表面蛋白的表达;利用转染有TGF-β应答性纤溶酶原激活物抑制剂1(PAI-1)启动子片段并融合萤火虫荧光报告基因片段的转化貂肺上皮细胞(TMLC)作为TGF-β活性报告细胞检测活化的TGF-β。结果: (1) 10 μmol/L LPA诱导2 h后,ITGB6 mRNA在上皮细胞中表达显著增加,具有明显的时间依赖性。(2) 10 μmol/L LPA诱导4 h后,上皮细胞表面ITGB6蛋白表达明显增加。(3) 抗α_Vβ₆抗体可阻断LPA诱导的活化TGF-β,但不能阻断LPA诱导的ITGB6 mRNA的表达。结论: (1) LPA可诱导上皮细胞ITGB6 mRNA和细胞表面蛋白的表达。(2) LPA诱导的ITGB6 mRNA 表达不依赖LPA 诱导的TGF-β活化。     

三七皂苷单体R1 抑制低氧高二氧化碳诱导的肺动脉平滑肌细胞p38 MAPK信号通路的活化

目的: 探讨三七皂苷单体R₁(R₁)减轻低氧高二氧化碳(CO₂)性肺动脉收缩的作用及其与p38 MAPK信号通路的关系。方法: 原代培养雄性SD大鼠肺动脉平滑肌细胞(PASMCs),取第2至5代对数生长期细胞至低氧高CO₂(1% O₂, 6% CO₂)条件下继续培养,并分别用8、40、100 mg/L R₁ 孵育24 h后收集细胞,采用免疫印迹法测定p38 MAPK磷酸化蛋白表达,半定量RT-PCR检测p38 MAPK mRNA的表达。结果: Western blotting和RT-PCR结果显示,低氧高CO₂组p-p38 MAPK蛋白和p38 MAPK mRNA表达明显高于对照组(N)组(P<0.01)。与低氧高CO₂组相比,R₁(8、40、100 mg/L)不同程度抑制了p-p38 MAPK蛋白和p38 MAPK mRNA的表达(P<0.01),并呈剂量依赖关系。结论: 低氧高CO₂诱导PASMCs p38 MAPK活化,三七皂苷单体R₁可能通过抑制p38 MAPK通路减轻低氧高CO₂性肺动脉收缩。     

干扰素诱导蛋白p204表达变化对大鼠血管平滑肌细胞增殖及p21表达的影响

目的: 观察干扰素诱导蛋白p204对鼠血管平滑肌细胞(VSMCs)增殖及p21表达的影响,探讨p204在VSMCs增殖调控中的作用及可能机制。方法: 应用干扰素 α(IFN-α)处理和p204基因( Ifi204 )的小干扰RNA(siRNA)瞬时干预体外培养的VSMCs,用MTT法测定细胞活力反映细胞增殖,流式细胞仪分析细胞周期,用半定量RT-PCR 法和Western blotting 分别检测p204和p21的mRNA和蛋白表达。结果: IFN-α可诱导鼠VSMCs p204 mRNA和蛋白表达上调,降低VSMCs细胞活力和细胞周期G₁/S转换,伴p21 mRNA和蛋白表达上调。转染 Ifi204 siRNA可抑制p204和p21表达,提高VSMCs细胞活力和促进细胞周期G₁/S转换。结论: p204表达可抑制鼠VSMCs的增殖,该效应可能与激活p21表达有关。     

2型糖尿病患者CD14CD16单核细胞的水平及其对LPS和IL-15刺激的反应

目的: 研究2型糖尿病(T2DM)患者外周血CD14⁺CD16⁺单核细胞的比例及脂多糖(LPS)联合白细胞介素 15(IL-15)对其的影响,以了解炎症性免疫反应在T2DM中的可能作用机制。方法: 对28例T2DM患者和20例健康志愿者外周血用流式细胞术检测CD14⁺CD16⁺单核细胞的比例,并分离其外周血单个核细胞(PBMC),用LPS和IL-15干预4 h,收集PBMC和培养上清。分别采用Western blotting检测PBMC内STAT5和p-STAT5蛋白表达。免疫荧光检测p-STAT5蛋白表达,ELISA法检测外周血25-羟维生素D₃ 和IL-6浓度,免疫比浊法检测其外周血C-反应蛋白(CRP)水平, ELISA法检测LPS和IL-15干预后细胞培养上清IL-6和单核细胞趋化蛋白-1(MCP-1)的浓度。结果: T2DM组外周血CD14⁺CD16⁺单核细胞数量明显高于正常对照组(P<0.01),并与血清CRP和IL-6水平呈正相关(r=0.394,P<0.05和r=0.741,P<0.01),与25(OH)D₃浓度呈负相关(r=0.409,P<0.01),且25(OH)D₃水平与CRP和IL-6水平均呈负相关(r=-0.479和r=-0.774,均P<0.01),LPS联合IL-15刺激后PBMC的p-STAT5蛋白表达水平和PBMC培养上清IL-6、MCP-1浓度明显高于正常对照组(P<0.01),且T2DM患者p-STAT5的表达存在激活现象。结论: T2DM患者体内存在单核细胞功能紊乱,这种功能紊乱可能与活性维生素D₃不足有关,二者可能参与了T2DM患者体内免疫炎症反应并与微炎症互为因果,从而导致了T2DM及其并发症的发生发展;其作用机制可能与STAT5信号通路的激活有关。     

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。     

Surface roughness modulates the response of MG63 osteoblast-like cells to 1,25-(OH)(2)D(3) through regulation of phospholipase A(2) activity and activation of protein kinase A.

Implant surface roughness influences osteoblast proliferation, differentiation, and local factor production. Moreover, the responsiveness of osteoblasts to systemic hormones such as 1, 25-(OH)(2)D(3) is altered by the effects of surface roughness; on the roughest Ti surfaces the effects of roughness and 1, 25-(OH)(2)D(3) are synergistic. Prostaglandin E(2) (PGE(2)) appears to be involved in mediating the effects of surface roughness on the cells, as well as in the response to 1,25-(OH)(2)D(3). However, it is not yet known through which signaling pathways surface roughness exerts its effects on the response of osteoblasts to 1, 25-(OH)(2)D(3). The present study examined the potential role of protein kinase A (PKA), phospholipase A(2)(PLA(2)), and protein kinase C (PKC) in this process. MG63 osteoblast-like human osteosarcoma cells were cultured on cpTi disks with R(a) values of 0. 54 microm (PT), 4.14 microm (SLA), or 4.92 microm (TPS). PKA was inhibited by adding H8 to the cultures; similarly, PLA(2) was inhibited with quinacrine or activated with melittin, and PKC was inhibited with chelerythrine. Inhibitors or activators were included in the culture media through the entire culture period or for the last 24 h of culture. In addition, cultures were treated for 24 h with inhibitors or activators in the presence of 1,25-(OH)(2)D(3). The effects on cell number and alkaline phosphatase specific activity were determined after 24 h; PKC activity was determined after 9 min and at 24 h. Cell number was reduced on rough surfaces, and alkaline phosphatase activity was increased. 1,25-(OH)(2)D(3) had a synergistic effect with surface roughness on alkaline phosphatase. However, neither surface roughness nor 1,25-(OH)(2)D(3) had an effect on PKC. H8 treatment for 24 h inhibited cell number and alkaline phosphatase on all surfaces; however, when it was present throughout the culture period, the PKA inhibitor had no effect on cell number, but decreased alkaline phosphatase-specific activity. H8 reduced the 1,25-(OH)(2)D(3)-mediated effect on cell number and alkaline phosphatase. Quinacrine inhibited cell proliferation and alkaline phosphatase on all surfaces and further reduced the 1,25-(OH)(2)D(3)-dependent decreases in both parameters. Melittin had no effect when applied for 24 h and did not modify the 1,25-(OH)(2)D(3) effect; however, when present throughout the culture period, it caused a decrease in proliferation and an increase in enzyme activity. Chelerythrine, the PKC inhibitor, only inhibited cell proliferation when it was present throughout the entire culture period. However, it decreased alkaline phosphatase in cultures treated for 24 h, but increased enzyme activity when it was present for the entire culture period. The results indicate that surface roughness and 1,25-(OH)(2)D(3) both mediate their effects through PLA(2) which catalyzes the rate-limiting step in PGE(2) production. Further downstream, PGE(2) activates PKA. Surface roughness-dependent effects are also mediated through PKC, but only after the cells have reached confluence and are undergoing phenotypic maturation. The effect of surface roughness on responsiveness to 1,25-(OH)(2)D(3) is mediated through PLA(2)/PKA and not through PKC.     

Influence of particle size in the effect of polyethylene on human osteoblastic cells

The influence of two different sizes of polyethylene particles (< 30 and 20-200 microm) on osteoblastic function has been studied in primary human bone cell cultures. Cells were obtained from trabecular bone fragments of patients undergoing knee reconstructive surgery. On reaching confluency, cells were subcultured in three flasks: < 30 microm polyethylene particles were added to the first flask, 20-200 microm particles to the second flask and none to the third flask, which was the control. The resulting subcultures were incubated until confluence. Osteoblastic function was evaluated by assaying the secretion of osteocalcin, alkaline phosphatase, and C-terminal type I procollagen (PICP), with or without 1.25(OH)2D3 stimulation in the cell-conditioned medium. Adding < 30 microm polyethylene particles to these osteoblastic cell cultures increased the levels of osteocalcin secreted after 1,25(OH)2D3 stimulation. Treating stimulated or basal osteoblastic cultures with either polyethylene particle size did not affect alkaline phosphatase secretion. However, the addition of <30 microm polyethylene particles decreased PICP levels in the basal and stimulated cultures. A parallel series of osteoblastic cultures was treated with < 30 microm polyethylene particles and stimulated or not with 1,25(OH)2D3 to determine the effect on osteocalcin mRNA expression using RT-PCR amplification. Polyethylene particle-treated cultures had higher osteocalcin mRNA expression regardless of whether they had been stimulated with 1,25(OH)2D3 or not. We conclude that particle size affects the influence of polyethylene on osteoblastic function markers. Particles with a diameter of less than 30 microm increase osteocalcin expression and secretion.     

糖皮质激素受体β对糖皮质激素受体α转录激活功能的影响

目的:通过研究糖皮质激素受体β对糖皮质激素受体α转录激活功能的影响,探讨糖皮质激素受体β存在的生物学意义。方法:将糖皮质激素受体α的报告基因CAT和糖皮质激素受体β共转染入人骨肉瘤细胞系HOS-8603,激素处理后,用ELISA方法检测CAT活性;用定量RT-PCR方法检测糖皮质激素受体β的稳定过度表达对糖皮质激素诱导p21mRNA表达的影响。结果:糖皮质激素受体β抑制报告基因CAT的表达,随着糖皮质激素受体β转染量的增加,抑制效果越明显;糖皮质激素受体β同样可以抑制糖皮质激素对糖皮质激素受体α内源性靶基因p21mRNA的诱导。结论:糖皮质激素受体β对糖皮质激素受体α的转录激活功能有抑制作用,糖皮质激素受体β可能是糖皮质激素受体α的内源性抑制因子。     

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.     

Regulation of matrix vesicle metabolism by vitamin D metabolites

Matrix vesicles are membrane organelles found in the extracellular matrix of calcifying cells. Vitamin D-responsive alkaline phosphatase specific activity has been localized to matrix vesicles in chondrocyte and osteoblast cultures. The effect of hormone is both metabolite and cell specific. Alkaline phosphatase in matrix vesicles produced by resting zone chondrocytes is stimulated by 24,25(OH)2D3 whereas alkaline phosphatase in matrix vesicles produced by growth zone chondrocytes is responsive to 1,25(OH)2D3. However, mesenchymal cell cultures, which exhibit a chondrogenic phenotype when exposed to bone inductive proteins in vitro, produce vesicles with alkaline phosphatase activity that is unaffected by either 1,25(OH)2D3 or 24,25(OH)2D3. Incorporation and release of arachidonic acid into phosphatidylethanolamine is also differentially regulated by 1,25(OH)2D3 and 24,25(OH)2D3 in chondrocytes. These data suggest that vitamin D metabolites may regulate endochondral ossification by altering matrix vesicle enzyme activities, perhaps through changes in membrane phospholipid metabolism.     

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。     

Biological significance of glucocorticoid receptor beta

Biological significance of glucocorticoid receptor beta