钙调素依赖蛋白激酶Ⅱ调控SSH-1L活性的作用

目的 探讨钙(Ca~(2+))/钙调素依赖蛋白激酶Ⅱ(CaMKⅡ)对Slingshot-1L(SSH-1L)活性的调控作用.方法 用脂质体法将含Myc-SSH-1L的重组质粒转染至MG63细胞,经不同浓度的钙离子载体A23187诱导10 min,蛋白印迹实验,观察P-cofilin、P-SSH1L、P-CaMK Ⅱ水平的变化;体内及体外实验检测CaMKⅡ对SSH-1L的磷酸化及活性调控作用.结果 当 A23187浓度为1 μmol/L时CaMK Ⅱ活性达到了最大,同时此浓度下P-cofilin水平以及SSH-1L的978位点丝氨酸的磷酸化水平升高.体外实验证实CaMK Ⅱ可使SSH-1L(WT)磷酸化,但是对其突变体SSH-1L(2 SA)无作用;同时CaMK Ⅱ明显抑制了SSH1L(WT)的活性,但对SSH-1L(2 SA)的活性无作用.结论 CaMKⅡ对SSH-1L的活性具有明显调控作用,且与SSH-1L的丝氨酸位点密切相关.     

1型多聚ADP核糖聚合酶激活NF-κB途径调节基质金属蛋白酶活性的机制研究

目的:探讨1型多聚ADP核糖聚合酶(PARP-1)通过激活NF-κB调节体外培养乳鼠心脏成纤维细胞基质金属蛋白酶(MMP)-2,MMP-9酶活性的机制.方法:使用10 μmol/L去甲肾上腺素 (NE)刺激体外培养的乳鼠心脏成纤维细胞24 h,利用荧光基团DCF-DA检测心脏成纤维细胞内活性氧(ROS)水平,使用明胶酶谱法检测MMP-2,MMP-9的酶活性水平,凝胶阻滞实验检测NF-κB的DNA结合能力,Western-blot检测PARP-1蛋白表达水平;使用PARP-1抑制剂3-氨基苯甲酰胺(3AB),α、β受体阻滞剂及抗氧化剂vitC干预后,观察上述指标的变化.结果:NE诱导心脏成纤维细胞内ROS产生增加,PARP-1蛋白表达增加,NF-κB的核转录能力明显增强,MMP-2、MMP-9酶活性明显增加.使用3AB抑制PARP-1活性,或使用抗氧化剂vitC及α、β受体阻滞剂等预先处理后可显著抑制NF-κB的DNA结合能力,进而减少NE诱导的MMP-2,MMP-9酶活性.结论:NE诱导心脏成纤维细胞内ROS产生明显增多,ROS激活PARP-1并促使其蛋白表达显著增高, PARP-1通过增强NF-κB的DNA结合能力调控MMP-2和MMP-9酶活性.     

Fibulin-2在亚剂量血管紧张素Ⅱ诱导心室重塑中的作用及机制

目的蛋白磷酸酶4(PP4)是白细胞介素6(IL-6)信号通路中新发现的调控因子。本文主要观察白细胞介素6诱导的肝脏胰岛素抵抗模型中PP4的表达及活性变化,并探讨PP4对白细胞介素6诱导的肝脏胰岛素抵抗模型中信号通路的影响。方法用白细胞介素6(10μg/L)刺激人肝癌细胞系HepG2细胞18 h,建立胰岛素抵抗模型。氧化酶法测HepG2细胞糖异生,应用蒽酮法测细胞内糖元合成;Western Blotting和定量PCR检测细胞内PP4的表达;免疫沉淀及磷酸酶活性分析检测PP4的活性;使用RNAi技术抑制PP4表达,并用Western Blotting检测胰岛素信号通路中一些关键蛋白,如IRS1、P-IRS1、JNK和P-JNK的表达。结果白细胞介素6刺激HepG2细胞后,导致HepG2细胞糖异生能力增强,细胞内糖元合成显著降低,出现明显胰岛素抵抗状态。Western Blotting和定量PCR结果均表明白细胞介素6刺激HepG2细胞后,PP4表达较正常组明显升高,且免疫沉淀及磷酸酶活性分析结果证明PP4活性也显著升高。同时,Western Blotting结果显示,白细胞介素6刺激后,IRS1表达下降,P-I...     

RhoA/ROCK与糖尿病肾病

Rho具有与GTP及GDP结合的能力,同时具有GTP酶活性,通过与GTP、GDP结合形式的转换调控许多细胞内信号转导.糖尿病时在高糖环境下肾小球系膜细胞RhoA/Rho激酶活性增加,诱导激活蛋白(AP)-1、转化生长因子(TGF)-β、血管内皮生长因子(VEGF)等上调,抑制内皮型一氧化氮合酶(eNOS),增强Ca2+依赖的血管平滑肌收缩,在肾脏缺血、纤维化中起重要作用.他汀类药物亦通过抑制RhoA活性,对肾脏结构和功能起保护作用.     

mTOR抑制剂PP242对高糖诱导足细胞损伤保护机制的实验研究

目的探讨高糖诱导的小鼠肾足细胞增殖、凋亡和Podocin蛋白表达的变化以及哺乳动物雷帕霉素靶蛋白(mTOR)抑制剂PP242的干预效应。方法以体外培养的条件永生性的小鼠肾足细胞为研究对象,将足细胞分为4组。正常组给予葡萄糖5.5 mmol/L;高糖组给予葡萄糖30 mmol/L;PP242干预1组给予葡萄糖30 mmol/L、1μmol/L PP242; PP242干预2组给予葡萄糖30 mmol/L、10μmol/L PP242。用四甲基偶氮唑蓝(MTT)法检测PP242对高糖诱导的足细胞增殖的影响,用流式细胞仪检测PP242对高糖诱导的足细胞凋亡的影响,用Western Blot方法检测PP242对高糖诱导的足细胞Podocin蛋白表达。结果高糖诱导足细胞48 h后,与正常组比较,高糖组细胞增殖减少,细胞凋亡增加,Podocin蛋白表达降低,差异均有统计学意义(P0.05)。高糖诱导经PP242干预后,与高糖组比较,PP242干预1组、PP242干预2组足细胞增殖增加,足细胞凋亡减少,Podocin蛋白表达明显升高,差异均有统计学意义(P0.05)。结论 PP242可以部分减少高糖诱导的足细胞损伤,具有潜在的足细胞保护作用。     

利培酮抑制结肠癌细胞株SW480生长的作用机制

目的:探讨利培酮对人结肠癌细胞株SW480生长抑制的作用机制及其相关研究.方法:表皮生长因子和利培酮单独或联合作用于SW480细胞,通过蛋白印迹实验观察蛋白激酶B(protein kinase B,PKB)及细胞外信号调节激酶1/2(extracellular signal-regulated kinase1/2,ERK1/2)磷酸化程度,利用RT-PCR判断细胞因子信号转导抑制因子基因表达水平,利用显微镜及细胞计数方法判断细胞生长情况.结果:利培酮抑制人表皮生长因子诱导的结肠癌细胞株SW480细胞的生长;其机制是通过激活ERK1/2的活性并诱导SOCS3的基因的表达,从而抑制PKB的磷酸化,最终抑制SW480的生长.结论:利培酮具有抑制表皮生长因子对人结肠癌细胞株的生长作用.     

维甲酸对HL-60细胞及NB4细胞端粒酶活性的调节

目的 :探讨全反式维甲酸 (ATRA)对白血病细胞端粒酶活性的调节作用及其机制。方法 :采用半定量端粒重复扩增 -银染法对 ATRA作用后 HL- 6 0、NB4细胞的端粒酶活性进行检测 ,并分析相对端粒酶活性与细胞分化比例之间的关系。结果 :1μmol/L ATRA在诱导两种细胞分化成熟的同时 ,端粒酶活性显著下调 ,84～ 96h变为阴性。相对端粒酶活性 (Y)与细胞分化比例 (X)呈对数相关关系 ,其拟合对数曲线方程 ,HL- 6 0细胞 Y=0 .46 - 49.32 lg X ,NB4细胞 Y=2 - 44 .79lg X。 0 .1～ 10μmol/L ATRA在诱导两种细胞分化成熟的同时 ,随药物浓度增加 ,对端粒酶活性的抑制作用逐渐增强。 ATRA直接作用于端粒酶提取液对酶活性无显著影响。结论 :白血病细胞中存在失活的端粒酶活性负调控机制。恢复端粒酶活性抑制开关的正常功能可能是 ATRA治疗白血病的分子机制之一。     

红霉素对香烟烟雾刺激的人巨噬细胞组蛋白去乙酰化酶2表达降低的抑制作用

目的 探讨红霉素对香烟烟雾刺激的人巨噬细胞组蛋白去乙酰化酶2（H DAC2）表达的影响及其机制研究.方法 体外培养人类单核细胞系U937细胞,用佛波脂将其诱导分化为人巨噬细胞.按传统方法制备好香烟烟雾提取物（CSE）用于实验.将传代后的细胞分组：对照组、CSE组、红霉素＋CSE组、HDAC的特异性抑制剂曲古霉素A（TSA）组.应用流式细胞仪检测人巨噬细胞活性氧（ROS）的含量;应用蛋白印迹实验（Western blot）检测HDAC2和核因子κB（NF-κB）蛋白表达;通过酶联免疫吸附试验检测细胞上清液中肿瘤坏死因子α（TNF-α）的浓度.结果 CSE可刺激人巨噬细胞释放ROS,具有浓度依赖性和时间依赖性;红霉素（1 mg/L）预孵育24 h可以增强1％ CSE抑制的人巨噬细胞HDAC2蛋白表达;红霉素（1 mg/L）预孵育24 h可以下调1％ CSE诱导的NF κB的活性;红霉素（1 mg/L）预孵育24 h可以抑制1％CSE诱导的TNF-α的合成和释放.结论 红霉素通过提高HDAC2蛋白表达进而抑制香烟烟雾诱导氧化应激导致的NF-κB活性增强和炎症介质TNF-α的合成和释放.     

Diplacone通过抑制氧化应激作用及NF-κB信号通路活性降低Hcy诱导的血管内皮细胞损伤

目的采用高同型半胱氨酸血症(HHcy)诱导体外培养的人脐静脉内皮细胞(HUVEC),建立血管内皮细胞炎性损伤模型,通过Diplacone预处理,在之前实验的基础上进一步探讨Diplacone对血管内皮细胞可能的保护作用机制。方法以HUVEC为研究对象,经过不同浓度的Diplacone预孵育2 h后,加入2 mmol/L同型半胱氨酸(Hcy)继续处理12 h,流式细胞术检测细胞凋亡率;通过检测活性氧(ROS)细胞比率和抗氧化指标超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)的活性及MDA含量的变化反应细胞氧化应激水平。Western blot和qRTPCR检测核因子κB(NF-κB)蛋白和mRNA表达水平的变化。结果与对照组比较,2 mmol/L Hcy模型组细胞凋亡率显著增加,ROS细胞比率和MDA含量显著升高,抗氧化指标SOD、GSH-Px的活性显著降低,NF-κB的蛋白和mRNA表达水平逐渐升高。与2 mmol/L Hcy模型组比较,0.1～10μmol/L Diplacone预处理后细胞凋亡率逐渐降低;氧化应激指标ROS细胞比率和MDA含量降低,抗氧化指标SOD、GSH-Px的活性增加; NF-κB的蛋白和mRNA表达水平逐渐降低。结论 Diplacone具有抑制Hcy诱导的HUVEC内皮损伤的作用,且作用机制可能是通过降低氧化应激作用和抑制NF-κB信号通路活性而降低细胞凋亡率,抑制细胞凋亡。     

LIMK1/Cofilin在瘦素诱导的结肠癌SW620细胞迁移中的作用

目的 观察瘦素对结肠癌迁移的影响,以及LIMK1/Cofilin信号通路的作用.方法 贴壁培养SW620细胞,分别转染YFP-LIMK1及pSUPER-LIMK1.以浓度为1 μmol/L的瘦素孵育细胞为实验组,通过Western印迹实验检测Cofilin磷酸化水平的变化;通过细胞迁移实验观察LIMK1在瘦素诱导的细胞迁移中的作用.结果 经瘦素诱导可使SW620细胞中Cofilin磷酸化水平增加,被LIMK1调控;细胞迁移实验显示LIMK1参与瘦素诱导的细胞迁移.结论 瘦素/LIMK1/Cofilin信号通路对结肠癌细胞的迁移具有明显的促进作用,可能与结肠癌的发生、发展相关.     

Celecoxib suppresses proliferation and metastasis of pancreatic cancer cells by down-regulating STAT3 / NF-kB and L1CAM activities

Objective

To explore the molecular mechanisms of celecoxib-induced pancreatic cancer suppression in vivo and in vitro.

Protein kinase A activates protein phosphatase 2A by phosphorylation of the B56delta subunit

Our previous studies of DARPP-32 in striatal slices have shown that activation of D1 receptors leads to cAMP-dependent dephosphorylation of Thr-75, the Cdk5 site in DARPP-32. In the current study, we have elucidated a mechanism whereby protein phosphatase 2A (PP2A) is activated by a cAMP/PKA-dependent pathway, leading to dephosphorylation of Thr-75. PP2A consists of a catalytic C subunit that associates with the scaffolding A subunit and a variety of B subunits. We have found that the A/C subunits of PP2A, in association with the B56delta (or PPP2R5D) regulatory subunit, is an active DARPP-32 phosphatase. The B56delta subunit expressed in HEK293 cells forms a heterotrimeric assembly that catalyzes PKA-mediated dephosphorylation at Thr-75 in DARPP-32 (also cotransfected into HEK293 cells). The B56delta subunit is phosphorylated by PKA, and this increases the overall activity of PP2A in vitro and in vivo. Among four PKA-phosphorylation sites identified in B56delta in vitro, Ser-566 was found to be critical for the regulation of PP2A activity. Moreover, Ser-566 was phosphorylated by PKA in response to activation of D1 receptors in striatal slices. Based on these studies, we propose that the B56delta/A/C PP2A complex regulates the dephosphorylation of DARPP-32 at Thr-75, thereby helping coordinate the efficacy of dopaminergic neurotransmission in striatal neurons. Moreover, stimulation of protein phosphatase activity by this mechanism may represent an important signaling pathway regulated by cAMP in neurons and other types of cell.     

Protein phosphatase 2A impairs IFNα‐induced antiviral activity against the hepatitis C virus through the inhibition of STAT1 tyrosine phosphorylation

Mammalian cells have developed several mechanisms to sense viruses and initiate adequate responses such as production of interferons. Interferons activate the antiviral response through the Jak‐STAT signalling pathway. To establish a chronic infection, viruses need to counteract this barrier of defence. The hepatitis C and hepatitis B viruses are known to up‐regulate the expression of protein phosphatase 2A (PP2A). In this study, we show that PP2Ac associates with Jak1/Tyk2/STAT1 and reduces Jak1/Tyk2/STAT1 phosphorylation resulting in an impairment of the IFNα‐induced HCV antiviral response. Using the fully infectious HCV cell culture system (HCVcc), we demonstrate that the PP2A catalytic activity is not required to block the antiviral effect of IFNα, although it is needed to support HCVcc replication. Our data suggest an important contribution of virus‐induced PP2Ac up‐regulation in the establishment of a chronic infection.     

Erythropoietin Administration May Potentiate Mobilization of Storage Iron in Patients on Oral Iron Chelation Therapy

Five, repeatedly transfused, patients with refractory anemia (RA) or RA with ringed sideroblast (RARS) subtypes of myelodysplastic syndrome (MDS), with serum ferritin (SF) levels of >2,000 μg/L, and one female with Hb E [β26(B8)Glu→Lys]/β⁰-thalassemia (thal) with an SF level of 1,760 μg/L, were treated with deferiprone (L1) at the dose of 4–6 g per day for at least 26 months. Beginning in the second month, all patients received recombinant human erythropoietin (rHuEPO) at the dose of 150 IU/kg thrice weekly, subcutaneously for 24 months. A significant increase in iron excretion after combined administration of L1 and rHuEPO compared to treatment with L1 as a single agent, was observed in all patients. The amount of excreted iron in urine ranged from 7.5 to almost 20 mg per day. In one patient, a response to rHuEPO resulted in transfusion independence and her SF decreased from 2086 to 879 μg/L. In four MDS patients, who remained dependent on red blood cell (RBC) transfusions, simultaneous administration of L1 and rHuEPO enabled the stabilization of SF levels, despite continuing iron load from the transfusions. Combined administration of rHuEPO and oral iron chelators may potentiate mobilization of storage iron and maintain iron balance in transfusion-dependent iron overloaded early MDS patients.     

蛋白磷酸酶2A抑制剂对胰腺癌细胞活力的影响及其机制

目的 研究蛋白磷酸酶2A(PP2A)抑制剂对胰腺癌细胞系PANC-1活力的影响及其机制.方法 用PP2A抑制剂斑蝥素和冈田酸处理PANC-1细胞.通过Western印迹检测核因子(NF) -κB通路的激活程度.通过转染PP2A活性亚基cα(PP2A cα)表达质粒、NF-κB抑制蛋白激酶(IKK)α和NF-κB抑制蛋白(IκB)α的显性负性突变体、p65干扰质粒,分别从各环节阻断NF-κB通路,通过四甲基偶氮唑盐比色法(MTT法)检测细胞活力.结果 PP2A抑制剂可使IKKa磷酸化,进一步使IκBa磷酸化并降解,继而释放p65入核.过表达PP2Acα、IKKα显性负性突变体、IκBα显性负性突变体或干扰p65可分别使斑蝥素对细胞活力的抑制率下降(31.85±13.37)％、(23.48±8.98)％、(22.63±5.81)％、(20.88±3.24)％,使冈田酸对细胞活力的抑制率下降(40.17±11.65)％、(27.34±14.28)％、(24.85±3.39)％、(27.08±3.81)％.结论 PP2A抑制剂通过PP2A/IKKα/IκBα/p65依赖性通路发挥抗胰腺癌作用.     

A Practical Chelation Protocol Based on Stratification of Thalassemic Patients by Serum Ferritin and Magnetic Resonance Imaging Cardiac T2*

Our previous study showed that combined therapy with deferiprone (L1) and deferoxamine (DFO) was safe and efficacious in reducing iron overload in poorly-chelated thalassemia major patients for the short-term but the magnetic resonance imaging (MRI) T2* evaluation was not available at that time. Since October 2006, we applied a standardized chelation protocol by stratifying transfusion-dependent thalassemic patients into three groups, namely well-chelated group (A), poorly-chelated group without (B) or with (C) risk of cardiac complications, based on their serum ferritin (SF) levels and magnetic resonance imaging (MRI) cardiac T2* measurements. The patients in each group were given options of chelation regimens to improve their iron overload status. Chelation regimens included continuation or intensification of DFO alone (Regimen Ic or Ii, respectively), L1 alone (Regimen II), and combined therapy with L1 and DFO (Regimen III). Group A patients continued with Regimen Ic. Group B patients could opt for either Regimen Ii or II/III. Group C patients could opt for either Regimen Ii or III. Serum ferritin levels and MRI cardiac and liver T2* measurements were evaluated after 1 year of treatment. Fifty-seven patients (27 males, 30 females; age range 5–34 years, median: 25 years) were categorized into Group A (n = 3), B (n = 20) and C (n = 34). All Group A patients continued with DFO treatment. In Group B, seven were on Regimen Ii, five on Regimen II and five on Regimen III. In Group C, five were on Regimen Ii, two on Regimen II and 26 on Regimen III. Significant improvement was noted only for Group C patients using Regimen III (combined therapy) in SF levels, cardiac T2* and liver T2* measurements.     

抑制性消减杂交技术克隆和筛选丙型肝炎病毒F蛋白的反式调节基因

目的 构建丙型肝炎病毒（HCV）F蛋白反式激活相关基因差异表达的差异cDNA，克隆HCV-F蛋白反式激活相关基因。方法 以HCV-F表达质粒pcDNA3．1（-）-F转染HepG2细胞，以空载体pcDNA3．1（-）为对照；制备转染后的细胞裂解液，从中提取mRNA并合成cDNA，经RsaI酶切后将实验组cDNA分成两组，分别与两种不同的接头衔接，再与对照组cDNA进行两次消减杂交及两次抑制性聚合酶链反应，将产物与T／A载体连接，构建cDNA消减文库，并转染大肠杆菌进行文库扩增，随机挑选克隆聚合酶链反应后进行测序及同源性分析。结果 成功构建人HcVF蛋白反式激活相关基因差异表达的cDNA。扩增后得到56个200～1000bP插入片段的克隆，随机挑选其中28个插入片段测序，并通过生物信息学分析获得其全长基因序列，结果共获得19种编码基因，其中2个为未知功能的新基因。结论 筛选到的cDNA全长序列，包括一些与细胞生长调节、物质代谢和细胞凋亡密切相关的蛋白编码基因。