PI3K在白细胞趋化中的作用

磷酸肌醇3-激酶（Phosphoinositide 3-kinase，PI3K）也称磷脂酰肌醇3-激酶（Phosphatidyl inositol 3-kinase，PI3K），是指能够磷酸化磷脂酰肌醇（Phosphatidyl inositol，PtdIns）及其衍生物肌醇环3位羟基的磷酸激酶。其产物的主要功能是作为膜引导信号介导质膜募聚相应的选择性蛋白。在真核细胞中，PI3K及其调节蛋白转位的功能是相当保守的。基因阻断或增强PI3K活性均对细胞和机体的功能产生较大影响心。大部分关于PI3K的早期研究主要集中在鉴定能够活化该酶的受体，以及观察激酶活化后继发的细胞应答。后来，学者们对PI3K的下游效应分子进行了鉴定。     

磷脂酰肌醇3激酶β(PI3Kβ)和PI3Kδ在KIT突变介导的细胞转化中起不同作用

目的 探究磷脂酰肌醇3激酶(PI3K)的不同亚型在三型酪氨酸激酶受体KIT突变介导的信号传递及细胞增殖中的作用.方法 在BaF3细胞中稳定表达野生型KIT及胃肠间质瘤中常见的KIT突变V560D、W557K558del,分别用PI3Kα、PI3Kβ、PI3Kδ亚型特异性抑制剂或者广谱PI3K抑制剂处理细胞,免疫沉淀法和...     

PI3K信号通路调控动脉粥样硬化发生发展的研究进展

磷脂酰肌醇3激酶(PI3K)信号通路参与动脉粥样硬化、肿瘤及免疫系统疾病的发生、发展。调节PI3K可以调控细胞凋亡、自噬、炎性反应等病理生理过程,为疾病的治疗提供了新思路。     

PI3K对心梗大鼠TNF-α表达的影响

目的 探讨磷脂酰肌醇-3激酶(phosphatidylinositol 3 kinase,PI3K)抑制剂LY-294002对心梗大鼠心肌肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)表达的影响。方法 45只SD大鼠,随机分为假手术组、心肌梗死组和PI3K抑制剂组。分别用Western blot和RT-PCR方法检测各组大鼠梗死区及其周围心肌TNF-α蛋白和TNF-α mRNA表达变化。结果 心肌梗死组心肌梗死区及其周围心肌TNF-α蛋白、TNF-α mRNA表达水平显著高于假手术组(p<0.05),PI3K抑制剂组心肌梗死区及其周围心肌TNF-α蛋白、TNF-α mRNA表达水平则明显低于心肌梗死组(p<0.05)。结论 PI3K能够上调心肌梗死大鼠TNF-α的表达。     

PI3K信号途径在哮喘患者外周血单个核细胞中的表达及意义

目的初步了解磷脂酰肌醇3激酶(PI3K)在急性发作期哮喘患者外周血单个核细胞(PBMC)中的活化情况,并观察其特异性抑制剂(Wortmannin)对Th1、Th2型细胞因子IFN-γ和IL-4表达的影响,探讨PI3K信号途径在哮喘T细胞免疫紊乱中可能的作用机制.方法以20例急性发作期哮喘患者作为实验组,15例健康人为对照组.PBMC提取采用Ficoll密度梯度离心法,半定量RT-PCR法测定PBMC中PI3KmRNA的表达,ELISA法测定不同浓度组Wortmannin处理的PBMC培养上清液中IFN-γ、IL-4水平.结果与对照组相比,急性发作期哮喘患者PI3KmRNA表达增高(P＜0.05).哮喘患者PBMC培养上清液IFN-γ水平低下、IL-4水平升高,与对照组比较差异均有统计学意义(P＜0.05和P＜0.01);同时加入不同浓度Wortmannin共培养后,实验发现均浓度依赖性地抑制了哮喘组及对照组IL-4的产生(P＜0.05或P＜0.01),而同组不同浓度梯度组间比较IFN-γ产生升高,但差异无统计学意义.结论哮喘患者急性发作时可能存在PI3K信号途径的过度活化,其过度活化对Th1型细胞因子IFN-γ影响不明显,有可能主要通过介导Th2型细胞因子IL-4的产生而参与Th1/Th2的失衡.     

PI3K/Nrf2通路在内毒素休克兔肾损伤中的作用

 目的: 探讨磷脂酰肌醇3-激酶/核因子E2相关因子2(PI3K/Nrf2)信号通路在内毒素休克兔急性肾损伤中的作用。方法: 健康清洁级雄性新西兰大白兔50只,随机分为5组(每组10只):对照组(C组)、内毒素休克模型组(L组)、渥曼青霉素+内毒素休克组(WL组)、渥曼青霉素组(W组)和二甲基亚砜组(D组)。W组、WL组经耳缘静脉注射渥曼青霉素0.6 mg/kg,D组注射二甲基亚砜0.08 mL/kg,C组和L组注射等容量生理盐水。30 min后,L组和WL组静脉注射脂多糖5 mg/kg(溶于2 mL生理盐水),C组、W组和D组注射等容量生理盐水。注射脂多糖或生理盐水后6 h处死兔,取肾组织进行肾损伤评分(HSK),测定血尿素氮(BUN)、肌酐(Cr)和尿α1-微球蛋白(α1-MG)浓度,检测肾组织MDA含量及SOD活性,检测肾组织Nrf2和HO-1的mRNA总Akt蛋白、p-Akt蛋白、总Nrf2蛋白、p-Nrf2蛋白、核Nrf2蛋白和HO-1蛋白水平。结果: 与C组、W组及D组比较,L组和WL组HSK、BUN、Cr、α1-MG及MDA含量升高,SOD活性降低,肾组织Nrf2和HO-1的mRNA、p-Akt蛋白、Nrf2总蛋白、p-Nrf2蛋白、Nrf2核蛋白及HO-1蛋白的水平上调(P<0.05)。C组、W组和D组之间上述指标差异无统计学显著性。与L组比较,WL组HSK、BUN、Cr、α1-MG及MDA含量升高,SOD活性降低,肾组织Nrf2和HO-1的mRNA、p-Akt蛋白、Nrf2总蛋白、p-Nrf2蛋白、Nrf2核蛋白及HO-1蛋白的水平降低(P<0.05)。结论: PI3K/Nrf2通路激活是内毒素休克诱发兔急性肾损伤时机体的适应性调节反应机制之一。     

磷脂酰肌醇3激酶在支气管哮喘大鼠气道平滑肌细胞中的表达

 目的： 探讨磷脂酰肌醇3激酶(PI3K)在支气管哮喘大鼠气道平滑肌细胞（ASMC）的表达。方法: 根据随机化分配原则将16只Wistar大鼠随机均分成2组，即哮喘组和正常对照组；模型建立后分别从每只大鼠分离培养ASMC，流式细胞检测方法检测ASMC的生长分数，用免疫细胞化学荧光染色及Western blotting方法进行PI3K表达的检测，并利用图像分析系统进行半定量对比分析。结果: 流式细胞仪检测发现哮喘组ASMC的S+G2/M期细胞所占细胞总数的百分比 (27.90±3.44) %显著高于正常对照组(13.00±1.56)%,P<0.05；免疫细胞化学荧光染色及Western blotting方法检测发现哮喘组和正常对照组ASMC胞浆内均有PI3Kp85阳性表达，且哮喘组的表达明显高于正常对照组；大鼠ASMC中PI3K的表达与大鼠ASMC的生长分数呈显著正相关。 结论: PI3K表达的增加可能在哮喘ASMC增殖中起重要作用。     

糖尿病大鼠肺组织PI3K/Akt表达的变化及意义

目的研究糖尿病大鼠肺组织磷脂酰肌醇3激酶(PI3K)/丝氨酸-苏氨酸激酶(Akt)表达的变化及意义。方法 40只大鼠随机分为对照组(10)和糖尿病组(30),通过高糖、高脂饮食加腹腔注射小剂量链脲佐菌素(STZ)的方法建立2型糖尿病大鼠模型。糖尿病模型建立后,检测血糖、血脂的改变;采用免疫组织化学方法和Western blot方法检测各组大鼠肺组织中PI3K、Akt和p-Akt表达的变化情况。结果与对照组比较,糖尿病模型组大鼠血糖、血脂值明显增高(0.01)。免疫组织化学和Western blot结果显示,糖尿病模型组大鼠模肺组织PI3K、Akt和pAkt的表达量均显著高于对照组。结论 PI3K/Akt信号通路可能参了与糖尿病肺组织病变的发生发展。     

P2Y嘌呤受体活化的PI-3K信号通路在前列腺癌细胞生长和侵袭中的作用

目的研究P2Y嘌呤受体激动剂对前列腺癌细胞PI-3K信号通路的激活及对肿瘤细胞生长和侵袭的影响。方法以高转移的人前列腺癌细胞系1E8为研究对象,以Western blot法,用特异性识别磷酸化Akt的抗体检测PI-3K信号通路的活化情况;以细胞计数、流式细胞术、Matrigel侵袭实验等方法检测P2Y受体激活的PI-3K／Akt信号通路在前列腺癌细胞生长和侵袭中的作用。结果P2Y嘌呤受体激动剂以时间和剂量依赖的方式激活了1E8细胞中的PI-3K／Akt信号通路。其持续刺激导致的生长抑制作用主要通过将细胞阻滞在S期实现（刺激组S期细胞分数比对照组提高22．3％）。应用特异性抑制剂LY294002阻断PI-3K通路明显影响1E8细胞的生长,但不能逆转P2Y受体介导的生长抑制作用。P2Y受体瞬时激活对前列腺癌细胞体外侵袭的促进作用主要通过增强细胞的运动能力（刺激组划痕修复率比对照组提高21．2％）,而非提高基质金属蛋白酶（MMP）-2和MMP-9的酶解活性实现,且此促侵袭作用可被LY294002明显抑制。结论P2Y嘌呤受体可以激活人前列腺癌细胞的PI．3K信号通路,并通过这条通路促进肿瘤细胞的运动能力进而促进侵袭;该通路是前列腺癌细胞生长的重要调节通路,但不参与P2Y嘌呤受体对前列腺癌细胞生长抑制的调节。     

Classical swine fever virus NS5A regulates viral RNA replication through binding to NS5B and 3'UTR

In this report, classical swine fever virus (CSFV) NS5A inhibit viral RNA replication when its concentration reached and surpassed the level of NS5B. Three amino acid fragments of CSFV NS5A, 137-172, 224-268 and 390-414 individually were shown to be essential to NS5B binding. The former two fragments were independently necessary for regulation of viral RNA replication and correlated with NS5B and 3′UTR binding activity. We also found that amino acids W143, V145, P227, T246, P257, K399, T401, E406 and L413 of CSFV NS5A were essential to NS5B binding activity. Furthermore, these amino acids were shown to be necessary for viral RNA replication and infection and conserved in NS5A proteins of CSFV, BDV, BVDV and HCV. These results indicated that NS5A may regulate viral RNA replication by binding to NS5B and 3′UTR. NS5A can still regulate viral RNA synthesis through binding to 3′UTR when binding to NS5B is not available.     

对两种第三代丙肝试剂检测的不同功能区抗体组份研究

目的　比较第二代和第三代丙肝试剂对丙肝不同功能区抗体的检出能力。方法　用两种第二代抗 Ｈ Ｃ Ｖ Ｅ Ｉ Ａ 试剂（ Ｋ２ 中国制造, Ａ２ 美国制造） 及两种第三代主流试剂（ Ａ３ 德国制造, Ｏ３ 美国制造） 对１２１ 份来源于全国各地的供血员血样进行了检测。结果　该１２１ 份样品经 Ｒ Ｉ Ｂ Ａ Ｈ Ｃ Ｖ３ ．０ 试剂及 Ｐ Ｃ Ｒ 方法确证, 其中４３ 份为阳性样品,７８ 份为阴性样品。两种第二代试剂 Ｋ２ , Ａ２阳性检出符合率为３６／４３ 和３７／４３ , 两种第三代试剂 Ａ３ , Ｏ３ 阳性检出符合率分别为４０／４３ 和３９／４３ 。在４３ 份阳性样品中,２０ 份既含有抗 Ｈ Ｃ Ｖ 核心抗体, 也含有抗 Ｈ Ｃ Ｖ Ｎ Ｓ３ 抗体,用 Ｋ２ , Ａ２ , Ａ３ , Ｏ３ 四种试剂分别检出１８ 、１８ 、１９ 和２０ 份。１４ 份含有抗 Ｈ Ｃ Ｖ Ｎ Ｓ３ 抗体而不含有抗 Ｈ Ｃ Ｖ 核心抗体的样品中,用两种第二代试剂（ Ｋ２ , Ａ２） 分别检出９ 份和１０ 份, 而用两种第三代试剂（ Ａ３ , Ｏ３） 则全部检出。与此相反,９ 份含有抗 Ｈ Ｃ Ｖ 核心抗体而不含抗 Ｈ Ｃ Ｖ Ｎ Ｓ３ 抗体的样品, 用两种第二代试剂（ Ｋ２ , Ａ２）均可检出, 而用两种第三代试剂（ Ａ３ , Ｏ３） 却     

The hepatitis C virus core protein interacts with NS5A and activates its caspase-mediated proteolytic cleavage

Viral proteins interact with one another during viral replication, assembly, and maturation. Systematic interaction assays of the hepatitis C virus (HCV) proteins using the yeast two-hybrid method have uncovered a novel interaction between core and NS5A. This interaction was confirmed by in vitro binding assays, and coimmunoprecipitation in mammalian cells. Core and NS5A are also colocalized in COS-7 cells. Interestingly, NS5A is cleaved to give specific-size fragments, when core is coexpressed in mammalian cells. Overexpression of core produced many dying and rounded cells and effects such as DNA laddering and the truncation of poly(ADP-ribose) polymerase 1 (PARP1), both indicators of apoptosis. These observations led us to investigate the link between the induction of apoptosis by core and the cleavage of NS5A. The proteolysis of NS5A and these apoptotic events can be inhibited by caspase inhibitor, Z-VAD, indicating that core induces apoptosis and the cleavage of NS5A by caspases. In cells infected by the HCV, core may provide the intrinsic apoptotic signal, which produces truncated forms of NS5A. The biological function of core-NS5A interaction and the downstream effect of NS5A cleavage are discussed.     

Nonstructural protein 5A does not contribute to the resistance of hepatitis C virus replication to interferon alpha in cell culture

The hepatitis C virus (HCV) subgenomic replicon system was used to study a possible involvement of nonstructural protein 5A (NS5A) in the mechanisms of HCV resistance to interferon alpha (IFN-alpha). A series of chimeric HCV replicons was constructed. In these replicons, the NS5A gene in the backbone of the Con1 replicon was swapped by corresponding fragments obtained from four IFN-alpha responder and four IFN-alpha nonresponder patients that had been infected with the same HCV AD78 strain. Experiments with transfected Huh7 cells did not reveal significant differences in sensitivity of HCV RNA replication to IFN-alpha in cell clones, bearing chimeric Con1/AD78 replicons with NS5A sequences from IFN responders and nonresponders. Thus, these data provide no evidence that the NS5A protein contributes to the resistance of HCV replication to IFN-alpha.     

庚型肝炎病毒NS5区部分基因的表达及其在EIA中的初步应用

目的　在大肠杆菌中表达HGVNS5抗原 ,以建立HGV抗体血清学检测方法。方法　利用反转录PCR(RT PCR)法从人血浆中分离庚型肝炎病毒NS5部分基因 ,克隆到pRSETA载体 ,经酶切初步鉴定后进行DNA序列分析 ,结果表明克隆序列正确。以pPROEX 1为表达载体 ,转化DH5α ,诱导表达后进行SDS PAGE和Westernblot分析。结果　克隆的基因片段在大肠杆菌中表达出相对分子质量 (Mr)约 2 0× 10 3 的可溶性蛋白 ,其氨基端含有 6个组胺酸肽段 ,可经Ni NTA亲和层析纯化。免疫印迹分析表明 ,该表达产物可与HGV感染患者体内的HGV抗体特异性结合。结论　克隆了HGVNS5部分基因并获得初步表达 ,表达的HGVNS5蛋白具有特异抗体结合活性 ,为建立检测HGVNS5抗体的血清学方法奠定了基础     

The hepatitis C virus NS5A inhibitor (BMS-790052) alters the subcellular localization of the NS5A non-structural viral protein

The hepatitis C virus (HCV) non-structural (NS) 5A protein plays an essential role in the replication of the viral RNA by the membrane-associated replication complex (RC). Recently, a putative NS5A inhibitor, BMS-790052, exhibited the highest potency of any known anti-HCV compound in inhibiting HCV replication in vitro and showed a promising clinical effect in HCV-infected patients. The precise mechanism of action for this new class of potential anti-HCV therapeutics, however, is still unclear. In order to gain further insight into its mode of action, we sought to test the hypothesis that the antiviral effect of BMS-790052 might be mediated by interfering with the functional assembly of the HCV RC. We observed that BMS-790052 indeed altered the subcellular localization and biochemical fractionation of NS5A. Taken together, our data suggest that NS5A inhibitors such as BMS-790052 can suppress viral genome replication by altering the proper localization of NS5A into functional RCs.     

Cell culture-adaptive mutations in the NS5B gene of hepatitis C virus with delayed replication and reduced cytotoxicity

Remarkable advances have been made through recent demonstrations of hepatitis C virus (HCV) replication in cultured cells transfected with in vitro synthesized viral genome RNA. From the HCV JFH1 (genotype 2a) subcultured successively in Huh-7 cells we have identified several missense mutations near the junction of NS5A and NS5B genes. Reverse genetic analysis indicated that two mutations in the N-terminal region of NS5B replicase caused delayed viral RNA replication and protein expression in the early stage of infection. However, the mutant viruses showed significantly alleviated effects on cell growth inhibition, proteolysis of viral proteins, apoptotic DNA cleavage, and induction of antiviral responses, giving rise to a 100-fold higher titer compared to the parental JFH1 virus in a more extended time period. These results suggested that delayed replication and reduced cytotoxicity can be characteristic features of cell culture-adaptive mutants with enhanced infectivity.     

HCV核心蛋白在HepG2细胞中对COX-2表达的影响

目的研究丙型肝炎病毒(HCV)核心蛋白是否影响环氧化酶-2(COX-2)的表达。方法用PCR从含HCVH77株全长基因组序列质粒中扩增HCV核心蛋白基因,并克隆至pcDNA3.1载体中,构建HCV核心蛋白基因的真核表达载体HCV-C/pcDNA3.1。用HCV-C/pcDNA3.1和含COX-2启动子的荧光素酶报告载体COX2pro1.5kb/luc瞬时共转染HepG2细胞,测定萤火虫荧光素酶的活性,并用Westernblot检测COX-2蛋白的表达水平。结果成功地构建了HCV-C/pcD-NA3.1重组质粒。瞬时转染后的HepG2细胞中,COX2启动子荧光素酶的活性显著增强。Westernblot检测,发现COX-2的表达明显升高。结论HCV核心蛋白在HepG2细胞中激活COX-2启动子,并且明显诱导COX-2的表达,为进一步研究COX-2与HCV致病性的关系提供了新的实验依据。