病毒对宿主PKR信号转导途径抑制作用的研究进展

干扰素(interferon,IFN)诱导的双链RNA激活的蛋白激酶(double-stranded RNA-activated protein kinase,PKR)的激活是细胞抵御病毒感染的重要机制之一,活化的PKR可磷酸化真核起始因子2(eukaryotic initiation factor2,elF2)的α亚基,导致病毒蛋白合成停止,因此,PKR信号转导途径异常与病毒持续感染密切相关.@@1 PKR的结构与功能@@人类PKR基因位于染色体2p21-22,属于丝/苏氨酸蛋白激酶亚家族的成员,为一种双链RNA (double-stranded RNA,dsRNA)结合蛋白,相对分子质量为68kDa,含551个氨基酸.PKR由氨基末端的调节结构域和羧基末端的催化结构域构成,后者含有11个保守的激酶亚结构域,其中位于296位的赖氨酸在磷酸化反应时结合ATP.     

异源蓝舌病毒dsRNA与PolyI:C诱导人源体细胞产生IFN-β的比较研究

目的 探讨异源动物蓝舌病毒dsRNA诱导人源细胞产生干扰素(IFN)的能力.方法 借助体外培养系统,利用ELISA技术检测IFN诱导剂多聚次黄苷酸-胞苷酸(PolyI:C)、蓝舌病毒(BTV)及BTV dsRNA 3种不同处理因素作用后,培养细胞上清液中IFN-β的含量,以比较研究其诱导人肺癌细胞A549和人胚肺细胞HEL产生IFN-β的能力和特征.结果 这3种因子均能诱导人肺癌A549细胞和人正常肺细胞HEL产生IFN-β,BTV dsRNA诱导IFN-β产生的能力显著高于Poly I:C和BTV.浓度高的BTV dsRNA诱导细胞产生的IFN-β水平高,说明二者之间存在剂量依赖性.BTVdsRNA对不同性质的人源细胞(人肺癌A549细胞、人正常肺细胞HEL)具有不同的诱导产生IFN-β的能力,诱导人正常肺细胞HEL产生IFN-β的能力显著地高于诱导人肺癌细胞A549产生IFN-β的能力(P<0.05).结论 裸露的BTV dsRNA分子能有效地诱导人源细胞产生IFN-β;结合BTV和dsRNA对人的安全性以及人类正在努力寻找新的内源性IFN诱导剂的事实,可以推断BTV dsRNA具有发展成内源性IFN诱导剂之潜能.     

补中益气汤含药血清诱导肺癌A549细胞凋亡及抗氧化能力的研究

目的研究补中益气汤含药血清诱导肺癌A549细胞凋亡及其抗氧化能力的分子机制。方法利用补中益气汤含药血清干预体外培养的肺癌A549细胞,分别应用MTT法检检测细胞增殖,流式细胞术检测细胞凋亡发生,蛋白质免疫印迹实验检测Caspase 3活化,细胞活性氧实验检测细胞中活性氧(reactive oxygen species,ROS)的产生。结果补中益气汤含药血清能抑制A549细胞增殖,诱导Caspase 3依赖的细胞凋亡,且细胞凋亡发生与ROS活化相关。结论补中益气汤含药血清诱导A549细胞凋亡可能与ROS信号通路相关     

二甲基亚砜通过激活caspase-3以及抑制PARP-1引起A549细胞凋亡

目的 检测肺上皮癌细胞（A549）细胞经二甲基亚砜（DMSO）诱导发生凋亡后聚腺苷酸二磷酸核糖转移酶（PARP-1）的活性是否受到抑制。方法 通过MTT法检测caspase-3的活性升高以及TUNEL等方法验证二甲基亚砜可引起A549细胞凋亡后,采用western blotting 验证PARP-1是否被切割成俩个片段。结果 DMSO可诱导A549细胞发生凋亡,凋亡过程中caspase-3的活性上调,其下游底物PARP-1大量被切割成小片段。结论 DMSO诱导的A549细胞凋亡受到caspase-3-PARP-1信号分子的调节。     

LOC152742通过TLR4信号通路和炎症反应调控结核分枝杆菌对Ⅱ型肺泡上皮细胞的感染

目的 探讨长链非编码RNA(lncRNA)LOC152742对结核分枝杆菌感染的人Ⅱ型肺泡上皮细胞炎症反应和Toll样受体4(TLR4)信号通路的影响。方法 在A549细胞中转染LOC152742 siRNA，实验分为对照（Con）组、感染（H37Rv）组、转染对照（H37Rv+si-NC）组和转染（H37Rv+si-LOC152742）组。RT-PCR分析LOC152742表达水平变化，MTT法分析A549细胞增殖率，流式细胞术检测细胞凋亡情况，蛋白免疫印迹（Western blot）法检测活化的半胱氨酸天冬氨酸蛋白酶-3(cleaved caspase-3)、活化的半胱氨酸天冬氨酸蛋白酶-9(cleaved caspase-9)、TLR4、髓样分化因子（MyD88）蛋白表达，ELISA分析细胞上清中白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)的含量。在H37Rv+si-LOC152742组A549细胞中添加TLR4特异性抑制剂TAK242，检测TAK242对炎症反应的影响。结果 与Con组相比，H37Rv组A549细胞凋亡率、LOC15...     

热应激对A549细胞HSP70、XPA修复酶表达的影响

目的研究热应激作用下A549细胞热休克蛋白70（HSP70）和XPA修复酶的表达规律及其可能的意义。方法利用免疫印迹方法（Western blot）检测A549细胞中HSP70、XPA修复酶的表达水平。结果A549细胞接受不同温度（39、41、42、43℃）处理2h后,HSP70表达水平随温度的增高而逐渐增加,并在42℃应激时表达最高,与对照组相比有统计学意义（P〈0.05）;细胞接受42℃应激1～4h,HSP70表达水平随应激时间的增加而递增,与对照组相比,细胞在42℃应激2～4h时HSP70表达显著上调（P〈0.05）。两处理组细胞XPA修复酶表达水平均无明显变化（P〉0.05）。结论高温能诱导HSP70的表达,并呈一定的温度-效应和时间-效应关系,而对XPA修复酶未见明显影响。     

基于PPARα基因为靶序列的RNA干涉作用

目的利用RNAi技术研究PPARα基因表达对骨骼肌脂代谢相关基因的表达的影响。方法设计合成针对过氧化物酶体增殖蛋白激活性受体α(PPARα)的dsRNA并将其转染到鼠骨骼肌C2C12细胞中,用RT-PCR测定PPARα及其应激基因mRNA水平的变化。结果dsRNA浓度的升高和PPARαmRNA水平的降低之间存在剂量线性关系。PPARα应激基因(包括乙酰辅酶A氧化酶ACO;长链脂酞辅酶A合成酶LCAS;脂蛋白脂肪酶LPL;LDL受体)mRNA水平也随之降低,进一步证明PPARαRNAi可以特异性的降低PPARαmRNA水平。然而PPARγ和PPARδmRNA水平不受影响,而且PPARαRNAi不能降低GAPDHmRNA和18srRNA水平,说明PPARαRNAi并不诱导一般mRNA的降解。结论PPARαRNAi能够特异性的降低骨骼肌细胞中PPARα及其应激基因的mRNA水平。     

含I-A~dα和β链cDNA的三顺反子逆转录病毒载体构建及真核表达

为了协同表达MHCII类分子的两个链α和 β ,利用脑心肌炎病毒和脊髓灰质炎病毒的内核糖体进入位点 ,构建了含MHCII类分子α和 β链及选择标记新霉素磷酸转移酶基因的三顺反子逆转录病毒载体。经包装细胞包装成重组逆转录病毒后 ,感染NIH3T3、MM45T Li、COS7细胞 ,经PCR、RT PCR、Southern印迹、Northern印迹及流式细胞术在多水平上证实了α链和 β链的基因表达和MHCII类分子的正确组装。     

Mir－526 b可促进顺铂诱导非小细胞肺癌凋亡

目的：观察microRNA分子miR－526 b对非小细胞肺癌细胞A549的化疗敏感性的影响。方法将miR－526 b基因转染到A549细胞中;通过细胞活性检测测定A549细胞对顺铂（ diamminedichloroplatinum, DDP）的敏感性,通过流式细胞技术以及Western印迹检测细胞凋亡水平。结果筛选获得稳定表达miR－526 b的克隆细胞。细胞活性检测显示miR－526 b可明显提高非小细胞肺癌细胞A549对顺铂的敏感性,流式细胞技术显示DDP处理后miR－526 b稳定转染细胞的凋亡细胞率（10．2％±1．1％）较对照组A549细胞（3．5％±0．3％）以及DDP未处理对照A549细胞（0．4％±0．1％）明显升高。 Western印迹实验表明DDP处理后miR－526 b非小细胞肺癌细胞A549凋亡指标cleavaged caspase－3、 Bax明显升高。结论 MiR－526 b可促进顺铂诱导非小细胞肺癌凋亡。     

含Ⅰ-Adα和β链cDNA的三顺反子逆转录病毒载体构建及真核表达

为了协同表达MHCⅡ类分子的两个链α和β,利用脑心肌炎病毒和脊髓灰质炎病毒的内核糖体进入位点,构建了含MHCⅡ类分子α和β链及选择标记新霉素磷酸转移酶基因的三顺反子逆转录病毒载体.经包装细胞包装成重组逆转录病毒后,感染NIH3T3、MM45T.Li、COS7细胞,经PCR、BT-PCR、Southern印迹、Northern印迹及流式细胞术在多水平上证实了α链和β链的基因表达和MHCⅡ类分子的正确组装.     

A functional study of low molecular weight IgM from patients with autoimmune disease

High levels of low molecular weight (LMW) IgM in certain diseases are associated with clinical and laboratory indices which reflect the severity of the disease. These associations suggest that LMW IgM may play an important role in the immunopathogenesis of these diseases. To further approach the question concerning the functional activity of LMW IgM in disease, a panel of LMW IgM and high molecular weight (HMW) IgM preparations with or without rheumatoid factor (RF) activity were used to investigate their antibody binding activity and their effector function. It was found that LMW IgM-RF and HMW IgM-RF had a similar binding capacity to Fc fragment as there was no significant difference in the affinity index between them. It further showed that the rate of activation and total amount of utilization of complement by LMW IgM and HMW IgM was similar, although the mean fluorescence of C3 deposition by IgM-RF and HMW IgM-RF was slightly higher than that of LMW IgM-RF and other control RF antibodies. However, the current study demonstrated that LMW IgM had strong neutrophil activating properties when compared with HMW IgM. These findings suggest that one mechanism of LMW IgM contributing to the immunopathogenesis of RA may be due to the formation of circulating immune complex ( CIC) by LMW IgM with subsequent activation of neutrophils. Whether LMW IgM has other functional activity in disease is unclear and needs further investigation.     

Biochemical analyses of multiple fractions of PKR purified from Escherichia coli.

PKR is a cellular protein kinase activated by double-stranded RNA (dsRNA) that phosphorylates eukaryotic initiation factor alpha (eIF2alpha) and inhibits protein translation. Activation of PKR is accompanied by Ser/Thr autophosphorylation on multiple sites. Because PKR negatively regulates cell growth, overexpression and purification of PKR are difficult to achieve. Here, we describe overexpression and purification of recombinant PKR protein from Escherichia coli under native conditions at the milligram level. Affinity, ion exchange, and gel filtration chromatographies revealed multiple fractions of PKR with distinctive biochemical characteristics. During gel filtration, a small amount of PKR was found in a high molecular weight (>300 kDa) fraction that also contained endogenous bacterial RNA. The PKR in this fraction has a constitutive substrate phosphorylation activity. The majority of PKR is found in fractions of lower molecular weight and is free of RNA but is differentially phosphorylated as examined by isoelectric focusing electrophoresis and can be further separated by gradient anion exchange chromatography. PKR eluted with low salt has a lower level of basal autophosphorylation, and its kinase activity can be induced by dsRNA. With an increasing NaCl gradient, the purified PKR exhibits an increased level of autophosphorylation and constitutive kinase activity but reduced dsRNA inducibility. The highest salt eluent of PKR exhibits little dsRNA-induced activation. The inducible activation of high salt eluent PKR by dsRNA can be partially restored by treatment with protein phosphatase 1. The production of multiple fractions of PKR with different biochemical properties in E. coli suggests that the spectrum of PKR activity and regulation in mammalian cells is likely to be similarly complex.     

miR-206及Bcl-2蛋白在非小细胞肺癌中的表达及意义

目的探讨mi R-206及Bcl-2蛋白在非小细胞肺癌(NSCLC)的表达,以及抑制或过表达mi R-206对Bcl-2蛋白表达的影响。方法实时定量PCR方法检测NSCLC组织mi R-206的表达水平,Western blot方法检测NSCLC组织Bcl-2蛋白表达;将mi R-206 inhibitors及mi R-206 mimics转染至A549细胞,通过CCK-8法测定A549细胞增殖能力的变化,Western blot方法检测转染后Bcl-2蛋白表达变化。结果 mi R-206在非小细胞肺癌组织中的表达量较癌旁组织明显降低(0.01),Bcl-2蛋白在癌组织中较癌旁组织表达明显增高(0.05);转染mi R-206 inhibitor的细胞增殖能力与对照组相比显著升高,细胞内Bcl-2蛋白水平亦明显升高(0.05),转染mi R-206 mimics的细胞增殖能力与对照组相比显著降低,细胞内Bcl-2蛋白水平亦明显降低(0.05)。结论mi R-206在NSCLC组织中低表达,mi R-206抑制能够促进A549细胞增殖和Bcl-2蛋白表达。     

Activation of topoisomerase II-mediated excision of chromosomal DNA loops during oxidative stress

Hydrogen peroxide (H2O2), a reactive oxygen species (ROS), is known to induce oxidative stress and apoptosis. U937 cells treated with H2O2 were shown to produce high molecular weight (HMW) DNA fragments approximately 50 to 100 kb in size in <1 min. The formation of these HMW DNA fragments is reversible and shown to be mediated by DNA topoisomerase II (TOP2). Following this initial event, formation of irreversible HMW DNA fragments and nucleosomal ladders occurs. Our results thus demonstrate a potential role of TOP2 in oxidative damage of DNA and apoptotic cell death.     

Abnormal response to a human B cell growth factor in patients with common variable immunodeficiency (CVI)

Patients with common variable immunodeficiency (CVI) generally fail to produce antigen-specific IgG. We have identified a lymphokine called high molecular weight B cell growth factor (HMW BCGF) that expands an IgG-producing subpopulation of B cells. The B cells from 15 of 16 patients with CVI evaluated in this study failed to proliferate to HMW BCGF, although they proliferated normally to another BCGF, low molecular weight BCGF (LMW BCGF). Nevertheless, 11 patients had more than normal numbers of B cells expressing HMW BCGF receptors. The HMW BCGF receptors on the B cells of three patients with CVI studied were the same molecular weight as the normal HMW BCGF receptor. Examination of B cells from four patients with CVI for intracellular signals produced in normal B cells after stimulation with HMW BCGF revealed that B cells from patients with CVI failed to developed significant increases in cyclic adenosine monophosphate or phosphoinositides after HMW BCGF stimulation. However, cytoplasmic phosphoinositides in the B cells from all four patients with CVI were already increased above what is observed in normal B cells before stimulation with HMW BCGF (either freshly isolated or Staphylococcus aureus Cowan I-activated B cell). Thus, the failure of B cells from patients with CVI to respond to HMW BCGF may be related to their abnormal activation in vivo. Since HMW BCGF expands a subpopulation of memory B cells, the inability of CVI B cells to respond to HMW BCGF may contribute to their abnormal secondary responses to antigens.     

Effect of magnesium on fibrin formation from lower molecular weight (LMW) fibrinogen.

Fibrinogen circulating in human blood is comprised of high molecular weight (HMW) and lower molecular weight (LMW) fractions. As previously documented by means of SDS-polyacrylamide gel electrophoresis (PAGE), LMW fraction was significantly increased in patients with cardiovascular disease and with diabetes mellitus (DM). We have recently observed that the values of fibrinogen measured by thrombin clotting time (the method of Clauss) were consistently lower in EDTA plasma than those obtained with citrated plasma. However, supplementation of EDTA plasma with magnesium (Mg) ions gave comparable results. In this study we documented by SDS-PAGE that fibrin formed with thrombin alone in EDTA plasma originated from HMW fibrinogen, whereas that formed after addition of Mg was derived from LMW fibrinogen. Thus, measurement of thrombin clotting time in EDTA plasma with and without Mg may serve as a quick method for the determination of HMW and LMW fibrinogens in human blood. Preliminary result obtained with this new method revealed that LMW fibrinogen was significantly increased in DM patients.We have therefore concluded that measurement of this fraction of fibrinogen may prove to be of clinical diagnostic significance.     

FRMD8抑制肺癌细胞的增殖与迁移

目的 探讨FERM domain-containing protein 8（FRMD8）在肺癌发生发展中的作用。 方法 用Western blotting检测肺癌组织及相应癌旁正常组织中FRMD8的表达情况;敲低FRMD8后平板克隆形成实验检测细胞克隆形成能力,MTT法检测细胞增殖情况;过表达FRMD8后流式细胞术检测细胞凋亡情况,Western blotting检测过表达FRMD8后某些细凋亡相关蛋白表达变化;过表达及敲低FRMD8后Transwell细胞迁移实验检测对A549细胞迁移能力的影响。 结果 1. 与正常组织相比,肺癌组织中FRMD8表达下调（P＜0.05）; 2. FRMD8敲低,细胞克隆形成能力及增殖速度提高; 3. FRMD8可诱导细胞凋亡,并影响细胞凋亡相关基因的表达或活化： FRMD8过表达可下调Caspase-3、8和bax表达,而p53、激活型Caspase-3、8以及bcl-2 的表达上调; 4. FRMD8过表达抑制细胞迁移能力（P＜0.01）,而敲低FRMD8则促进细胞迁移（P＜0.001）。 结论 FRMD8可抑制肺癌的发生发展,并具有肿瘤抑制子的功能。     

Hyaluronic-acid-based semi-interpenetrating materials

In order to enhance the mechanical performances of hyaluronic acid (HA) without compromising its biological activity, HA has been interpenetrating with a fibrillar collagen scaffold. The semi-interpenetrating materials were obtained by mixing HA with different molecular weight and a pepsin-solubilized collagen (atelocollagen) solution, and then by inducing collagen fibrillogenesis. Results indicate that molecular weight of HA significantly influences the mechanical properties of the semi-interpenetrating materials and more specifically stronger material results from the use of low-molecular-weight (LMW) HA. According to the dynamic mechanical data the composite collagen-LMW HA has a higher elastic modulus than collagen, whereas the opposite is true for the high-molecular-weight (HMW) HA. This result highlights the role of specific interactions that occur between collagen and HA during the gel formation in controlling the network mechanical stability. LMW HA may, probably, interact more strongly with collagen during the fibrillogenesis process than HMW HA due to the higher mobility of the chains and the weaker homologous interactions. Moreover, morphological observations showed that LMW HA is intimately interdispersed within the collagen network and completely coated the fibrils, which act as mechanical support.     

Hyaluronic-acid-based semi-interpenetrating materials

In order to enhance the mechanical performances of hyaluronic acid (HA) without compromising its biological activity, HA has been interpenetrating with a fibrillar collagen scaffold. The semi-interpenetrating materials were obtained by mixing HA with different molecular weight and a pepsin-solubilized collagen (atelocollagen) solution, and then by inducing collagen fibrillogenesis. Results indicate that molecular weight of HA significantly influences the mechanical properties of the semi-interpenetrating materials and more specifically stronger material results from the use of low-molecular-weight (LMW) HA. According to the dynamic mechanical data the composite collagen-LMW HA has a higher elastic modulus than collagen, whereas the opposite is true for the high-molecular-weight (HMW) HA. This result highlights the role of specific interactions that occur between collagen and HA during the gel formation in controlling the network mechanical stability. LMW HA may, probably, interact more strongly with collagen during the fibrillogenesis process than HMW HA due to the higher mobility of the chains and the weaker homologous interactions. Moreover, morphological observations showed that LMW HA is intimately interdispersed within the collagen network and completely coated the fibrils, which act as mechanical support.     

包裹E1A基因的纳米粒子制备及转染人肺腺癌细胞A549实验研究

目的制备包裹E1A基因（腺病毒早期表达基因）的纳米粒子,并观察其介导E1A基因转染人肺腺癌细胞A549的可行性和效率。方法应用聚乳酸聚乙醇酸共聚物和聚乙烯醇包载E1A基因,制备纳米级粒子混合物,检测其包埋率、体外释放情况及粒径大小。用制备的包裹DNA纳米粒子转染人肺腺癌细胞A549,并以阳离子脂质体为对照,用PCR、RT-PCR方法分别检测转染细胞中E1A基因DNA整合和mRNA表达。结果制备的纳米粒子粒径为150～280nm,包埋率为0.78%,体外释放约为22d;在转染相等质量的DNA情况下,纳米组所得克隆数较脂质体组多（P〈0.05）;PCR、RT-PCR结果表明纳米粒子和脂质体转染细胞均有E1A基因整合和mRNA表达。结论成功制备了纳米粒子,纳米粒子可携带外源基因进行基因转染。