EDU 免疫荧光法检测整合素连接激酶（ILK）高表达对新生大鼠心肌细胞DNA 合成的影响

目的:通过EDU免疫荧光法观察整合素连接激酶(Integrin-Linked Kinase,ILK)在新生大鼠心肌细胞内高表达后对心肌细胞DNA合成的影响。方法:取新生(1-3天内)大鼠原代心肌细胞,培养72小时后随机分为正常对照组、ILK转染组。对照组转染重组腺病毒载体(adeno-GFP),ILK组转染重组腺病毒载体+ILK基因(adeno-ILK)。转然成功后48小时将两组心肌细胞分别通过5-乙基-2’-脱氧尿嘧啶核苷(EDU)免疫荧光法测定心肌细胞DNA合成。结果:ILK转染组心肌细胞内DNA合成较对照组明显增加(P<0.05)。结论:ILK高表达具有促进新生大鼠心肌细胞的DNA合成的能力。     

整合素连接激酶在IgA肾病肾组织中的表达及其意义

目的探讨整合素连接激酶(ILK)在IgA肾病患者肾组织中的表达及其意义.方法采用间接免疫荧光双标记法检测不同病变程度IgA肾病患者肾组织ILK、纤维连接蛋白(Fn)和整合素(Integrin)的表达.结果在正常组肾组织,ILK主要表达于肾小球脏层上皮细胞.随着IgA肾病病变程度的加重,ILK的表达逐渐增强,Ⅳ级病变时,ILK的表达最强,正常组及IgA肾病Ⅰ-Ⅴ级的荧光强度分别为:11.67±4.82、12.39±7.71、19.50±7.79、26.26±9.27、35.83±7.01和15.40±5.80(P<0.01);Integrin的表达与ILK平行.Fn的表达随病变程度的加重进行性增强.双标记免疫荧光染色显示,Ⅰ-Ⅳ级,ILK与Fn的表达成正相关(r=0.85,P<0.01);ILK与Integrin的表达也呈正相关(r=0.89,P<0.01).结论 ILK可能通过介导Integrin参与IgA肾病的异常细胞外基质的沉积,尤其在IgA肾病早中期起着重要作用.     

RNAi沉默整合素连接激酶基因表达对膀胱癌细胞BIU-87增殖的影响

为了研究整合素连接激酶(integrin-linked kinase,ILK)基因沉默对膀胱癌BIU-87细胞系增殖产生的影响,应用RNA干扰(RNA interference,RNAi)技术,合成了4条针对ILK的miRNA干扰载体pcDNATM6.2-GW/EmGFP-ILK-miR(miR-1、miR-2、miR-3、miR-4)作为实验组,另设1条为阴性对照组,分别转染膀胱癌BIU-87细胞系.经杀稻瘟菌素持续压力筛选和有限稀释法培养获得稳定转染细胞株.采用RT-PCR和Western-blot检测各组对ILK基因的抑制作用,实验组miRNA对BIU-87细胞ILK mRNA和蛋白的表达均有明显的抑制作用,以miR-3组抑制效应最强.通过流式细胞术检测发现,miR-3组细胞周期G0/G1期细胞所占比例较未转染组明显增加,而S期则明显减少(P0.05).四甲基偶氮唑盐(MTT)比色法检测发现,转染组细胞在体外的增殖能力较对照组明显下降(P0.05).证明RNA干扰技术能有效抑制靶基因ILK的表达,进而抑制了膀胱癌细胞系BIU-87的增殖.     

整合素连接激酶和基质金属蛋白酶-9在非小细胞肺癌表达的研究

目的探讨整合素连接激酶(integrin-linked kinase,ILK)和基质金属蛋白酶(matrix metalloproteinase-9,MMP-9)在非小细胞肺癌(non-small cell lung cancer,NSCLC)中的表达及其与临床病理特征的关系.方法应用SP免疫组织化学方法检测74例手术切除NSCLC标本和10例正常肺组织中ILK和MMP-9蛋白的表达.结果在NSCLC组织中ILK和MMP-9蛋白的阳性表达率分别为74.5%(55/74)和71.6%(53/74),均明显高于正常肺组织(P<0.05).ILK 阳性表达率与原发灶大小,组织学类型,分化程度,淋巴结转移,临床分期均无密切关系(P>0.05);MMP-9阳性表达率与原发灶大小,组织学类型,分化程度,淋巴结转移,临床分期均有密切关系(P<0.05).结论 ILK和MMP-9蛋白过度表达可能分别在NSCLC的发生和发展过程中起重要作用.     

低氧对新生大鼠脾单个核细胞DNA合成及转化的影响

本研究以荧光法测定脾单个核细胞ＤＮＡ合成及ＭＴＴ比色法测定的脾单个核细胞对ＣｏｎＡ的增殖反应,观察模拟高原低氧对出生后１４天大鼠上述两指标的影响,同时也观察了交感神经和副交感神经的活动状态,以初步探讨低氧对上述两指标的作用是如何介导的。结果表明：５ｋｍ海拔高度低氧作用２４ｈ不抑制脾单个细胞ＤＮＡ合成及脾单个核细胞转化,而作用５天时则抑制ＤＮＡ合成及脾单个核细胞转化,分别为对照组的５６．６％（Ｐ＜０．０１）和８６．８％（Ｐ＜０．０５）;７ｋｍ海拔高度低氧作用２４ｈ,ＤＮＡ合成及脾单个核细胞转化均受抑制,分别为对照组的６１．０％（Ｐ＜０．０１）和８１．２％（Ｐ＜０．０１）;７ｋｍ海拔２４ｈ低氧导致脾脏中乙酰胆碱下降,儿茶酚胺升高;用ＤＳＰ－４中枢药理性损毁ＮＥ神经元,可使脾单个核细胞ＤＮＡ合成的抑制程度减弱,脾脏中儿茶酚胺含量下降。这些结果表明低氧可抑制新生大鼠脾单个核细胞的ＤＮＡ合成及转化,并可能与交感神经兴奋及副交感神经抑制有关     

钙调素对DNA合成的影响

利用钙调素ｃａｌｍｏｄｕｌｉｎ,ＣａＭ）拮抗剂─三氟拉嗪（ｔｒｉｆｌｕｏｐｅｒａｚｉｎｅ,ＴＦＰ）对Ｇ０期小鼠Ｃ３Ｈ１０Ｔ１／２成纤维细胞进入Ｓ期和ＤＮＡ合成进行了研究．Ｇ０期细胞进入Ｓ期时,大量钙调素进入细胞核,其水平为Ｇ０期的２倍。ＴＦＰ处理的细胞被阻抑在Ｇ１期,不仅使Ｓ期细胞群体下降,而且３Ｈ－ＴｄＲ掺入ＤＮＡ强度受到明显抑制．同时,ＴＦＰ处理的细胞胸腺嘧啶核苷激酶（ｔｈｙｍｉｄｉｎｅｋｉｎａｓｅ,ＴＫ）基因表达及ＴＫ活性亦明显下降,但不影响Ｓ期细胞核内的钙调素水平,结果表明钙调素功能之抑制不仅阻抑细胞从Ｇ１期至Ｓ期的进程,而且对细胞ＤＮＡ合成强度亦有抑制作用．     

尾加压素对新生大鼠心肌细胞一氧化氮合成的影响

应用半定量逆转录－多聚酶链反应法,观察尾加压素（urotensin Ⅱ,UⅡ）对培养的新生SD大鼠心肌细胞内皮型一氧化氮合酶（endothelial nitric oxide synthase,eNOS)mRNA表达的影响,并测定UⅡ对心肌细胞内一氧化氮合酶（nitric oxide synthase,NOS)活性和一氧化氮（nitric oxide,NO)释放的影响。结果显示：UⅡ抑制培养的新生大鼠心肌细胞eNOS mRNA表达、抑制NOS的活性及NO释放;0.1μmol/L浓度的UⅡ呈时间依赖性抑制心肌细胞NOS的活性及NO生成。上述实验结果提示UⅡ的心血管作用可能与NO合成系统有关。     

猴泡沫病毒间接免疫荧光检测方法的建立及初步应用

目的建立检测血清中猴泡沫病毒（SFV）抗体的间接免疫荧光方法,为检测实验用猴群中SFV的感染情况提供参考依据。方法用SFV-1病毒感染BHK-21细胞,待50%细胞出现病变时,用胰蛋白酶消化细胞后以2×107/mL浓度40μL的细胞滴到10孔镀膜的玻片上,丙酮固定。利用制备的抗原片通过间接免疫荧光法对34份猴血清标本进行检测。结果建立了检测SFV抗体的间接免疫荧光染色方法,SFV抗体阳性19例,15例血清检测为阴性。结论本方法具有良好的特异性,可作为SFV检测的可靠方法。     

Molecular Mechanisms of Mitochondrial DNA Depletion Diseases Caused by Deficiencies in Enzymes in Purine and Pyrimidine Metabolism

Mitochondrial DNA depletion syndrome (MDS), a reduction of mitochondrial DNA copy number, often affects muscle or liver. Mutations in enzymes of deoxyribonucleotide metabolism give MDS, for example, the mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) genes. Sixteen TK2 and 22 dGK alterations are known. Their characteristics and symptoms are described. Levels of five key deoxynucleotide metabolizing enzymes in mouse tissues were measured. TK2 and dGK levels in muscles were 5- to 10-fold lower than other nonproliferating tissues and 100-fold lower compared to spleen. Each type of tissue apparently relies on de novo and salvage synthesis of DNA precursors to varying degrees.     

Cell Cycle-Specific Requirement for Mevalonate, but Not for Cholesterol, for DNA Synthesis in Glial Primary Cultures

The requirement for the sterol biosynthetic pathway for the occurrence of DNA synthesis in glial cells and, in particular, the relative roles of cholesterol and of mevalonate have been studied. Primary cultures of developing glial cells were synchronized by reducing the content of fetal calf serum (FCS) in the culture medium from 10% to 0.1% (vol/vol) for 48 h between days 4 and 6 in culture. Reversal of the resulting quiescent state by the return of the cultures to 10% serum caused after 24 h a marked increase in DNA synthesis, and this increase was prevented by the simultaneous addition of mevinolin, a specific inhibitor of the sterol biosynthetic pathway at the 3-hydroxy-3-methylglutaryl coenzyme A reductase step, at the time of serum repletion. A dose-dependent reversal of the mevinolin inhibition of DNA synthesis occurred with simultaneous addition of mevalonate to the culture medium. The induction of DNA synthesis by serum repletion, its inhibition by mevinolin, and the reversal of the inhibition by mevalonate were unaffected by a 95% reduction in exogenous cholesterol produced by utilization of lipoprotein-poor serum (LPPS) rather than FCS. Similarly, return of quiescent cultures to 10% LPPS containing mevinolin and sufficient low-density lipoprotein (LDL) to raise the cholesterol concentration 80-fold failed to restore DNA synthesis. In addition, reversal of the mevinolin inhibition of DNA synthesis by mevalonate occurred despite the continuous presence of mevinolin if mevalonate was added as late as 12 h after serum repletion, but not if added after 16 h or more.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of DNA in excised watermelon cotyledons grown in water and benzyladenine

Excised watermelon cotyledons were grown in water and benzyladenine, which greatly promotes growth, breakdown of reserves and development of organelles. In order to investigate the involvement of DNA synthesis in these benzyladenine-induced effects, [³H]thymidine was applied continuously (for 3 d) or administered briefly (5 h) to excised cotyledons at various stages of development. Autoradiographic analysis of squashed and sectioned cotyledons showed that both the cytoplasm (mainly in the region of the plastids) and most of the nuclei were labelled. Both types of labelling were promoted by benzyladenine treatment. The highest percentage of labelled nuclei was found in the early stages of growth (first day after excision of cotyledons), long before the burst of enzymatic activities involved in the germination processes. The possible meaning of the increase of nuclear DNA, apart from the normal replicative synthesis preceding cell division, is discussed.Abbreviations BA N⁶-benzyladenine - DNase deoxyribonuclease - EtBr ethidium bromide - FUdR fluorodeoxyuridine - [³H]T [methyl-³H]thymidine     

In Vivo measurement of unscheduled DNA synthesis and S-phase synthesis as an indicator of hepatocarcinogenesis in rodents

Measurement of chemically induced DNA repair as unscheduled DNA synthesis in rodent liver following in vivo treatment is a useful screen for potential hepatocarcinogens. In addition to measurement of unscheduled DNA synthesis, examination of S-phase synthesis provides an indicator of chemically induced cell proliferation in the liver, which may be a basis for hepatic tumor promotion. Several chemicals and classes of chemicals have been examined using these endpoints. The pyrrolizidine alkaloid riddelline is a potent genotoxic agent in vitro, and in vivo studies confirm this response as riddelline induces significant elevations in unscheduled DNA synthesis and S-phase synthesis in rat liver. Conversely, H. C. Blue dyes #1 and #2 are both potent genotoxic agents in vitro but fail to express this genotoxicity in vivo. H. C Blue #1 induces significant increases in S-phase synthesis in B6C3F1 mouse liver, which correlates with the observed carcinogenicity of this compound. Halogenated hydrocarbons likewise fail to induce unscheduled DNA synthesis in vivo, but many of these compounds do increase hepatic cell proliferation in mice, which may be the principal mechanism of hepatocarcinogenesis in this species.Abbreviations BCMEE bis(2-chloro-l-methylethyl)ether - dThd thymidine - HCB1 H.C. Blue #1 - HCB2 H.C. Blue #2 - UDS unscheduled DNA synthesis     

The Tyrosine Kinase c-Abl Promotes Homeodomain-interacting Protein Kinase 2 (HIPK2) Accumulation and Activation in Response to DNA Damage

The non-receptor tyrosine kinase c-Abl is activated in response to DNA damage and induces p73-dependent apoptosis. Here, we investigated c-Abl regulation of the homeodomain-interacting protein kinase 2 (HIPK2), an important regulator of p53-dependent apoptosis. c-Abl phosphorylated HIPK2 at several sites, and phosphorylation by c-Abl protected HIPK2 from degradation mediated by the ubiquitin E3 ligase Siah-1. c-Abl and HIPK2 synergized in activating p53 on apoptotic promoters in a reporter assay, and c-Abl was required for endogenous HIPK2 accumulation and phosphorylation of p53 at Ser⁴⁶ in response to DNA damage by γ- and UV radiation. Accumulation of HIPK2 in nuclear speckles and association with promyelocytic leukemia protein (PML) in response to DNA damage were also dependent on c-Abl activity. At high cell density, the Hippo pathway inhibits DNA damage-induced c-Abl activation. Under this condition, DNA damage-induced HIPK2 accumulation, phosphorylation of p53 at Ser⁴⁶, and apoptosis were attenuated. These data demonstrate a new mechanism for the induction of DNA damage-induced apoptosis by c-Abl and illustrate network interactions between serine/threonine and tyrosine kinases that dictate cell fate.     

Epidermal Growth Factor Induces Proliferation and DNA Synthesis in Ciliate Cells

We studied the effect of murine epidermal growth factor on cell proliferation and DNA synthesis in macronuclei of ciliate Tetrahymena pyriformis Gl. Mitogenic effect of epidermal growth factor on proliferation-induced tetrahymena cells has been revealed. This effect is due to the induced progression of cells at G ₁ and, consequently, their earlier entering DNA synthesis phase of the first cell cycle. Epidermal growth factor had no mitogenic effect on the resting cells in a stationary culture (G ₀ phase) whose development is independent of the growth factors in the medium.     

The HIV-1 Nef Protein Inhibits Extracellular Signal-Regulated Kinase-Dependent DNA Synthesis in a Human Astrocytic Cell Line

Abstract: The role of nonproductive infection of astrocytes by human immunodeficiency virus type 1 (HIV-1), characterized by the overexpression of nef, in brain disease progression is largely unknown. We investigated the consequences of stable expression of nef from the HIV-1 strain LAI in the human astrocytic cell line U373. DNA synthesis induced by endothelin-1 (ET-1) was largely decreased by nef. Stable expression of nef did not affect the ET-1-induced tyrosine phosphorylation of focal adhesion kinase, an adhesion-dependent pathway known to participate in DNA synthesis in astrocytes. Conversely, the activation of extracellular signal-regulated kinase (ERK) by ET-1 was largely inhibited in cells stably or transiently expressing nef. A similar inhibitory action of nef on ERK activation was observed after direct stimulation of G proteins. Furthermore, the inhibitory action of nef did not require protein kinase C (PKC) and affected mainly the PKC-independent pathway of ERK activation. Following chemokine receptor CXCR4-mediated infection of U373 cells stably expressing CXCR4 with the T-tropic HIV-1 strain m7-NDK, ET-1-induced activation of ERK was also inhibited. Altogether, these results indicate that intracellular signaling pathways associated with the growth factor activity of ET-1 are impaired in nef-expressing and HIV-1-infected astrocytes, suggesting that infection of astrocytes may play a significant role in the neuropathogenesis of HIV-1 encephalopathy.