ATP对人不死化成纤维细胞DNA的合成、膜蛋白表达及离子通道的影响

目的：探讨ATP抑制不死化人成纤维细胞增殖过程中对其DNA合成，细胞膜蛋白的表达以及离子通道的影响，方法：将正常人TIG－7和OUMS－36细胞株，不死化人KMST－6和SUSM－1细胞株在不同浓度的ATP，ADP，AMP条件下分别进行24和96h的常规细胞培养，观察存活细胞数目，DNA的合成，[32P]ATP标记膜蛋白的表达以及不死化KMST－6的细胞分别在无钙离子培养基和应用Ca^2 和K^ 通道拮抗剂时，细胞增殖率的改变，结果：培养96h后，0．4mmol/L ATP对不死化细胞KMST－6的抑制率为77％，且DNA合成明显地被抑制，而正常OUMS－36细胞抑制率为41％，且DNA合成无明显改变，在1mmol/L ATP时，多数KMST－6细胞发生死亡，而正常OUMS－36细胞的增殖无明显影响，当ADP，AMP和腺苷或磷酸处理的细胞，仅有ADP处理的不死化细胞存活数目减少（P＜0．01），与正常细胞比较，不死化细胞30，31，33和40kD的[^32P]-ATP标记膜蛋白呈高表达，0．4mmol/L ATP与KMST－6细胞共培养时分别加入Ca^2 以及K^ 通道拮抗剂，这些药物在某种程度上可以降低ATP的增殖抑制作用，当ATP处理的细胞液中无钙离子时，它们的增殖不受影响。结论：ATP对不死化人成纤维细胞的DNA合成有明显的抑制作用。并且使磷酸化细胞膜蛋白的表达增高，其过程中有钙通道和钾通道的参与。     

ATP对人不死化成纤维细胞增殖及其细胞膜蛋白表达的影响

目的探讨ATP对不死化人成纤维细胞增殖及其细胞膜蛋白表达的影响。方法将正常人TIG-7和0UMS-36细胞株，不死化人KMST-6和SUSM-1细胞株在不同浓度的ATP，ADP，AMP条件下分别进行24和96h的常规细胞培养，观察存活细胞数目，DNA的合成和[     

ATP及其衍生物影响不死化人成纤维细胞增殖的实验研究

目的:探讨ATP及其衍生物对不死化人成纤维细胞的增殖抑制作用,并通过P₂嘌呤能受体从而了解ATP发挥细胞毒性作用的途径。方法:使用ATP及其衍生物ATP-Na₂,ATP-Mg,ADP,MeATP,BzATP,ATPγS,2-MeSATP和UTP在不同条件培养KMST-6人不死化成纤维细胞,检测细胞增殖状况,Westernblot分析、流式细胞仪检测细胞增殖周期以及Hoechst33258特异性细胞染色和DNA电泳检测细胞凋亡。结果:ATP及其衍生物对细胞增殖抑制作用的程度依次为:ATP=ADP＞ATPγS＞MeATP=BzATP;而2-MeSATP和UTP却未显示任何细胞毒性作用;0.4mmol/LATP培养48h时P²¹表达未见增高,细胞增殖停止于G₁／S期;1mmol/LATP培养48h时未发现细胞凋亡。结论:通过与P_2X或P_2Y嘌呤能受体相结合,ATP激活细胞内某些信号传导发挥细胞增殖的抑制作用;ATP引起增殖抑制不是通过细胞凋亡或者是周期素／CDK激酶抑制剂P²¹所致。     

二氢杨梅素联合丝裂霉素对胃癌细胞的生长抑制作用

目的: 探讨二氢杨梅素(AMP)联合细胞化疗药物丝裂霉素(MMC)对胃癌细胞SGC-7901的抑制作用。方法: 体外培养人胃癌细胞株SGC-7901,分别设空白对照组、AMP组、MMC组以及AMP+MMC组。用MTT法观察细胞生长;流式细胞术分析细胞凋亡率;Western blotting检测细胞Bcl-2及survivin的表达情况。结果: 在AMP组中,当浓度在2.2 mg/L-14.84 mg/L时,AMP对胃癌细胞生长均有抑制作用,以14.84 mg/L抑制作用最明显(60.85%±1.13%,P<0.05)。MMC随着浓度由1×10^-3g/L增加至1×10g/L,其抑制率也由17.40%±0.30%增加至72.23%±1.36%,呈递增趋势。AMP联合MMC的抑制作用随着MMC浓度由1×10^-3 g/L增加至5×10^-3g/L,抑制率也由21.83%±2.50%增加至46.70%±1.45%。联合治疗组优于单独给药组。AMP、MMC及AMP+MMC均可以下调Bcl-2及survivin的表达,其中AMP+MMC组下调作用最明显。结论: AMP联合MMC可以增强对胃癌细胞的抑制作用,联合应用可以减低MMC的剂量;其抑制肿瘤作用可能与下调Bcl-2及survivin蛋白的表达有关。     

ATP抑制不死化人成纤维细胞增殖信号转导机制的研究

目的:探讨ATP抑制不死化人成纤维细胞增殖信号转导机制。方法:观察0.4mmol/LATP与不死化人成纤维细胞共培养不同时间下,细胞内游离Ca²⁺浓度([Ca²⁺]i)和三磷酸肌醇(IP₃)浓度的变化特点和意义,以及分别应用Ca²⁺和K⁺通道拮抗剂时,细胞增殖率的改变。结果:ATP与不死化人成纤维细胞共培养时,[Ca²⁺]i明显升高,IP₃浓度明显降低(均P＜0.01)。分别应用Ca²⁺和K⁺通道拮抗剂时,细胞增殖率都显著增高(均P＜0.01)。结论:ATP抑制不死化人成纤维细胞增殖过程中有钙通道和钾通道以及IP₃的参与。     

洛伐他汀对NB4细胞ras基因p21Ras膜结合作用影响的体外试验

目的:探讨洛伐他汀(LOV)对人白血病NB4细胞的作用及其机制。方法:以MTT比色法首先观察LOV对NB4细胞增殖的影响;利用逆转录-聚合酶链反应半定量测定LOV作用于NB4细胞不同时间H-ras、K-ras、N-ras癌基因mRNA表达水平;同时采用流式细胞术测定NB4细胞p21^Ras总蛋白、膜蛋白的表达。结果:①LOV抑制NB4细胞增殖,IC50为12.59μmol/L。②NB4细胞H、K、N-ras基因表达均为阳性。③LOV处理不同时间的NB4细胞H、K、N-ras基因转录水平无明显变化;p21^Ras总蛋白水平也无变化,而细胞膜表面p21^Ras蛋白水平随时间进行性下降。结论:LOV抑制NB4细胞增殖。LOV靶向HMG-CoA还原酶,抑制p21^Ras蛋白异戊二烯化、阻滞p21^Ras蛋白与细胞膜结合;不影响ras癌基因以及p21^Ras总蛋白的表达。     

丹参制剂对家兔缺血心肌腺苷酸类代谢的影响

本文以结扎家兔冠状动脉左室支为心肌缺血模型、应用阴离子交换高效液相色谱方法(HPLC)测定了心肌缺血40分钟以及预先给丹参制剂再行缺血,心肌游离腺苷酸的含量变化。从而观察了丹参对心肌的保护作用。结果表明,缺血心肌ATP、ADP、AMP、AN(总腺苷酸量)以及ATP/ADP均明显减少。预先给予丹参制剂后再缺血40分钟,各类腺苷酸下降幅度与单纯缺血组相比明显增加。AMP及AN值近于正常。ATP、ADP及ATP/ADP值与正常心肌比虽亦下降,但与单纯缺血组比却明显上升,差异显著。说明丹参制剂对心肌具有明显的保护作用。     

索拉非尼抑制人肝星状细胞胶原合成

目的: 研究索拉非尼(sorafenib)对人肝星状细胞胶原合成的影响。方法: 应用人肝星状细胞株LX-2进行体外研究,采用 -脯氨酸掺入法测定胶原的合成,采用免疫细胞化学法检测I型胶原蛋白表达,采用real-time PCR法测定I型胶原α1 mRNA表达。结果: 免疫细胞化学研究显示血小板源性生长因子(PDGF)刺激可引起LX-2细胞胶原合成增加,10.0 μmol·L^-1索拉非尼作用于LX-2细胞 24 h能明显抑制I型胶原蛋白的合成。无论有无PDGF的刺激,索拉非尼均呈剂量与时间依赖性地抑制LX-2细胞胶原合成(P<0.01);在10.0 μmol·L^-1浓度下,索拉非尼作用于LX-2细胞 12 h、24 h和48 h对胶原合成的抑制率为22.69%、37.52%和71.74%。索拉非尼剂量依赖性地抑制PDGF诱导的I型胶原α1 mRNA表达上调;在2.5 μmol·L^-1、5.0 μmol·L^-1和10.0 μmol·L^-1 索拉非尼作用下,I型胶原α1 mRNA表达较PDGF刺激组分别下调58.66%、 67.06%和81.64%。结论: 索拉非尼在体外能抑制人肝星状细胞胶原的合成,抑制I型胶原的表达,有可能成为一种新型的治疗肝纤维化药物。     

BCL-6在经典型霍奇金淋巴瘤细胞株L428和过表达CD99的L428中的表达差异及其意义

目的: 探究BCL-6在经典型霍奇金淋巴瘤(classical Hodgkin's lymphoma,cHL)细胞株L428和过表达CD99的L428(L428-CD99⁺)中的表达差异及其意义。方法: 应用免疫细胞化学和免疫荧光共聚焦检测BCL-6和CD99在cHL细胞株L428和L428-CD99⁺中的表达;运用实时荧光定量PCR和Western blotting检测细胞株L428和L428-CD99⁺中BCL-6和CD99的表达水平;MTT实验检测L428和L428-CD99⁺细胞增殖能力的差异;流式细胞术检测L428和L428-CD99⁺细胞的凋亡差异。结果: 免疫细胞化学和免疫荧光共聚焦显示CD99在L428细胞中为阴性表达,在L428-CD99⁺细胞中为阳性表达,并定位于细胞膜;BCL-6在L428细胞为阴性表达,在L428-CD99⁺细胞株中为阳性表达,并定位于细胞核。Western blotting显示CD99和BCL-6在L428细胞为阴性表达,在L428-CD99⁺细胞为阳性表达。实时荧光定量PCR结果显示,与L428细胞相比,CD99和BCL-6 mRNA在L428-CD99⁺细胞中的表达量增高(P<0.01)。MTT显示L428细胞增殖能力强于L428-CD99⁺细胞(P<0.01);流式细胞术显示L428-CD99⁺细胞凋亡较L428细胞增多(P<0.01)。结论: 过表达CD99诱发BCL-6表达增高,可导致L428细胞增殖能力下降及凋亡增加。     

抗人F1-F0 ATP合成酶beta亚基单抗的制备及其抗肿瘤活性研究

目的：制备鼠抗人F1-F0ATP合成酶beta亚基（hATP5B）单抗,并对其特异性和抗肿瘤活性进行研究。方法：以原核表达人hATP5B免疫BALB/c小鼠,通过杂交瘤技术,筛选分泌抗hATP5B单抗的杂交瘤细胞株。采用Protein A亲和层析法纯化抗体腹水,SDS-PAGE检测纯化产物纯度。利用Western blot、细胞免疫荧光对其特异性进行鉴定,并通过抑制乳腺癌细胞MCF-7及其耐药株MCF-7/Adr表面ATP生成,细胞毒性实验进行抗肿瘤活性研究。结果：获得一株稳定表达抗hATP5B单抗的杂交瘤细胞株Mab3B8,Western blot和细胞免疫荧光结果表明,该抗体与乳腺癌MCF-7及其耐药细胞MCF-7/Adr细胞天然抗原结合；可明显抑制MCF-7及其耐药株MCF-7/Adr细胞膜表面ATP合成；与化疗药物多柔比星（曾用名：阿霉素）联合作用,可降低化疗药物多柔比星对肿瘤细胞的IC50。结论：成功建立了一株可特异性识别天然hATP5B的单抗,有阻断肿瘤细胞膜表面ATP合成活性并可明显增强MCF-7及其耐药株MCF-7/Adr细胞对多柔比星敏感性的作用。此项研究对膜表达F1-F0ATP合成酶功能探讨及肿瘤治疗研究都具有重要意义。     

Growth inhibitory effects of ATP and its derivatives on human fibroblasts immortalized with 60Co-gamma rays

In our previous study (Katayama B et al, Int J Mol Med 2: 603-606, 1998), cell growth inhibition caused by ATP added to cultures was found to be greater in immortalized human fibroblasts than in the normal human fibroblasts. Since it has been reported that ATP affects cells via P2-purinergic receptors, growth inhibitory effects of ATP and its derivatives on immortalized human fibroblasts were investigated in the present study in order to learn what type of receptors are involved in ATP cytotoxicity. The ATP derivatives used in this study were: ATP, ADP, beta, gamma-methyleneadenosine 5'-triphosphate (MeATP), 2' & 3'-o-(4-benzoylbenzoyl) adenosine, triethylammonium salt (BzATP), adenosine 5'-o-(3-thiotriphosphate) (ATPgammaS), 2-methylthioadenosine 5'-triphosphate (2-MeSATP) and UTP. The extent of cytotoxicity induced by these drugs was found to be in the order of: ATP=ADP>ATPgammaS>MeATP=BzATP. On the other hand, neither 2-MeSATP nor UTP showed any cytotoxicity. These findings indicate that ATP may exert the cell growth inhibition by certain kinds of signal transduction via P2x or P2y purinergic receptors which affect intrinsic channels/pores of cell membrane and/or G protein activation. As a result, intracellular elevation in the concentrations of ions such as calcium and potassium, membrane depolarization, loss of endogenous ions/metabolites, and activation of inositol phospholipid-specific phospholipase C may occur. Actually, a dihydropyridine calcium channel blocker, nifedipine, and an ATP-sensitive K+-channel blocker, glybenclamide, reduced the growth inhibitory effects of ATP on the cells to some extent. The growth inhibition caused by ATP was not due to apoptosis or induction of a cyclin/CDK kinase inhibitor, P21.     

The Effect of ATP and Related Compounds on Spontaneous Mechanical Activity in the Rat Portal Vein

The effects of ATP, ADP, AMP and adenosine were studied on the spontaneous mechanical activity of the rat portal vein. It was found that ATP and ADP, in concentrations higher than 300 μM, caused a transient tetanus, followed by inhibition, and at lower concentrations an increase in the frequency and amplitude of the spontaneous contractions. AMP and adenosine on the other hand, inhibited spontaneous activity, by reducing the amplitude of contractions and increasing their frequency. The effects were dose-dependent. ATP was found to be 2.2 times more potent than ADP, while AMP and adenosine were equipotent. Weak inhibitory effects were obtained with GMP, guanosine and adenine, while GTP, 3′5′-cyclic AMP and guanine had no effect. ATP and ADP increased the K-contracture, while AMP and adenosine relaxed it. The effects of ATP were augmented in Mg-free solutions and partially inhibited in Mg-high solutions in the normally polarized muscle, while Mg had no influence on the ATP-induced contraction in the depolarized muscle. Theophylline potentiated the inhibitory response to AMP and adenosine. Adrenergic and cholinergic blockers had no influence on the response to ATP, ADP, AMP or adenosine. It is suggested that the effects of ATP and ADP are linked with Ca⁺⁺ movements across the membrane, while AMP and adenosine might stimulate intracellular metabolism causing increased intracellular Ca⁺⁺ binding.     

外源性ATP诱导PC12细胞的膜孔形成

 目的：探讨外源性三磷酸腺苷(ATP)诱导PC12细胞的膜孔形成及关键分子靶标。方法：用不同浓度的ATP处理培养的PC12细胞,采用倒置相差显微镜观察形态,CCK-8法检测细胞存活率,YO-PRO-1染色检测细胞膜通透性,Western blotting和real-time PCR检测P2X7 受体和pannexin 1（Panx1）表达的变化。结果：（1）ATP（1 mmol/L、3 mmol/L、5 mmol/L）作用3 h,可见随着ATP浓度升高,PC12细胞变圆,脱壁细胞增多;当ATP浓度为3 mmol/L或5 mmol/L时,PC12细胞活力较对照组显著下降（P<0.05）。（2）不同浓度的ATP（0、1、3、5 mmol/L）作用1 h,PC12细胞摄入YO-PRO-1的荧光强度随着浓度增加而增加;同一浓度的ATP作用不同时间（15、30、60 min）,随着时间的增加,胞内的荧光强度也增加。（3）亮蓝G（P2X7受体的抑制剂）预处理可明显拮抗ATP引起的细胞活力下降和胞内荧光强度增强（P<0.05）,而生胃酮（Panx1的抑制剂）预处理不改变细胞活力和胞内的荧光强度（P>0.05）。（4）ATP作用3 h使PC12细胞 P2X7受体的mRNA和蛋白表达明显升高（P<0.05）,而Panx1 mRNA和蛋白表达变化不大（P>0.05）。结论：胞外高浓度ATP引起PC12细胞的膜孔形成可能主要与P2X7受体的表达和激活有关。     

Biochemical background of the development of gastric mucosal damage in pylorus-ligated plus aspirin-treated rats

Gastric mucosal damage was produced in rats after pyloric ligation by intragastric administration of 200 mg/kg aspirin diluted in 2 ml 150 mmol/l HCl. The animals in the control group received 2 ml saline solution, or submitted to pyloric ligation only. The animals were killed 4 h after the pyloric ligation, when the number and severity of gastric lesions (ulcers), and the gastric fundic mucosal level of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), cyclic adenosine monophosphate (cAMP) and lactate, were noted and measured. The adenylate pool (ATP + ADP + AMP) and the energy charge (ATP + 0.5ADP). (ATP + ADP + AMP)-1 were calculated. It was found that: the gastric H+ output decreased significantly in the pylorus-ligated plus aspirin-treated animals; the number and severity of gastric lesions increased significantly in the pylorus-ligated aspirin-treated animals; the extent of ATP transformation into the ADP decreased significantly in the pylorus-ligated aspirin-treated animals; the extent of ATP transformation into the cAMP decreased significantly during the aspirin treatment; the values of adenylate pool and of "energy charge" remained unchanged in the different groups of animals. It is concluded that: the decreased H+ output in the pylorus-ligated plus aspirin-treated group can be obtained by the decreased extent of ATP transformation into the ADP by membrane ATPase, and the biochemical changes in the gastric mucosa indicate a decreased energy turnover.     

Expression of CYP3A4 by an immortalized human hepatocyte line in a three-dimensional culture using a radial-flow bioreactor

Cytochrome P450 (CYP) 3A is responsible for about 50% of drug metabolizing activity in the liver. The present study was undertaken to establish a CYP3A4-active model for in vitro analysis of human drug metabolism. The cells used were immortalized normal human fetal hepatocytes (OUMS-29) and its HNF4alpha-introduced subline (OUMS-29/H-11). The cells were cultivated under high-density three-dimensional conditions in a radial-flow bioreactor (RFB). The number of OUMS-29 cells increased 15-fold over 49 days and their apical surfaces were covered with abundant microvilli, a characteristic of hepatocytes in vivo. The amount of albumin secreted by OUMS-29 cells in the three-dimensional RFB culture was 6-fold higher than those in a monolayer culture. CYP3A4 protein and an intermediate metabolite of testosterone by CYP3A4 were detected in OUMS-29/H11 cells cultivated in RFB >29 days. These results indicate that the RFB culture of OUMS-29/H-11 cells is useful for screening and developing new drugs.     

Adenine Nucleotide Degradation by the Obligate Intracellular Bacterium Rickettsia typhi

Adenosine 5′-triphosphate (ATP) was catabolized by whole cells and cell-free extracts of Rickettsia typhi to adenosine 5′-diphosphate (ADP) and then to adenosine 5′-monophosphate (AMP), the end product of ATP catabolism under the experimental conditions used. The only intermediate of the pathway from ATP to AMP which was identified by thin-layer chromatography and quantitated by the ¹⁴C content was ADP, whereas products such as adenine, adenosine, hypoxanthine, inosine, and inosine 5′-monophosphate were not detected. The enzymes which could be theoretically responsible for the catabolism or the anabolism of AMP were not detected by standard assay procedures. Most importantly, 5′-nucleotidase or nonspecific phosphatase and AMP nucleosidase activities were undetectable under a variety of experimental conditions. Although these two enzymes remove AMP from the adenylate pool in other cells, they are apparently nonfunctional in R. typhi. The biosynthesis of ATP was initiated by adenylate kinase because no adenine phosphoribosyltransferase or adenosine kinase could be detected. Furthermore, AMP was transported intact without prior dephosphorylation. These observations suggest that for R. typhi the in vivo activity of adenine nucleotide interconversion was limited to the nucleotides, with AMP being the end product of ATP catabolism, and that the salvage of purine bases and nucleosides was not an essential feature of purine metabolism. These results elucidate the findings of a previous study which showed that in the absence of glutamate as a source of energy, the adenylate energy charge of resting cells of R. typhi is drastically lowered by the high proportion of AMP.     

Reaction kinetics and inhibition of adenosine kinase from Leishmania donovani

The reaction kinetics and the inhibitor specificity of adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) from Leishmania donovani, have been analysed using homogeneous preparation of the enzyme. The reaction proceeds with equimolar stoichiometry of each reactant. Double reciprocal plots of initial velocity studies in the absence of products yielded intersecting lines for both adenosine and Mg2+-ATP. AMP is a competitive inhibitor of the enzyme with respect to adenosine and noncompetitive inhibitor with respect to ATP. In contrast, ADP was a noncompetitive inhibitor with respect to both adenosine and ATP, with inhibition by ADP becoming uncompetitive at very high concentration of ATP. Parallel equilibrium dialysis experiments against [3H]adenosine and [gamma-32P]ATP resulted in binding of adenosine to fre enzyme. Tubercidin (7-deazaadenosine) and 6-methyl-mercaptopurine riboside acted as substrates for the enzyme and were found to inhibit adenosine phosphorylation competitively in vitro. 'Substrate efficiency (Vmax/Km)' and 'turnover numbers (Kcat)' of the enzyme with respect to specific analogs were determined. Taken together the results suggest that (a) the kinetic mechanism of adenosine kinase is sequential Bi-Bi, (b) AMP and ADP may regulate enzyme activity in vivo and (c) tubercidin and 6-methylmercaptopurine riboside are monophosphorylated by the parasite enzyme.     

The effect of ATP and related compounds on spontaneous mechanical activity in the rat portal vein.

The effects of ATP, ADP, AMP and adenosine were studied on the spontaneous mechanical activity of the rat portal vein. 2It was found that STP and ADP, in concentrations higher than 300 mug M, caused a transient tetanus, followed by inhibition, and at lower concentrations an increase in the frequency and amplitude of the spontaneous contractions. AMP and adeosine on the other hand, inhibited spontaneous activity, by reducing the amplitude of contractions and increasing their frequency. The effects were dose-dependent. ATP was found to be 2.2 times more potent than ADP, while AMP and adenosine were equipotent. Weak inhibitory effects were obtained with GMP, guanosine and adenine, while GTP, 3K-cyclic AMP and guanine had no effect. ATP and ADP increased the K-contracture, while AMP and adenosine relaxed it. The effects of ATP were augmented in Mg-free solutions and partially inhibited in Mg-high solutions in the normally polarized muscle, while Mg had no influence on the ATP-induced contraction in the depolarized muscle. Theophylline potentiated the inhibitory response to AMP and adenosine. Adrenergic and cholinergic blockers had no influence on the response to ATP, ADP, AMP or adenosine. It is suggested that the effects of ATP and ADP are linked with Ca++ movements across the membrane, while AMP and adenosine might stimulate intracellular metabolism causing increased intracellular Cs-++ binding.