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96 h的常规细胞培养,观察存活细胞数目,DNA的合成和[³²P]-ATP标记膜蛋白的表达。结果：培养96 h后,0.4 mmol/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1 mmol/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 kD、31 kD、33 kD和40 kD的[³²P]-ATP标记膜蛋白呈高表达。结论：ATP对不死化人成纤维细胞的增殖有明显的抑制作用,并且使磷酸化细胞膜蛋白表达增高。     

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。     

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。     

人肺癌单克隆抗体的制备及其抗原分子的鉴定

用人肺癌细胞株A549的细胞膜蛋白作为免疫原,采用杂交瘤技术成功制备了抗人肺癌细胞单克隆抗体McAb4E7,并利用双向电泳、免疫印迹杂交、肽指纹图谱和MALDI-TOF-MS串连质谱分析鉴定其相关抗原分子,发现其相关抗原在人肺癌细胞株A549及人肝癌细胞株HepG2中有表达,且在A549细胞中表达量较高。最后得到一个在A549细胞中表达的与单克隆抗体McAb4E7特异性结合的蛋白,并鉴定出这个蛋白是ATP合成酶β亚基,从而证明了ATP合成酶β亚基就是肺癌单克隆抗体McAb4E7的靶抗原,其可作为肺癌具有潜在治疗意义的靶点。     

cAMP对人胃腺癌细胞系SGC-7901的影响——微绒毛和Na~+—K~+—ATP酶活性变化的电镜观察

本实验通过外源性 cAMP 和茶碱对人胃腺癌细胞系 SGC-7901的连续作用,观察到外源性 cAMP 和茶碱对该细胞株的增殖,具有明显的抑制作用。在透射电镜和扫描电镜下,均可见到细胞表面较光滑,微绒毛减少或消失。同时见到细胞膜表面上的 Na~+—K~+—ATP 酶活性受到抑制。本文讨论了外源性 cAMP 对人胃腺癌 SGC-7901细胞系增殖的抑制作用,以及膜表面微绒毛和Na~+—K~+—ATP 酶活性变化的关系。     

分化抗原6A8在B淋巴细胞膜和胞浆中的表达

采用间接免疫荧光染色、流式细胞仪、激光扫描共聚焦显微镜、Ｎｏｒｔｈｅｒｎ ｂｌｏｔ及Ｗｅｓｔｒｎ ｂｌｏｔ分析研究分化抗原６Ａ８在人淋巴细胞的表达。流式细胞仪分析显示，单抗６Ａ８与所检测的Ｂ细胞株３Ｄ５、ＣＥＳＳ和Ｄａｕｄｉ有反应，与人组织细胞瘤细胞株Ｕ９３７也有反应，与慢性髓性白血症细胞株Ｋ５６２及所检测的Ｔ细胞株Ｊｕｒｋａｔ和Ｐｅｅｒ不反应。Ｎｏｒｔｈｅｒｎ ｂｌｏｔ检测支持上述现象，３Ｄ５和     

汉族家族性高胆固醇血症患者永生化细胞株的构建及其LDL-R功能的研究

 目的：构建汉族家族性高胆固醇血症（FH）患者永生化细胞株;分析细胞表面低密度脂蛋白受体LDL-R活性。方法：将研究对象20例分为3组：（1）FH纯合组6人，为临床确诊的家族性高胆固醇血症纯合患者;（2）FH杂合组12人,为纯合患者的父母;（3）正常对照组2人，为血脂正常者；采用EB病毒转化外周血淋巴细胞；免疫组化法观察细胞表面LDL-R分布；流式细胞技术观察淋巴细胞LDL-R功能，4 ℃时检测其对LDL结合能力，37 ℃时检测其对LDL内化功能。结果：免疫组织化学法，在光镜下可见淋巴细胞表面散在分布的LDL-R，呈现棕褐色颗粒样；流式细胞技术对3组细胞株检测证实LDL-R功能存在差异，EB病毒转化的细胞株具有永生化特性;正常组LDL-R平均表达量111.35，高于杂合组（97.17）及纯合组（60.62）；纯合组37 ℃内化量228.61，4 ℃ 结合量95.20，结合量为正常人的41.6%，杂合组37 ℃内化量91.28，4 ℃结合量67.42，结合量为正常人的73.8%。结论：采用EB病毒转化外周血淋巴细胞，成功地构建了汉族家族性高胆固醇血症患者的永生化细胞株，此细胞株的功能研究表明其具有稳定的永生化特性，完整地保存了FH患者的细胞种质。     

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。     

稳定敲除Mcart-1下调RAW264.7巨噬细胞增殖能力和氧化磷酸化水平

目的 基于CRISPR/Cas9原理构建Mcart-1稳定敲除的小鼠单核巨噬细胞白血病细胞株RAW264.7,检测其细胞增殖能力和能量代谢水平。方法 用Cas9慢病毒和sgRNA慢病毒分两步感染RAW264.7巨噬细胞，并挑选单克隆细胞：qPCR和Western blot检测Cas9表达水平和Mcart-1敲除效果；单细胞测序分析突变位点；细胞能量代谢分析仪Seahorse检测细胞耗氧速率(OCR)和能量代谢表型；试剂盒测定细胞内总ATP含量；细胞计数仪计数和羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)染色检测细胞增殖。结果 成功构建Mcart-1稳定敲除的RAW264.7细胞株(记为Mcart-1^-/--RAW264.7);该细胞株中Mcart-1发生移码突变；与野生型RAW264.7细胞系(记为RAW264.7)相比，Mcart-1^-/--RAW264.7细胞氧气消耗速率(OCR)下降，胞外酸化速率(ECAR)升高；与RAW264.7细胞相比，Mcart-1^-/--RAW264.7细胞内总ATP含量下降(P<0....     

亚油酸对成纤维细胞增殖和分泌炎症介质影响的实验研究

目的探讨亚油酸对人成纤维细胞增殖及其分泌炎症介质的影响及其可能的作用机制。方法体外培养人成纤维细胞,分为正常对照组（不作任何处理）和实验组（细胞培养液中分别加入4mmol/L、2mmol/L、1mmol/L、0.5mmol/L和0.25mmol/L的亚油酸）。不同培养时相点观察细胞的光镜和电镜形态学、台盼蓝染色法绘制生长曲线、MTT法检测增殖抑制、流式细胞仪检测生长周期,ELISA法检测培养上清液中TNFα、IL-1β、IL-6、IL-8的含量,改良Griess法测定NO含量。结果亚油酸培养48h后,细胞增殖明显降低（P〈0.05）,光镜下见细胞生长缓慢,形态改变;电镜下见胞内脂滴、胞核和线粒体肿胀,部分细胞胞膜与核膜溶解破裂;流式细胞分析显示,G0/G1期、G2/M期细胞增多,S期细胞明显减少。亚油酸浓度为1mmol/L和2mmol/L时,实验组细胞TNFα分泌量显著高于对照组（P〈0.05）;亚油酸浓度为4mmol/L时,IL-1β、IL-6的分泌量与对照组相比明显升高（P〈0.01）;亚油酸浓度为2mmol/L和4mmol/L时,细胞IL-8、NO分泌量与对照组相比明显升高（P〈0.05）。结论高浓度亚油酸抑制体外培养人成纤维细胞生长、增殖,并促进人成纤维细胞分泌炎症介质。     

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.     

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.     

Fibroblast proliferation factors in pulmonary granuloma induced by Trichosporon cutaneum in rabbits: presence of a lymphocyte-derived fibroblast proliferation factor and its functional specificity

The fibroblast proliferation activity (FPA) in pulmonary granulomatous lesions in rabbits which were exposed once (primary response) or twice (secondary response) to Trichosporon cutaneum was examined using R9ab, a rabbit fibroblast cell line cell, and fibroblasts from the lesions of the primary and secondary responses. The FPA in lung extracts and cell-free culture supernatants of bronchoalveolar lavage cells from the secondary response was more evident than that from the primary response. FPA from the primary response were recovered at about 60, 18, and 4.5 kD and those from secondary response at about 60, 26, 18, and 4.5 kD on Sephadex G-75 gel filtration. Among the FPA, the activity with a molecular weight of 26 kD and a pI of 7.0 was derived from lymphocytes, whereas the other activities were derived from macrophages. The macrophage-derived fibroblast proliferation factors (FPF) enhanced proliferation of fibroblasts from the lesions of both primary and secondary responses, while the lymphocyte-derived FPF enhanced only proliferation from the secondary response. It was further found that lymphocyte-derived FPF could chemotactically attract fibroblasts from the secondary but not the primary response, indicating functional specificity of lymphocyte-derived FPF on fibroblasts in the secondary response. The present results suggest that this lymphokine with fibroblast proliferation and chemotactic activity plays an important role in the granuloma formation in the secondary response to T. cutaneum.     

All-trans retinoic acid reduces membrane fluidity of human dermal fibroblasts. Assessment by fluorescence redistribution after photobleaching.

All-trans retinoic acid (RA) preserves human dermal fibroblast viability and stimulates proliferation in vitro. These effects are mediated, at least in part, by reducing the extracellular Ca2+ requirement. The same concentrations of RA that reduce the extracellular Ca2+ requirement also interrupt movement of Ca 2+ across the fibroblast plasma membrane. Based on these observations, we have examined the effects of RA on membrane properties that could influence Ca2+ movement. Fibroblasts were labeled with 1-acyl-2-(N-4- nitrobenzo-2-oxa-1,3 diazole)-amino-caproyl phosphatidyl-choline (a fluorescent phospholipid analogue) and examined for fluorescence redistribution after photobleaching (FRAP) with a pulse of intense light as a measure of membrane fluidity. Using this approach, we observed that membrane fluidity was higher when the cells were incubated in medium containing a low (sub-optimal) level of extracellular Ca2+ (0.15 mmol/L) than in a medium containing an optimal concentration (1.4 mmol/L). Treatment of the cells with 3 micromol/L RA reduced membrane fluidity of the cells under both high- and low-Ca2+ conditions. These findings demonstrate that RA has a direct effect on the plasma membrane of human dermal fibroblasts. This provides a possible mechanism for the previously identified inhibition of Ca2+ movement across the membrane of the same cells and for the previously identified protective effects against lysis under low-Ca2+ conditions.     

Establishment of proliferative tetraploid cells from telomerase‐immortalized normal human fibroblasts

Aneuploidy is observed in the majority of human cancers and is considered to be causally related to carcinogenesis. Although malignant aneuploid cells are suggested to develop from polyploid cells formed in precancerous lesions, the mechanisms of this process remain elusive. This is partly because no experimental model is available where nontransformed polyploid human cells propagate in vitro. We previously showed that proliferative tetraploid cells can be established from normal human fibroblasts by treatment with the spindle poison demecolcine (DC). However, the limited lifespan of these cells hampered detailed analysis of a link between chromosomal instability and the oncogenic transformation of polyploid cells. Here, we report the establishment of proliferative tetraploid cells from the telomerase‐immortalized normal human fibroblast cell line TIG‐1. Treatment of immortalized diploid cells with DC for 4 days resulted in proliferation of cells with tetraploid DNA content and near‐tetraploid/tetraploid chromosome counts. Established tetraploid cells had functional TP53 despite growing at almost the same rate as diploid cells. The frequency of clonal and sporadic chromosome aberrations in tetraploid cells was higher than in diploid cells and in one experiment, gradually increased with repeated subculture. This study suggests that tetraploid cells established from telomerase‐immortalized normal human fibroblasts can be a valuable model for studying chromosomal instability and the oncogenic potential of polyploid cells. © 2016 Wiley Periodicals, Inc.     

人外周血内皮祖细胞增殖、凋亡及丙二醛、抗氧化因子合成与波动性高糖的影响

背景：研究表明波动性高血糖较持续性高血糖对血管内皮功能的损害可能更严重。 目的：观察波动性高糖对人外周血内皮祖细胞增殖、凋亡及丙二醛、抗氧化因子合成的影响。 方法：密度梯度离心法获取人外周血单个核细胞。取经培养鉴定后的内皮祖细胞,细胞同化后分别给予5.5 mmol/L,20 mmol/L,5.5/20 mmol/L葡萄糖(5.5,20 mmol/L的葡萄糖培养液每8 h更换1次)及20 mmol/L甘露醇。干预72 h后,MTT法检测内皮祖细胞的增殖情况;流式细胞仪检测细胞凋亡率;比色法测定培养液中丙二醛水平及超氧化物歧化酶活力。 结果与结论：20 mmol/L和5.5/20 mmol/L葡萄糖作用72 h,内皮祖细胞增殖减少、凋亡率增高(P < 0.01）,培养液中丙二醛水平增高、超氧化物歧化酶活力降低(P < 0.01),均以5.5/20 mmol/L葡萄糖作用最明显(P  < 0.01)。说明波动性高糖较恒定性高糖更易抑制内皮祖细胞增殖并促进其凋亡,其机制可能与波动性高糖环境下氧化应激水平增高有关。