Staurosporine引起正常和肿瘤细胞内Ca~(2 )和CaM水平发生变化

低剂量Staurosporine可以使正常细胞可逆地阻断于G1期,但对肿瘤细胞的周期运行不发生任何影响。本文利用显微光度术,测定了单个细胞内Ca~(2 )、活化钙调素和总钙调素的含量,结果表明:5ng/mL staurosporine作用于细胞18h,使正常细胞2BS G1和S期总CaM含量降低;而BGC-823细胞各周期时相总钙调素含量不发生改变;钙活化钙调素增加。Staurosporine阻断2BS细胞于G1期而不影响BGC-823的周期运行可能与Stauro-Sporine使2BS G1期细胞的钙调素降低以及抑制了2BS细胞的P~(107)磷酸化有关。     

THE UPTAKE OF PURINES BY RAT BRAIN IN VIVO AND IN VITRO

Abstract— The uptake of [¹⁴C]guanine and some of its [¹⁴C]-labelled derivatives into rat brain was studied in vivo and in vitro. In vivo guanine, guanosine, and hypoxanthine penetrated the brain of adult rats to a very small extent. Inosine was taken up somewhat better. In young animals, also, guanosine was taken up poorly, but guanine was taken up fairly well. When guanine was administered to adult animals, only guanine was found in the brain. In young animals, by contrast, radioactivity from guanine appeared in guanosine and in guanine nucleotides, but no free guanine was found. In vitro guanine was taken up much better and, in fact, remained mostly as guanine in slices from 10-day-old rats. The in vitro conversion of guanine to GMP and its incorporation into RNA was unimpaired by the addition of unlabelled guanosine, an indication that guanine was converted directly to GMP. The uptake of guanine in vitro was not subject to competitive inhibition or influenced by the presence of dinitrophenol. This finding suggested that guanine entered the slice by simple diffusion.     

CHARACTERIZATION OF TAURINE UPTAKE BY NEURONAL AND GLIAL CELLS IN CULTURE

Uptake of [³⁵S]taurine was studied in parallel on glial and neuronal cells maintained in continuous culture, including transformed neuronal cells. Both glial and neuronal taurine uptake systems were concentrative, highly sodium-dependent and inhibited by closely related structural analogues such as hypotaurine, β-alanine and GABA. Strychnine was found to be a potent inhibitor of taurine uptake, especially in the glial cells, while parachloromercuriphenylsulphonate was more efficient on the neuronal clones. In contrast with uptake by neuroblastoma cells, the glial transport was dependent on the presence of calcium in the incubation medium.     

SH－SY5Y细胞胞内钙库特性研究

运用单细胞显微荧光测量技术测量了单个ＳＹ－ＳＹ５Ｙ细胞内游离钙离子浓度的变化。首次报道了ＳＨ－ＳＹ５Ｙ细胞内存在毒蕈碱敏感而非咖啡因敏感的钙库,并研究了它的动力学特征。     

钙调素拮抗剂与胞内钙离子浓度对离体培养的人蜕膜细胞活力的影响

应用妊娠 6～ 8周的人工流产蜕膜组织为材料进行离体人工蜕膜细胞培养 ,观察两种化学结构完全不同的特异性钙调素拮抗剂——三氟拉嗪、蝙蝠葛碱衍生物 ,以及 Ca2 + 螯合剂—— EGTA和 Ca2 +载体—— A2 3187对蜕膜细胞活力的影响。结果表明 :三氟拉嗪、蝙蝠葛碱衍生物与 EGTA对蜕膜细胞活力的抑制作用与药物的浓度及其作用时间密切相关 ,随着浓度加大及作用时间延长而增强。≥ 1 5 μmol/L的三氟拉嗪或≥ 2 5 μmol/L的蝙蝠葛碱衍生物 ,或2 mmol/L的 EGTA均可明显抑制蜕膜细胞活力。三氟拉嗪和蝙蝠葛碱衍生物作用 96小时后分别使细胞活力降至对照组的 8.7%和 1 2 .0 % ,EGTA作用 72小时后降至对照组的 2 8.6%。6μmol/L的 A2 3187可一定程度地刺激细胞活力上升 ,但这种刺激效应随作用时间的延长而逐渐减弱。EGTA可增强三氟拉嗪对细胞活力的抑制作用。由此推测 ,Ca2 + -钙调素系统可能直接参与子宫蜕膜的发育和维持 ,并在其中发挥重要作用。这可能也是钙调素拮抗剂抗早孕的主要机理之一。     

SH－SY5Y细胞胞内钙库特性研究

运用单细胞显微荧光测量技术测量了单个ＳＨ—ＳＹ５Ｙ细胞内游离钙离子浓度的变化。首次报道了ＳＨ—ＳＹ５Ｙ细胞内存在毒蕈碱敏感而非咖啡因敏感的钙库,并研究了它的动力学特征。Ｎ￣￣Ｃ￣ｈｅｔｗＭａｄＵｇｈ＊ＴＯｗｈｏｍＣｂ￣ｐｏｎｄｅｎｅｅｓｈｏｕｌｄｂｅ￣．     

利用离体大白鼠肝癌细胞的EROD诱导指示二恶英的复合毒性效应

离体条件下,以大白鼠肝癌细胞株H4IIE的7－乙氧基－3－异吩恶唑酮－脱乙基酶（EROD）活力诱导作为毒性指标,测定了2,3,7,8－TCDD单独存在以及与一定浓度的2,3,7,8,－TCDF,OCDD,PCB126和PCB77分别共存下的EROD活力,并用TEF和独立作用模型（independence)两种方法对实验结果进行了评估。利用TEF评估的结果表明实验的TEQ值和理论计算的TEQ值十分接近,复合毒性表现为加合作用（additivity),这一结果与用独立作用模型评估的结果完全一致。研究结果不仅证实了TEF评估方法的有效性和利用模型方法评估二恶英类化合物复合毒性的可行性,同时还表明离体条件下,大白鼠肝癌细胞株H4IIE的EROD酶活力诱导适用于化合物的复合毒性的研究。     

合成洗涤剂对人和哺乳动物细胞的诱变性研究

各种合成洗涤剂(洗衣粉,洗发膏,餐具洗涤剂等)产量日增。在日常生活中使用越来越普遍。洗涤剂直接或间接通过环境污染对人类健康产生影响,特别是潜在的致突变性引起公众的普遍注意。而现有的研究结果并不一致。本研究选用三种型号的合成洗涤剂,以小鼠生殖细胞染色体畸变和骨髓细胞微核率及离体的人类细胞和中国仓鼠细胞的染色体畸变和姐妹染色单体交换(SCE)为测定指标,系统地对合成洗涤剂的诱变活     

嗅神经鞘细胞的培养纯化及体外生长特性

采用原代培养的方法,从2,5月成年大鼠的嗅球分离培养嗅神经鞘细胞（OECs),培养6天后,用阿糖胞苷（Ara-C)抑制,差速贴壁,Forskolin和BPE营养物质处理,根据P75蛋白免疫细胞化学染色和形态学特征分析了所得细胞的纯度,同时对不同培养时期的OECs 的形态进行观察和纯化后的活力测定。实验结果显示：（1）这种纯化方法简单,经济,快捷,所得的OECS纯度可达95％以上,并且随培养时间延长,细胞仍保持较高的纯度。（2）在培养早期2天到5天主要以巨噬细胞状,多极状,不规则状为主,培养中期7天到20天主要以扁平的双极,三极为主。晚期20天以后呈现双极,三极形态,其起上有许多细小的棘突。93）其中以培养早中期细胞的活力较好,培养20天以后,细胞活力较差,本研究为以OECs 作为移植材料对促进神经再生的研究获得丰富的细胞来源奠定了基础。     

猪瘟病毒的形态结构与形态发生

建立了猪瘟病毒(CSFV)弱毒疫苗Thiverval株(T株)与中 国兔化弱毒疫苗C株在MPK细胞中的感染模式。使用MPK细胞增殖CSFV,其病毒滴度明显提高,从而为应用电镜超薄切片技术研究猪瘟病毒的形态结构与形态发生提供了可能性。猪瘟病毒呈圆形颗粒,直径约为70nm。其内部是电子致密的核心,直径约为40nm,有时呈六角形;外有包膜包裹。在CSFV感染的MPK细胞质中,可观察到处于不同发育阶段的子代病毒粒子。此外,猪瘟病毒的感染能够引起某些宿主细胞超微结构上的变化。     

阿片肽类物质对大鼠黄体细胞孕酮生成的影响

本文观察了外源性阿片肽对大鼠离体黄体细胞孕酮生成的影响,结果表明:β-内啡肽以剂量-反应依赖方式促进黄体细胞孕酮生成,有效浓度范围是10~(-8)-10~(-6) mol/L;强啡肽仅在浓度为10~(-6)mol/L时才显示刺激孕酮生成的作用;而甲硫-脑啡肽无明显作用。μ-阿片受体激动剂DAGO和乙基吗啡也能明显促进孕酮的生成。纳洛酮可完全阻断β-内啡肽,DAGO和乙基吗啡的作用。由于大鼠血液中β-内啡肽含量较低,而卵巢局部具有较高浓度的β-内啡肽。因此,我们认为,β-内啡肽可能在卵巢局部参与黄体细胞孕酮生成的调节,是卵巢内促黄体因子之一,这种作用可能是由μ-型阿片受体介导的。     

利用FRET技术在活细胞内观察EGF对PKA作用的时空成像

cAMP依赖的蛋白激酶(protein kinase A,PKA)在细胞生长与分化过程中扮演重要角色,特别是在调节Ras信号通路引起的细胞增殖效应中起着重要作用。为了在活细胞内动态观察表皮生长因子(epidermal growth factor,EGF)对PKA的作用,采用一种可以检测PKA酶活性的报告蛋白(A-kinase activity reporter,AKAR)——这种报告蛋白是利用荧光共振能量转移(fluorescence resonance energy transfer,FRET)原理设计的,使其在人类肺癌细胞(ASTC-a-1)中稳定表达。加入EGF刺激因子后,随时间变化的成像分析显示出在活细胞生理条件下被EGF作用的PKA酶活性变化的时空信息。这些资料为EGF作用PKA提供了直接的实时证据。     

甲胎蛋白在体外对人肝癌细胞生长的影响

本文用MTT比色法观察了甲胎蛋白(AFP)在体外对人肝癌细胞生长的影响。结果表明,AFP能促进SMMC-7721人肝癌细胞的生长。当AFP与AFP抗体合用时,AFP抗体能减弱AFP对SMMC-7721细胞生长的促进作用;AFP抗体单用对此种细胞的生长亦有抑制作用。另一方面,在相同的实验条件下,AFP和AFP抗体对HL-60人白血病细胞的生长无明显影响;提示AFP的促生长作用具有一定的肿瘤细胞特异性,并非一种蛋白质对培养细胞的非特异性营养作用。此外,AFP亦能促进MCF-7人乳腺癌细胞的生长,AFP抗体对此种细胞的生长有抑制作用。由于MCF-7细胞存在功能性AFP受体,也能合成和分泌AFP。这就提示,人肝癌细胞中的AFP很可能与其受体特异性结合,产生促生长效应。确切机制尚待进一步阐明。     

TRANSCELLULAR BIOSYNTHESIS OF CYSTEINYL LEUKOTRIENES BY KUPFFER CELL—HEPATOCYTE COOPERATION IN RAT LIVER

We previously proposed that an enzymatic cooperation between Kupffer cells and hepatocytes may play an important role in cysteinyl leukotriene (LT) production in rat liver. Anin vitrotranscellular synthesis cysteinyl LTs by a Kupffer cell—hepatocyte coculture system was characterized here. Kupffer cells alone, with A23187 stimulation, did not generate cysteinyl LTs until supplemented either with isolated hepatocytes or with LTC₄synthase and glutathione, indicating that Kupffer cells can synthesize LTA₄but not convert it into LTC₄. In contrast, hepatocytes converted the LTA₄into cysteinyl LTs and further degraded the cysteinyl LTs. Cysteinyl LT production by the Kupffer cell—hapatocyte coculture system was optimized by addition of 1–3% serum albumin to the culture and by bringing the cell—cell distance closer to less than 3μ. Tumour necrosis factor also stimulated cysteinyl LT production by the coculture system. From these results, it is expected that the Kupffer cell—hepatocyte transcellular system for cysteinyl LT production actually functionsin vivo.     

UPTAKE AND LOCALIZATION OF 3H-1-METHYLADENINE BY THE GONADAL EPITHELIAL CELLS OF SEA URCHINS IN VITRO

The meiotic inducing hormone, 1-methyladenine, first isolated from starfish has been implicated to play some role in the gamete maturation of a number of marine invertebrates. However, there have been controversial and sometimes opposite conclusions due to the fact that there is no direct bioassay system other than in starfish.
Using ³H-1-methyladenine, we demonstrated that the hormone was localized exclusively in the outer epithelial cells of gonads of sea urchins as revealed by autoradiography. Uptake by the testes differs from that by the ovaries. Competition data show that the uptake of the labelled hormone is real and also show the different rates of uptake by the gonads of male and female sea urchins.
It is concluded that 1-MA may exhibit differential effects on cell types due to their differentiated state and the seasonal variations of the organisms. The action of 1-MA is, therefore, multiple and not restricted to the gametes although its action would ultimately result in gamete maturation.     

MAGNETIC FIELD STIMULATION OF HYBRIDOMA CELLS IN VITRO

Non-ionizing physical field interactions with cells, both in situ and in vitro, is of current interest globally. This is from various directions—starting from their abilities to induce permanent modifications in cell behavior in situ, through carcinogenesis and mutagenesis, to utilizing field effects for possibly enhancing the viable cell population in vitro. This results in parallel increase in some high-value, low-volume biochemical production. In the present study, screening experiments were carried out with a unique cell line—hybridoma (OKT3) (secreting monoclonal antibodies [MAbs] against T3 surface antigens of human peripheral CD₄⁺ cells)—for a possible enhancement in the yield of extremely high value product (MAb). Overall, in the absence of any such data globally, there is apparently an urgent need for screening of such “field effects” on various other cell types in vitro for various reasons; e.g., low cost of manipulation, nonpolluting nature of interactions, distinct possibility of enhancement of produced biochemical titers, etc. In the present study, we observed various responses of the cell population both to magnetic fields alone and in combination with other known chemical stimulants of viable biomass (mono- and poly-lysine). Fifty hertz, 0.8 mT magnetic field and below, in conjunction with bulkier poly-lysine molecules, needs to be investigated further for a possible resonance-induced anti-interaction between these known mitogens and their cell surface receptors, which possibly could be extrapolated to other growth factor-receptor interactions in magnetic field environments, in situ.     

脂肪来源细胞体外增殖规律及定向诱导分化研究

脂肪组织由整形外科吸脂术获得(19例,31．5±5．8岁)。酶消化法分离抽吸物中细胞,体外扩增至第10代．测定细胞生长曲线、累计倍增数目,明确其体外生长规律和增殖能力;通过对表面抗原CD29、CD105、CD106、CD166、CD49d、CD34、CD31、3G5等的检测分析脂肪来源细胞的群体组成：分别向软骨、骨、脂肪定向诱导,进一步明确该细胞群体定向分化能力。实验表明,每300ml脂肪抽吸物平均可获得5×10~7个有核细胞,体外扩增10代,平均每代倍增数目为1．59±0．224．累计倍增数目为15．53。流式细胞学及免疫细胞化学检测显示,干细胞相关抗原CD29、CD105、CD106、CD166等表达率均＞60%,但与造血系相关的CD34、CD31表达率也分别达到7．3%、29．2%。ADC向软骨诱导可检测到Ⅱ型胶原表达;向成骨诱导可见矿化结节形成,并可检测到AKP、Osteonectin基因表达;向脂肪诱导可检测到PPARr2、GLU-4、Leptin基因表达,细胞内有脂滴形成。脂肪来源的细胞获得量大,体外增殖能力强,并含有具有多向分化潜能细胞,有可能作为组织构建的种子细胞。     

Synthesis of multifunctional chitosan with galactose as a targeting ligand for glycoprotein receptor

Chitosan-O-PEG-galactose was synthesized through hydroxyl groups of chitosan, which followed several steps including protection of amino group of chitosan, pegylation of chitosan, galactosylation of pegylated chitosan, and final removal of protection to obtain chitosan-O-PEG-galactose. The synthesized intermediates and final product were characterized and confirmed by ¹H NMR and FTIR, and the amounts of PEG and galactose conjugated with chitosan were measured. The pegylated chitosan possesses amphiphilic property in terms of soluble in both neutral aqueous (e.g., water) and organic solvents (e.g., DMF, dichloromethane). The corresponding critical micelle concentration is measured to be 0.56 mg/mL, and the size of micelles is 294.5 ± 2.3 nm with polydispersity 0.123 ± 0.021. The contents of PEG and galactose conjugated in chitosan-O-PEG-galactose are 98.09 ± 4.63% w/w and 3.06 ± 0.54% w/w, respectively. In terms of the degree of O-substitution of chitosan by PEG (DS_PEG) and the degree of substitution of PEG by galactose (DS_g) are 177.69% and 86.7%, respectively. Exclusively high DS_PEG indicates both C6–OH and C3–OH of chitosan are conjugated with PEG polymer chains. Further prominent attachment of galactose onto hydroxyl end group of PEG allows chitosan-O-PEG-galactose to possess sufficient quantity of targeting moieties for asialoglycoprotein receptor on hepatocytes.     

聚乙二醇支载有机小分子合成的研究进展

本研究综述了聚乙二醇（PEG）支载的有机小分子合成研究的进展,从PEG所支我的直链化合物包括胺化产物、酰胺化合物、硫脲及取代硫脲化合物、其他化合物,从PEG所支载的杂环化合物包括单杂原子杂环、多杂原子杂环等两大方面分类讨论了所支载的有机小分子的合成方法,在此基础上对聚乙二醇作为载体的研究前景进行了展望。