石墨碳纳米颗粒对体外培养细胞生长特性的影响

背景：有报道指出石墨碳纳米颗粒具有强大的吸附能力,只要尽可能将其控制在有效浓度范围内,石墨碳纳米颗粒会具有很好的细胞相容性及增敏效应。 目的：了解石墨碳纳米颗粒的形态特征,观察石墨碳纳米颗粒其对体外培养细胞增殖与超微结构的影响。 方法：取石墨碳纳米颗粒0.5 g,加100 mL三蒸水,振荡后微孔过滤,即为石墨碳纳米颗粒母液。取处于对数生长期的HepG2细胞、L02细胞、Hl7702细胞、3T3细胞,调整密度为5×107 L-1接种于6孔板,0.5 mL/孔,加入含胎牛血清、青霉素、链霉素的RPMI-1640培养基1.5 mL,培养24 h后弃去旧液,设1~5号孔为实验组,分别加入质量浓度为25,10,7.5,5,0.25 mg/L石墨碳纳米颗粒培养液2.0 mL,设6号孔为空白对照组,不加石墨碳纳米颗粒溶液,继续培养24 h后终止培养。用原子力显微镜测量石墨碳纳米颗粒的粒径,电子显微镜观察石墨碳纳米颗粒的形态特征;用细胞计数板在光学显微镜下计数不同浓度石墨碳纳米颗粒对细胞数量的影响;透射电镜观察7.5 mg/L石墨碳纳米颗粒对细胞超微结构的影响。 结果与结论：石墨碳纳米颗粒呈球形微粒,粒径约20 nm。与空白对照组比较,各浓度石墨碳纳米颗粒培养液组除HepG2细胞外,其余3种细胞数量基本都有所增加,其中7.5 mg/L石墨碳纳米颗粒培养液对L02细胞、Hl7702细胞、3T3细胞、HepG2细胞数量的影响最为显著(P < 0.05)。对7.5 mg/L石墨碳纳米颗粒作用后的细胞进行透射电镜观察,可见石墨碳纳米颗粒分布于细胞内部,如细胞质、细胞核、线粒体中,未见亚细胞结构受损及细胞凋亡坏死现象发生。证实石墨碳纳米颗粒对体外培养的细胞无损伤不良反应,且能够促进细胞生长增殖,其作用强度与质量浓度有关,7.5 mg/L为较佳质量浓度。 关键词：纳米石墨碳;人肝细胞株;人肝癌细胞株;超微结构;细胞生长 doi:10.3969/j.issn.1673-8225.2010.03.015     

脑脊液对体外培养骨髓基质细胞生长的影响

目的 观察脑脊液对体外培养的骨髓基质细胞的影响并探讨颅内移植骨髓基质细胞的可能性。方法 分离BALB/C小鼠的骨髓细胞进行体外培养，然后将贴壁的骨髓细胞传代，在含不同比例脑脊液的培养基中培养，观察其生长情况。结果 在含不同比例脑脊液的培养基中生长的骨髓基质细胞形态相似，呈多角形、梭形以及圆形；各组骨髓基质细胞计数差异无显性意义(P＞0．05)。结论 骨髓基质细胞在50％(体积百分浓度)的脑脊液培养基中仍可继续生长、增殖，提示这种细胞对生长环境有高度的适应性。     

纳米氧化铁颗粒表征分析及其对体外标记肿瘤细胞磁信号变化的影响

背景：超顺磁性纳米氧化铁颗粒的粒径小,且具有良好的水溶性、组织相容性、超顺磁性及表面积效应,使其应用于磁共振成像和作为生物大分子等药物载体成为可能。 目的：构建葡聚糖包被的超顺磁性四氧化三铁纳米颗粒(dextran coated iron oxide nanoparticles, DCIONP),分析其主要物理性质和磁学特性,并探讨其标记肿瘤细胞后对肿瘤细胞磁学特性的影响。 方法：通过化学共沉淀法制备葡聚糖包被的DCIONP。透射电镜和X射线粉末衍射法分析DCIONP的粒径和晶体结构,磁共振仪测定其弛豫率等主要物理和磁学参数。体外DCIONP分别标记人骨肉瘤MG63细胞、人肝癌HGP2细胞和鼠骨髓间充质干细胞后,用普鲁士蓝铁显色法和透射电镜观察DCIONP粒子在细胞内的分布,1.5T磁共振仪测量DCIONP体外标记肿瘤细胞后对肿瘤细胞磁信号的影响。 结果与结论：X射线衍射分析确定所制备的葡聚糖包被的磁性纳米粒子主要为Fe3O4晶体,氧化铁核心约为10 nm,具有超顺磁性,其弛豫率达3.936×106 mol/s。体外标记3种细胞后,见DCIONP主要分布在细胞核中,细胞浆中所占比率相对较小。体外磁标记后,肿瘤细胞的T2信号随着细胞数的增加而逐渐缩短,且2×109 L-1和2×1010 L-1磁标记细胞的磁信号呈明显的衰减状态。结果证实所制备的DCIONP粒子物理性能稳定,磁标记肿瘤细胞后在MR上能产生特征性的低信号。 关键词：超顺磁性四氧化三铁纳米颗粒;肿瘤;细胞;磁共振成像;衰减;纳米生物材料 doi:10.3969/j.issn.1673-8225.2010.08.017     

敲减CDKL5对人胶质母细胞瘤细胞生长和细胞周期的影响

目的探讨敲减周期蛋白依赖型激酶样5(cyclin-dependent kinase-like 5,CDKL5)对人胶质母细胞瘤细胞生长和细胞周期的影响,为胶质母细胞瘤的治疗提供新思路。方法本研究使用人胶质母细胞瘤细胞U87,使用慢病毒感染的方法敲减CDKL5基因。利用Western blot显示CDKL5的细胞定位以及特异性的小发卡RNA敲减效率。使用血细胞计数法绘制4 d U87的细胞生长曲线,同时利用MTT检测细胞活性。使用流式细胞仪技术检测细胞周期。结果在U87人胶质母细胞瘤细胞中,CDKL5在细胞核和细胞质当中都有表达,我们发现使用shRNA敲减CDKL5后,胞质中的CDKL5显著减少,细胞形态也发生明显改变。敲减CDKL5同时抑制人胶质母细胞瘤细胞的体外增,引起细胞周期停滞。结论敲减CDKL5可以显著抑制人胶质母细胞瘤细胞增殖,引起细胞周期停滞,为人胶质母细胞瘤的治疗提供了一个新的理论方法。     

缺血缺氧对体外培养星形胶质细胞细胞周期和周期相关蛋白的影响

目的 观察缺血缺氧损伤对星形胶质细胞细胞周期及细胞周期相关蛋白的影响。方法 用流式细胞仪及Brdu掺入法检测缺血缺氧后不同时间点星形胶质细胞细胞周期变化和细胞的增殖活力；用荧光免疫细胞化学技术测定增殖细胞核抗原(PCNA)及细胞周期蛋白cyclin D1的表达水平。结果 体外缺血缺氧损伤后S期星形胶质细胞较正常组明显增加，6h达高峰，Brdu掺入法显示损伤后6h星形胶质细胞的增殖活力最高，而随后S期细胞数目及细胞增殖活力都呈下降趋势。PCNA阳性反应损伤后表达增加，6h表达最高，而cyclin D1的表达在损伤后逐渐增加，在24h时达高峰。结论 缺血缺氧损伤激活星形胶质细胞，使其进入新的细胞周期，出现细胞的增殖反应；PCNA及cyclin D1参与了损伤后星形胶质细胞的修复和增殖；细胞周期事件与星形胶质细胞的增殖活化密切相关。     

脑脊液对体外培养骨髓基质细胞生长的影响

目的观察脑脊液对体外培养的骨髓基质细胞的影响并探讨颅内移植骨髓基质细胞的可能性。方法分离BALB/C小鼠的骨髓细胞进行体外培养,然后将贴壁的骨髓细胞传代,在含不同比例脑脊液的培养基中培养,观察其生长情况。结果在含不同比例脑脊液的培养基中生长的骨髓基质细胞形态相似,呈多角形、梭形以及圆形;各组骨髓基质细胞计数差异无显著性意义(P>0.05)。结论骨髓基质细胞在50%(体积百分浓度)的脑脊液培养基中仍可继续生长、增殖,提示这种细胞对生长环境有高度的适应性。     

125I对体外培养的胶质瘤细胞C-myc、p53基因表达及细胞增殖的影响

目的探讨125I对体外培养的胶质瘤细胞SHG-44C-myc、p53基因表达及细胞增殖的影响。方法利用免疫组化技术检测经125粒子放射处理3d后,SHG-44细胞的C-myc蛋白表达与p53蛋白表达水平。结果经1粒、3粒125粒子放射处理3d后,SHG-44细胞C-myc蛋白表达明显减弱,而p53蛋白表达显著增强。二者呈负相关。结论125I可以通过影响C-myc基因与p53基因的表达抑制人脑恶性胶质瘤细胞株SHG-44增殖,从而抑制胶质瘤的生长。     

Cyclopamine对神经母细胞瘤细胞系SK-N-SH细胞生长周期的影响及其机制

目的探讨Shh信号途径的抑制剂cyclopamine对人神经母细胞瘤细胞系SK-N-SH细胞生长周期的影响及其分子生物学机制。方法以不同浓度的cyclopamine处理SK-N-SH细胞不同时间,在相差倒置显微镜下观察药物作用后的该细胞形态学变化。用四甲基偶氮唑盐（MTT）分析法检测各组SK-N-SH细胞的增殖情况,流式细胞仪检测不同浓度cyclopamine作用后对细胞周期的影响,Westernblot检测药物作用前后细胞周期蛋白D1、P16和P21蛋白表达的变化。结果cyclopamine各处理组SK-N-SH细胞形态学有所不同。MTT法检测结果显示,cyclopamine对SK-N-SH细胞增殖的抑制是浓度依赖性和时间依赖性的（P〈0.05）。流式细胞仪检测发现随着药物浓度的增加,G0/G1期细胞百分比明显增加（P〈0.05）,而S期细胞百分比明显降低（P〈0.05）。Westernblot检测结果显示,随着药物浓度的增加,SK-N-SH细胞中细胞周期蛋白D1表达逐渐降低,P16和P21表达逐渐增高（P〈0.05）。结论cyclopamine抑制SK-N-SH细胞生长,阻滞细胞从G1期向S期转化,其机制可能与调控细胞周期蛋白表达有关。cyclopamine可能成为未来治疗神经母细胞瘤的药物之一。     

转染SV40永生化基因对正常成人肝细胞生长特性的影响

背景：生物型人工肝采用猪肝细胞或肝癌细胞作为移植物来源存在动物源性疾病和致瘤性的担心,而正常成人肝细胞也具有一定的局限性。 目的：通过检测转染前、后正常成人肝细胞的活率、生长曲线和细胞周期的变化,了解转染SV40永生化基因对正常成人肝细胞生长特性的影响。 方法：培养不同时间的正常人肝细胞和永生化正常成人肝细胞,采用胎盘蓝染色和AO-PI染色,于培养后1~8 d采用MTT染色法计数细胞活率;用MTT比色法测定细胞的A值并绘制生长曲线;采用流式细胞术检测细胞的生长周期。 结果与结论：3种染色法检测显示两种细胞的活率为95%~99%,存活率无明显差异。转染前、后的两种正常成人肝细胞的生长曲线无明显差异,均在培养 3~5 d时呈指数型生长,但转染后正常成人肝细胞较转染前增殖略快。采用流式细胞术测得的两种肝细胞的细胞周期：转染后肝细胞S期细胞占65.64%,G0~G1期细胞占34.36%,G2期细胞占0%;转染前肝细胞S期占21.27%,G0~G1期细胞占62.64%,G2期细胞占12.09%,提示转染后肝细胞的S期细胞明显增多,增殖能力增强。转染SV40永生化基因的正常成人肝细胞较转染前细胞的增殖活力增高,存活率无明显差异,提示转染后细胞增殖能力更强。     

维生素A对体外培养小鼠精原干细胞生长增殖的影响*

背景：维生素A在体内对精原干细胞生长具有重要作用,目前还没有发现在体外培养过程中能够很好促进精原干细胞生长与分化的诱导物质。 目的：探讨维生素A对体外培养小鼠精原干细胞生长增殖的影响。 方法：无菌收集5~7 d龄昆明雄性小鼠双侧睾丸,采用差速贴壁联合非连续性Percoll密度梯度离心法分离纯化精原干细胞。无菌取出12~15 d龄昆明雄性小鼠双侧睾丸,酶消化法分离纯化Sertoli细胞,贴壁并极化后作为饲养层,将精原干细胞接种在单层Sertoli细胞上。设立2组,实验组向DMEM/F12培养液中加入1 g/L维生素A,对照组不添加维生素A。采用酶联仪测定精原干细胞生长增殖情况,流式细胞仪检测精原干细胞生长周期。 结果与结论：共培养6,9,12,15 d时,实验组精原干细胞吸光度值明显高于对照组(P < 0.05或0.01)。随共培养时间的延长,实验组精原干细胞S期染色体含量逐渐增多,然后又逐渐下降,开始另一个分裂周期;与实验组比较,对照组精原干细胞S期染色体含量增长缓慢(P < 0.05)。小鼠精原干细胞在体外培养过程中,维生素A可促进其增殖分化。     

Effects of the vasoactive intestinal peptide (VIP) and related peptides on glioblastoma cell growth in vitro

The growth rate of numerous cancer cell lines is regulated in part by actions of neuropeptides of the vasoactive intestinal peptide (VIP) family, which also includes pituitary adenylate cyclase-activating peptide (PACAP), glucagon, and peptide histidine/isoleucine (PHI). The aim of this work was to investigate the effect of these peptides on the growth of the rat glioblastoma cell line C6 in vitro. We also sought to determine which binding sites were correlated with the effects observed. Proliferation studies performed by means of a CyQuant™ assay showed that VIP and PACAP strongly stimulated C6 cell proliferation at most of the concentrations tested, whereas PHI increased cell proliferation only when associated with VIP. Two growth hormone-releasing factor (GRF) derivatives and the VIP antagonist hybrid peptide neurotensin-VIP were able to inhibit VIP-induced cell growth stimulation, even at very low concentrations. Binding experiments carried out on intact cultured C6 cells, using ¹²⁵I-labeled VIP and PACAP as tracers, revealed that the effects of the peptides on cell growth were correlated with the expression on C6 cells of polyvalent high-affinity VIP-PACAP binding sites and of a second subtype corresponding to very high-affinity VIP-selective binding species. The latter subtype, which interacted poorly with PACAP with a 10,000-fold lower affinity than VIP, might mediate the antagonist effects of neurotensin-VIP and of both GRF derivatives on VIP-induced cell growth stimulation.     

Effects of different concentrations of celecoxib on proliferation and cell cycle of SH-SY-5Y cells and its mechanisms In vitro study

BACKGROUND:Highly selective cyclooxygenase-2(COX-2)inhibitors have recently been approved for the treatment of colon cancer,breast cancer,urinary bladder cancer,and skin cancer.For the highly selective COX-2 celecoxib,the mechanism of action for inhibiting neuroblastoma cells is still uncertain.OBJECTIVE:To observe the influence of different celecoxib concentrations on proliferation and cell cycle of SH-SY-5Y cells in vitro and to reveal potential COX-2-independent mechanisms of celecoxib on SH-SY-5Y cells.DESIGN:Controlled experiment. SETTING:Department of Hematology,Affiliated Hospital of the Qingdao Medical College,Qingdao University,Shandong Province.MATERIALS:The study was erformed at the Cerebrovascular Disease Institute of Shandong Province and the Laboratory of Molecular Biology,Affiliated Hospital of Qingdao Medical College,Qingdao University between September 2006 and June 2007.The SH-SY-5Y cell line was obtained from the Department of Molecular Biology,Qingdao Medical College,Qingdao University.Celecoxib was obtained from Pfizer Pharmaceuticals LLC,USA.Coulter DNA PREP reagent kit was purchased from Beckman Coulter,Inc.The antibodies against human CyclinD1,P21,and P16 were purchased from Santa Cruz Biotechnology. METHODS:SH-SY-5Y cells were treated with different concentrations of celecoxib(10,20,40,and 80 μ mol/L)for 48 hours and comprised the experimental groups.The same concentrations of DMSO (dimethyl sulphoxide)treatment for 48 hours served as the control group.MAIN OUTCOME MEASURES:①Cellular morphology of cells pre-treated and post-treated with celecoxib by inverted microscopy.②Methabenzthiazuron assay was used to measure cell proliferation. ③Cell cycle was measured by flow cytometry after incubation with different celecoxib concentrations for 48 hours.④CyclinD1,P16,and P21 protein expression was detected by Western blot analysis. RESULTS:①Cellular morphology:The shape of SH-SY-5Y cells pre-treated with celecoxib was a slender,fusiform shape.SH-SY-5Y cells became short or polygon after 48 hours of treatment with different celecoxib concentrations.②Cell proliferation:Cell absorbance decreased with increasing concentrations. The inhibitory effect of celecoxib on SH-SY-5Y cells was dose-dependent effect.③Cell cycle:Cell cycle analysis demonstrated a dose-dependent accumulation of cells in the G0-G1 phase.Heteroploid cells were significantly inhibited.④CyclinD1,P16,and P21 protein expression:CyclinD1 expression decreased with increasing celecoxib concentrations; P21 and P16 expression increased.CONCLUSION:Celecoxib inhibited SH-SY5Y cell growth.This effect was dose-dependent.The inhibitory mechanisms on SH-SY-5Y cell growth with Celecoxib resulted in the down-regulation of CyclinD1 expression,as well as the up-regulation of P16 and P21 expression in vitro.BACKGROUND:Highly selective cyclooxygenase-2(COX-2)inhibitors have recently been approved for the treatment of colon cancer,breast cancer,urinary bladder cancer,and skin cancer.For the highly selective COX-2 celecoxib,the mechanism of action for inhibiting neuroblastoma cells is still uncertain.OBJECTIVE:To observe the influence of different celecoxib concentrations on proliferation and cell cycle of SH-SY-5Y cells in vitro and to reveal potential COX-2-independent mechanisms of celecoxib on SH-SY-5Y cells.DESIGN:Controlled experiment. SETTING:Department of Hematology,Affiliated Hospital of the Qingdao Medical College,Qingdao University,Shandong Province.MATERIALS:The study was performed at the Cerebrovascular Disease Institute of Shandong Province and the Laboratory of Molecular Biology,Affiliated Hospital of Qingdao Medical College,Qingdao University between September 2006 and June 2007.The SH-SY-5Y cell line was obtained from the Department of Molecular Biology,Qingdao Medical College,Qingdao University.Celecoxib was obtained from Pfizer Pharmaceuticals LLC,USA.Coulter DNA PREP reagent kit was purchased from Beckman Coulter,Inc.The antibodies against human yclinD1,P21,and P16 were purchased from Santa Cruz Biotechnology. METHODS:SH-SY-5Y cells were treated with different concentrations of celecoxib(10,20,40,and 80 μ mol/L)for 48 hours and comprised the experimental groups.The same concentrations of DMSO (dimethyl sulphoxide)treatment for 48 hours served as the control group.MAIN OUTCOME MEASURES:①Cellular morphology of cells pre-treated and post-treated with celecoxib by inverted microscopy.②Methabenzthiazuron assay was used to measure cell proliferation. ③Cell cycle was measured by flow cytometry after incubation with different celecoxib concentrations for 48 hours.④CyclinD1,P16,and P21 protein expression was detected by Western blot analysis. RESULTS:①Cellular morphology:The shape of SH-SY-5Y cells pre-treated with celecoxib was a slender,fusiform shape.SH-SY-5Y cells became short or polygon after 48 hours of treatment with different celecoxib concentrations.②Cell proliferation:Cell absorbance decreased with increasing concentrations. The inhibitory effect of celecoxib on SH-SY-5Y cells was dose-dependent effect.③Cell cycle:Cell cycle analysis demonstrated a dose-dependent accumulation of cells in the G0-G1 phase.Heteroploid cells were significantly inhibited.④CyclinD1,P16,and P21 protein expression:CyclinD1 expression decreased with increasing celecoxib concentrations; P21 and P16 expression increased. CONCLUSION:Celecoxib inhibited SH-SY5Y cell growth.This effect was dose-dependent.The inhibitory mechanisms on SH-SY-5Y cell growth with Celecoxib resulted in the down-regulation of CyclinD1 expression,as well as the up-regulation of P16 and P21 expression in vitro.     

Basic fibroblast growth factor- and platelet-derived growth factor-mediated cell proliferation in B104 neuroblastoma cells: effect of ethanol on cell cycle kinetics

In vivo studies show (a) that early exposure to ethanol depletes neurons in the central nervous system (CNS) and (b) that a primary target of ethanol in the developing nervous system is proliferating neuronal precursors. We used a neuronal cell line (B104 neuroblastoma cells) as an in vitro model for the effects of ethanol on the proliferation of neuronal precursors to test the hypothesis that ethanol interferes with growth factor-regulated proliferation of neuron-like precursors. The effects of ethanol on the mitogenic activity of two growth factors, basic fibroblast growth factor (bFGF) and platelet-derived growth factor AA and BB (PDGF-AA and PDGF-BB), were examined. Cell proliferation was monitored by tracing the change in the numbers of cultured cells over 4–5 days and in the cell cycle kinetics was determined using a cumulative labeling technique with bromodeoxyuridine (BrdU). Western immunoblots and immunohistochemical preparations show that B104 cells expressed the high affinity receptors for bFGF, PDGF-AA and PDGF-BB. The three growth factors were potent mitogens for the B104 cells; they promoted an increase in cell number even when the cells were grown in serum-free medium. Ethanol depressed the bFGF-, PDGF-AA- and PDGF-BB-mediated cell proliferation without altering the incidence of cell death. These changes in proliferation were concentration-dependent; at a concentration of 100 mg/dl, ethanol partially, but significantly inhibited growth factor-stimulated proliferation and higher ethanol concentrations (400 mg/dl or more) completely abolished growth factor-regulated cell proliferation. The effects of ethanol on cell growth were a result of ethanol-induced changes in growth factor-regulated cell cycle kinetics, principally the total length of the cell cycle and the fraction of the population that was actively cycling (the growth fraction). Ethanol completely negated the action of bFGF, but only partially blocked PDGF-promoted cycling activity. Thus, B104 cells are a suitable model for studying the effects of ethanol on neuronal proliferation. The blockage of bFGF- and PDGF-mediated cell proliferation by ethanol supports the hypothesis that growth factors are a target of ethanol neurotoxicity. Furthermore, the differential actions and effects of ethanol on the two growth factors mirror effects observed in vivo.     

Effects of spontaneous bioelectric activity and gangliosides on cell survival in vitro

Chronic suppression of spontaneous bioelectric activity in spinal cord explants in the presence of tetrodotoxin (TTX) during network formation caused a large reduction in cell number (lowered DNA levels). The addition of gangliosides failed to protect against this cell loss. Conversely, the omission of galactose from the growth medium had no effect on DNA levels. It was concluded that the presence or absence of afferent selectivity is unlikely to require the survival of a regionally specific subpopulation of preferred dorsal root ganglion target cells. Neocortical explants also showed a large reduction in DNA levels following chronic TTX treatment, and morphometric analysis confirmed that neuronal survival was affected to the same degree. Chronic ganglioside supplementation failed to influence DNA and cell counts in either control or TTX-treated explants, but one of the added gangliosides (GD1a) stimulated extensive neuritic outgrowth in electrically silenced cultures. Particular ganglioside species, therefore, may exert a growth stimulating influence that can partially compensate for the absence of bioelectric self-stimulation during early development.     

Effects of insulin-like growth factors and growth hormone on the in vitro proliferation of T lymphocytes.

The insulin-like growth factors I and II (IGF-I and IGF-II) promote proliferation and differentiation of many cell types. We report that recombinant IGF-I and IGF-II augment both the lectin- and anti-CD3-induced proliferation of human peripheral blood mononuclear cells (PBMC) at concentrations proportional to their binding affinities. IGF-I and IGF-II also augmented the lectin-induced proliferation of purified T lymphocytes. Effects of IGF-I were found in cultures of T cells vigorously depleted for monocytes and supplemented with saturating concentrations of interleukin-1. The latter results indicate that the effect of IGF-I on the proliferation of T lymphocytes can occur independent of monocytes or monocyte-derived factors.