红景天苷对C6胶质瘤细胞系迁移及生长的影响

目的:研究红景天苷(salidroside,SLDS)对C6胶质瘤细胞系迁移和生长的影响.方法:通过CCK-8实验和LDH实验确定SLDS作用于C6胶质瘤细胞的最适浓度;通过细胞生长曲线、细胞形态图和划痕实验观察SLDS对C6胶质瘤细胞系生长和迁移的影响;通过Western印迹和qPCR检测C6胶质瘤细胞中MMP-9和周期蛋白A(Cyclin A)的表达水平.结果:CCK-8实验和LDH实验显示在12.5,25,50,100和200μmol/L这5个浓度中,100 μ.mol/L的SLDS对C6胶质瘤细胞的抑制最为显著;细胞形态图和生长曲线显示,加入SLDS后C6胶质瘤细胞的生长被显著抑制;划痕实验显示SLDS组肿瘤细胞侵袭力较对照组明显减弱;Western印迹和qPCR显示SLDS组MMP-9和Cyclin A的表达水平明显低于对照组.结论:SLDS对C6胶质瘤细胞的生长及迁移具有抑制作用.     

大鼠C6胶质瘤细胞的基因表达谱

目的:筛选比较大鼠C6胶质瘤细胞与正常星型胶质细胞中的差异表达基因。方法:利用Illumina Hi Seq4000技术对大鼠C6胶质瘤细胞和正常星形胶质细胞进行转录组测序并对测序结果进行功能注释。以|log2 Fold Change|≥1和q值0.05为标准筛选差异表达基因,并通过GO和KEGG数据库分析其功能和参与的信号通路。结果:较之正常星形胶质细胞,大鼠C6胶质瘤细胞中共筛选得到4363个差异表达基因,其中1603个基因表达上调,2760个基因表达下调;GO富集结果表明:4109个差异表达基因共映射到906个GO term,主要与GTP酶活性正性调节、基因表达正性调节、蛋白结合和钙离子结合等功能相关;KEGG富集结果表明:1535个差异表达基因共参与106条通路,富集最多的是PI3K-Akt信号通路和癌症发生通路。结论:成功获取大鼠C6胶质瘤细胞与正常胶质细胞的基因表达谱,并对其差异基因进行GO和KEGG注释,结果有助于胶质瘤发生发展的研究。     

突变型Axin基因在神经胶质瘤细胞系C6中的功能研究

目的研究突变型Axin基因对C6细胞增殖及细胞周期的影响,以期揭示突变型Axin基因是否影响胶质瘤的发生。方法采用噻唑蓝(MTT)比色法绘制细胞生长曲线,应用流式细胞仪(FCM)测定细胞周期的变化,并用平板克隆形成实验对细胞克隆形成能力进行了检测。采用SP法免疫细胞化学染色法检测Ki-67、cyclinD1表达。结果转染突变型Axin基因与转染野生型Axin基因的C6细胞在生长速度,克隆形成能力方面均显著降低,且组间无差别;C6、C6-Vector、C6-Vector-Axin、和C6-Vector-Axin806各组细胞经流式细胞仪检测S期的细胞比例分别为:27.2%、24.8%、18.9%和18.5%;Ki-67及cyclinD1在转染突变型Axin基因与转染野生型Axin基因的C6细胞中表达均降低,且组间无差别。结论突变型Axin基因明显抑制C6细胞的增殖能力,该位点突变可能并不是胶质瘤发生的关键因素。     

逆转录病毒介导IL-18基因在大鼠胶质瘤细胞C6中的表达

目的 :建立表达IL 18基因的大鼠胶质瘤细胞C6 /IL 18,并探讨外源性IL 18基因对C6细胞生长的影响。方法 :应用逆转录病毒载体 ,将IL 18基因导入C6细胞。经G4 18筛选后 ,获得表达IL 18分子的细胞克隆C6 /IL 18。用RT PCR法检测目的基因mRNA的表达 ;用流式细胞和免疫细胞化学染色法检测目的蛋白的表达。用ELISA法检测C6 /IL 18细胞培养上清诱导脾细胞分泌IFN γ的能力 ,以确定IL 18的生物学活性。用MTT比色法检测细胞体外增殖的状况 ,用流式细胞技术检测细胞的增殖活性 ,并建立大鼠胶质瘤模型 ,观察C6 /IL 18细胞体内致瘤性的改变。结果 :外源IL 18基因于mRNA水平和蛋白水平可获得稳定表达 ,并可诱生大鼠脾细胞分泌IFN γ。同时 ,该细胞系的体外增殖率及体内致瘤性 ,均较亲代C6胶质瘤细胞明显下降。结论 :外源性IL 18基因能部分地抑制C6细胞的体外增殖率和体内致瘤性。建立了可进一步用于相关肿瘤基因免疫和基因治疗研究的大鼠胶质瘤细胞系C6 /IL 18。     

Control of cell proliferation by cell volume alterations in rat C6 glioma cells

K+ and Cl- channels are involved in regulating the proliferation of a number of cell types. Two main hypotheses have been proposed to explain the mechanism by which these channels influence cell proliferation: regulation of membrane potential and regulation of cell volume. In order to test these hypotheses, we measured, under different experimental conditions, the volume, membrane potential and rate of proliferation of C6 glioma cells. Cells cultured in control medium for 1-4 days were compared with cells cultured for the same period of time in the presence of broad spectrum channel blockers: tetraethylammonium, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and Cs+, in hypertonic media (29% increased osmolarity with NaCl, KCl or sucrose), in hypotonic medium (23% decreased osmolarity with H2O) or in the presence of the specific channel blockers, i.e. mast cell degranulating peptide, charybdotoxin or chlorotoxin. In all of these conditions, we observed a close correspondence between the rate of proliferation and the mean cell volume. The proliferation decreased when volume increased. Moreover, whereas control cells were flattened, spindle-shaped, bipolar or multipolar, cells cultured in media supplemented with NPPB, KCl or CsCl were round with few processes. Of the agents tested, only KCl and Cs+ depolarized the cells. These results show that alterations of the rate of proliferation by K+ and Cl- channel blockers or anisotonia are closely related with changes in cell volume or form but are not correlated with changes in membrane potential.     

地塞米松对C6胶质瘤细胞细胞周期的影响

目的:研究地塞米松(Dex)对C6胶质瘤细胞细胞周期和增殖的影响。方法:在培养的C6胶质瘤细胞中加入不同浓度(0 mol/L、10-5mol/L、10-4mol/L和10-3mol/L)的Dex,作用不同的时间(6、12和24 h)后取材,运用细胞计数和流式细胞仪检测C6胶质瘤细胞的增殖情况、细胞周期及凋亡。结果:10-3mol/L的Dex诱发了C6胶质瘤细胞的凋亡。24 h时,4个组的C6胶质瘤细胞凋亡率依次为0.37、0.52、0.39和8.24;4个组的C6胶质瘤细胞个数依次为4.37×106、4.29×106、3.57×106和3.44×106,后3组与第1组有明显的差异;4个组的C6胶质瘤细胞增殖指数依次为17.58、6.79、6.29和22.48,前3组随浓度增加而增加,第4组例外。结论:Dex对C6胶质瘤细胞的细胞周期进程有明显的抑制作用。10-4mol/L以下的浓度抑制其由G1期向S期的转换,10-3mol/L剂量的Dex可能抑制C6胶质瘤细胞由G2期向M期的转换或阻滞细胞在S期。大剂量的Dex(10-3mol/L)可引起C6胶质瘤细胞凋亡。     

神经毒素6-OHDA干预后星形胶质细胞铁转运蛋白表达的变化

BACKGROUND: Previous studies have confirmed that 6-hydroxydopamine is capable to increase the expression of divalent metal transporter-1 and reduce the expression of ferroportin-1 in the neurons and microglia, which may lead to iron deposition in the substantia nigra after Parkinson’s disease. However, it is unclear whether 6-hydroxydopamine can play diverse roles in astrocytes. OBJECTIVE: To observe the effects of 6-hydroxydopamine on the expression of divalent metal transporter-1 and ferroportin-1 in rat C6 glioma cell lines. METHODS: C6 glioma cell lines from rats were cultured in 10 μmol/L 6-hydroxydopamine for 24 hours. Then, protein expressions of divalent metal transporter-1 and ferroportiner-1 were measured by western blot method. RESULTS AND CONCLUSION: The protein expressions of divalent metal transporter-1 and ferroportin-1 in C6 glioma cell lines were increased by 2.5 times (P < 0.01) and 1 time (P < 0.05), respectively, after treatment with 6-hydroxydopamine. These findings indicate that 6-hydroxydopamine can promote iron transport rate in astrocytes by increasing both divalent metal transporter-1 and ferroportin-1 expressions, and astrocytes has a different response to 6-hydroxydopamine from neurons and microglia.       

C6胶质瘤细胞体外诱导神经干细胞的迁移

目的：体外观察 C6胶质瘤细胞的培养上清是否有诱导神经干细胞迁移的作用。方法：实验组取处于指数生长期的 C6胶质瘤细胞或星形胶质细胞的无血清培养上清加入细胞培养室的下室,并加无血清培养基;培养室的上室加处于对数生长期的神经干细胞球悬液,培养箱中共培养,光镜下计数迁移的细胞球数。结果：C6胶质瘤细胞的上清引起神经干细胞球迁移的数目明显多于星形胶质细胞的上清和新鲜无血清培养基。结论：C6胶质瘤细胞的培养上清中存在诱导神经干细胞球迁移的物质,这将为研究诱导神经干细胞迁移的物质提供很大的便利。     

氯化三乙基锡对大鼠C6脑胶质瘤细胞增殖和细胞周期的影响

目的探讨氯化三乙基锡(triethyltin chloride,TETC)对体外培养大鼠C6脑胶质瘤细胞增殖和细胞周期的影响。方法采用MTT法检测0.5、1.0和2.0μmol/L TETC对体外培养大鼠C6脑胶质瘤细胞作用24h和48h后细胞增殖的影响、采用细胞形态学观察及流式细胞术(FCM)方法检测0.5、1.0和2.0μmol/L TETC对体外培养大鼠C6脑胶质瘤细胞作用48h后细胞增殖和细胞周期的影响。结果MTT法及细胞形态学观察均检测到0.5、1.0和2.0μmol/L TETC在体外均可抑制C6细胞增殖,C6细胞增殖率存在着剂量依赖性和时间依赖性降低趋势;TETC可将C6细胞阻滞在G0/G1期。结论TETC可抑制体外培养大鼠C6胶质瘤细胞增殖,其机制可能与C6细胞周期阻滞在G0/G1期有关。     

Delta9-tetrahydrocannabinol increases C6 glioma cell death produced by oxidative stress

(-)Delta9-tetrahydrocannabinol is a scavenger of free radicals. However, the activation of the CB1 receptor in cultured C6 glioma cells by (-)delta9-tetrahydrocannabinol in the presence of reagents generating reactive oxygen species leads to amplification of the cellular damage from oxidative stress. This was evident by increased loss of cell wall integrity, impaired mitochondrial function and reduction of glucose uptake. In addition, (-)delta9-tetrahydrocannabinol treatment was also found to be deleterious to the cells under conditions of glucose starvation. Free radicals have been implicated in various conditions leading to cell death and, as a routine, the Fenton reaction is utilized for modeling reactive oxygen species production. Our study was performed using a cell permeating Fe(III) chelating quinone that provides more physiological conditions for mimicking the naturally occurring oxidative stress within the cell and thus serves as a better model for natural reactive oxygen species formation.     

Protein growth factor(s) from C6 glioma cells that promote the growth of murine hybridomas

C6 glioma cell conditioned medium (C6CM) has been used as a growth supplement for murine hybridomas. At 10% C6CM has been found to increase cell proliferation by 3-4-fold. This effect is observed in the presence of saturating concentration of FCS. Clonal growth is also enhanced 6.7-fold. No growth promoting effect is seen on murine myelomas or spleen cells. The factor(s) appear to be protein in nature.     

Biased cell migration of fibroblasts exhibiting contact guidance in oriented collagen gels

We present here the first quantitative correlation for cell contact guidance in an oriented fibrillar network in terms of biased cell migration. The correlation is between the anisotropic cell diffusion parameter,D _A=D_x/D_y, and the collagen gel birefringence, Δn, a measure of axially biased collagen fibril orientation in thex-direction. The cell diffusion coefficients,D _x andD _y, measure the dispersal of cells in the directions coincident with and normal to the axis of fibril orientation, respectively. Three essential methodological components are involved: (i) exploiting the orienting effect of a magnetic field on collagen fibrils during fibrillogenesis to systematically prepare uniform axially oriented collagen gels; (ii) using a microscope/image analysis workstation with precise, computer-controlled rotating and translating stages to automate birefringence measurement and, along with rapid “coarse optical sectioning” via digital image processing, to enable 3-D cell tracking of many cells in multiple samples simultaneously; and (iii) employing a rigorous statistical analysis of the cell tracks to estimate the magnitude and precision of the direction-dependent cell diffusion coefficients,D _x andD _y, that defineD _A. We find that this measure of biased migration in contact guidance (D _A) increases with increasing collagen fibril orientation (Δn) due mainly to a rapid enhancement of migration along the axis of fibril orientation at low levels of fibril orientation, and to a continued suppression of migration normal to the axis of fibril orientation at high levels of fibril orientation.     

黏液瘤病毒体外对胶质瘤细胞杀灭作用的研究

目的了解C6细胞在体外对黏液瘤病毒的易感性,并测定黏液瘤病毒对体外C6胶质瘤细胞的杀灭作用。方法以黏液瘤病毒感染体外培养的C6胶质瘤细胞,以灭活病毒作为阴性对照,5-Fu作为阳性对照,DEMD液做空白对照,应用Western—Blot法、倒置显微镜及MTT法观察各组细胞的形态、成活率。结果病毒感染组及5-Fu组24h后细胞成活率明显低于空白对照组及阴性对照组,同时病毒感染组细胞出现明显细胞病变。结论C6胶质瘤细胞在体外对黏液瘤病毒易感,黏液瘤病毒在体外对C6胶质瘤细胞有杀灭作用。     

黏液瘤病毒体外对胶质瘤细胞杀灭作用的研究

目的 了解C6细胞在体外对黏液瘤病毒的易感性,并测定黏液瘤病毒对体外C6胶质瘤细胞的杀灭作用.方法 以黏液瘤病毒感染体外培养的C6胶质瘤细胞,以灭活病毒作为阴性对照,5-FU作为阳性对照,DEMD液做空白对照,应用Western-Blot法、倒置显微镜及MTT法观察各组细胞的形态、成活率.结果 病毒感染组及5-FU组24 h后细胞成活率明显低于空白对照组及阴性对照组,同时病毒感染组细胞出现明显细胞病变.结论 C6胶质瘤细胞在体外对黏液瘤病毒易感,黏液瘤病毒在体外对C6胶质瘤细胞有杀灭作用.     

Host-dependent temperature-sensitive growth of HVJ (Sendai virus) wild-type in rat glioma C 6 cells

Summary At non-permissive temperature viral specific RNA synthesis was not restricted in rat glioma (C 6) cells infected with HVJ (Sendai virus) wild-type. However, as has previously been shown (J Gen Virol [1984] 65: 639–643), the synthesis of M protein was reduced at non-permissive temperature, in contrast to the L, P, HN, F₀ and NP proteins which were synthesized in comparable amounts at permissive and non-permissive temperatures. In this report we show additionally that viral nucleocapsids (NC), which consist of L, P and NP proteins, were formed within the infected cells at both temperatures. Hemagglutinin and neuraminidase activities were also detected in samples incubated at non-permissive temperature. By membrane immunofluorescence and cell-surface immunoprecipitation it was shown that migration of HN and F₀ proteins to the cell surface occurred normally at non-permissive temperature. Additionally, the L, P and NP proteins, which were associated with the plasma membrane isolated from the infected cells maintained at permissive temperature, were absent from the membrane of cells incubated at non-permissive temperature. These results suggest that NC and glycoproteins synthesized at non-permissive temperature could not assemble effectively at the plasma membrane because of a lack of M protein. Thus, the host-dependentts lesion of HVJ in C 6 cells was considered to be mainly in M protein synthesis.With 3 Figures     

Androglobin knockdown inhibits growth of glioma cell lines

Globin family was famous for oxygen supply function of its members such as hemoglobin and myoglobin. With the progress of research, several members of this protein family have been proven to play roles in tumors including glioma. Androglobin (ADGB) is a recently identified member of globin family with very few studies about its function. In the present study, we show that ADGB plays an oncogene role in glioma. Lentiviral vector mediated ADGB knockdown inhibited the proliferation of glioma cell lines determined by MTT assay and colony formation assay. ADGB knockdown also increased the apoptosis of glioma cell line U251 assessed by flow cytometry. In addition, western blot showed that ADGB knockdown altered levels of several proteins related to proliferation, survival or apoptosis in U251 cells. These findings suggest ADGB is involved in the progression of glioma in vitro.     

Anti-inflammatory effects of serum isolated from animals on intermittent feeding in C6 glioma cell line

Multiple sclerosis (MS) is a demyelinating disease of the CNS. Early inflammation leads to later destruction of myelin in MS. Dietary restriction (DR) produces anti-inflammatory and immunomodulatory effects in many species. Based on the reported anti-inflammatory effects of DR, we investigated whether sera collected from rats fed on intermittent feeding (IF, a type of DR) diet could modulate cytokine secretion and matrix metalloproteinase (MMP-2) activity that are involved in MS pathogenesis. Cytokine levels (IL-6 and TGF-β1) were measured in supernatant from C6 glioma cell line cultures treated with IF and AL fed animals' sera by enzyme-linked immunosorbent assay (ELISA) and MMP-2 activity was detected by gelatin zymography. Our results indicated that sera of animals on IF diet significantly reduced IL-6 (p<0.05) and increased TGF-β1 (p<0.05) production by C6 glioma cells. A significant decrease (p<0.05) in MMP-2 activity was also found. These results indicate anti-inflammatory and immunomodulatory activity in the sera of animals on IF regimen on cells involved in multiple sclerosis pathogenesis. Further studies on the detection of factors responsible for such activities and their mechanism of action in MS pathogenesis are recommended.     

Effects of laser-modified polystyrene substrate on CHO cell growth and alignment

Biomaterial surface chemistry and nanoscale topography of biomaterials can significantly influence cell behavior in vitro. Polystyrene (PS) Petri dishes were subjected to Nd:YAG laser irradiation at 266 nm, which resulted in well-defined three-dimensional (3D) periodic nanoscale surface topographies and surface oxidation. The surface changes were analyzed by X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and a contact-angle goniometer. The samples were then used to investigate the cell behavior of Chinese hamster ovary (CHO) cells. The surface laser modification affected the CHO cell adhesion and alignment, and caused morphological changes in comparison with unmodified PS. The results obtained from the cell-behavior studies revealed that nanoscale hydrophilic surface topography cues affected the adhesion, extension, alignment, and morphology of cells.     

鼠神经干细胞对胶质瘤C6细胞侵袭能力的影响

目的探讨鼠神经干细胞对鼠胶质瘤C6细胞侵袭能力的影响及机制。方法取新生1～2天SD大鼠大脑皮层组织,于无血清培养基中分离培养神经干细胞并进行NES免疫细胞化学染色,并将神经干细胞和鼠胶质瘤C6细胞于体外共培养,利用2D、3D生长实验和Transwell实验分别检测与鼠神经干细胞共培养鼠胶质瘤C6细胞(实验组)和单独培养鼠胶质瘤C6细胞(对照组)的侵袭能力。结果 2D实验中C6细胞的对照组和实验组的平均直径分别为(18.53±1.32)μm和(13.44±1.45)μm,差异有统计学意义(P<0.01)。3D实验中C6细胞的对照组和实验组的平均直径分别为(20.34±1.25)μm和(15.52±1.43)μm,差异有统计学意义(P<0.01)。Transwell侵袭实验发现胶质瘤C6细胞组中对照组和实验组平均视野侵袭细胞为(36.45±1.36)个和(22.73±1.66)个,差异有统计学意义(P<0.01)。结论与神经干细胞共同培养后C6细胞的侵袭能力明显降低。