Bone marrow stromal cell versus neural stem cell transplantation in a C6 glioma rat model

BACKGROUND： Embryonic neural stem cells （NSCs） have provided positive effects for the treatment of glioma. However, the source for embryonic NSCs remains limited and high amplification conditions are required. Bone marrow stromal cells （BMSCs） have been proposed for the treatment of glioma. OBJECTIVE： To investigate biological changes in NSCs and BMSCs following transplantation into rat models of glioma. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Embryonic Stem Cell Research Laboratory of Yunyang Medical College from February 2006 to August 2008. MATERIALS： The rat C6 glioma cell line was purchased from Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; mouse anti-bromodeoxyuridine （BrdU） monoclonal antibody and Cy3-1abeled goat anti-mouse IgG antibody was purchased from Upstate, USA. METHODS： A total of 95 Sprag6ue Dawley rats were randomly assigned to three groups： NSC （n = 35）, transplanted with 〉 6 × 10^6 NSCs via left medial hind limb; BMSC （n = 35）, transplanted with 〉 1 × 10^6 BMSCs via left medial hind limb; model group （n = 25）, injected with the same volume of 0.1 mmol/L phosphate buffered saline. MAIN OUTCOME MEASURES： Gliomal growth and size were assessed by nuclear magnetic resonance, and glioma morphological features were observed following hematoxylin-eosin staining and BrdU immunohistochemistry 3 and 4 weeks following transplantation. RESULTS： The average survival of rats in the BMSC, NSC, and model groups was 4.03, 4.28, and 3.88 weeks. At 3 weeks, there was no significant difference in the average glioma diameter between the BMSC and model groups （P 〉 0.05）. However, gliomal diameter was significantly decreased in the NSC group compared with the model group （P 〈 0.05）. At 4 weeks, there was no statistical difference between the groups （P 〉 0.05）. BrdU immunohistochemistry revealed that BMSCs and NSCs appeared to migrate to the gliomas. CONCLUSION： NSCs inhibited glioma cell growth and prolonged rat survival. BMSCs did not significantly suppress glioma cell growth.

Effects of antigliomatin from the scorpion venom of Buthus martensii Karsch on chloride channels on C6 glioma cells

Using whole-cell patch-clamp recordings, the effects of antigliomatin were observed on chloride channels on C6 glioma cells cultured in vitro. Antigliomatin was extracted from the venom of the scorpion Buthus martensii Karsch. Chloride channels are closed under normal osmotic pressure. When osmotic pressure was reduced to 120, 110 and 100 mV, the cell volume enlarged, chloride channels opened, and the chloride channel current increased. Three minutes after antigliomatin treatment, the chloride channel current decreased in a dose-dependent manner. These results show that antigliomatin extracted from the venom of the scorpion Buthus martensii Karsch diminishes chloride channel currents on C6 glioma cells.

Inhibitory effects of Pseudomonas aeruginosa mannose-sensitive hemagglutinin on rat C6 glioma cell proliferation

BACKGROUND： Pseudomonas aeruginosa mannose-sensitive hemagglutinin （PA-MSHA） parenteral injection is used as a broad-spectrum immunomodulator. It remains unclear whether PA-MSHA exhibits inhibitory effects on tumor cell growth. OBJECTIVE： To investigate inhibitory mechanisms of PA-MSHA-induced proliferation in rat C6 glioma cells in vitro. DESIGN, TIME AND SETTING： Comparative observation and in vitro experiments were performed at the Key Laboratory of Natural Medicine, Kunming Medical College, China from July 2008 to April 2009. MATERIALS： Rat C6 glioma cell line （Shanghai Institute of Cell Biology, Chinese Academy of Sciences, China） and PA-MSHA parenteral injection （Beijing Wanteer Bio-Pharmaceutical, China） were used in the present study. METHODS： Rat C6 glioma cells in logarithmic growth phase were harvested in vitro. Adherent monolayer cells were respectively treated with PA-MSHA at final colony-forming units （cfu） of 1 ×10^8 cfu/mL, 2 × 10^8 cfu/mL, 4 × 10^8 cfu/mL, 6 × 10^8 cfu/mL, and 8 ×10^8 cfu/mL following 24 hours of conventional culture. MAIN OUTCOME MEASURES： MTT colorimetric assay was utilized to determine the inhibitory rate of C6 glioma cells following treatment with various concentrations of PA-MSHA at different times. Cell apoptosis was detected by fluorescent microscopy following Hoechst 33258 staining. Flow cytometry was used to measure PA-MSHA effects on C6 cell cycle. RESULTS： Inhibitory rate of C6 glioma cells increased with prolonged time and increased dose. Hoechst 33258 staining revealed obvious morphological changes in apoptotic C6 glioma cells. Flow cytometry revealed hypodiploid peaks, Le., apoptotic peak, and the apoptotic rate in cells during S-phase significantly increased with increased concentrations in the experimental groups. CONCLUSION： With in vitro experiments, PA-MSHA preparations inhibited C6 glioma cell proliferation in a time- and dose-dependent manner. These mechanisms are likely associated with cell apoptosis induction and inhibition of the S phase.

Temporal dynamic changes of connexin 43 expression in C6 cells following lipopolysaccharide stimulation

Connexin 43, a gap junction protein, is expressed mainly in glia in the central nervous system. Neuroinflammation plays an important role in central nervous system injury. Changes to glial connexin 43 levels and neuroinflammation may trigger brain injury and neurodegenerative diseases To illustrate the relationship between connexin 43 and neuroinflammation, this study investigated how connexin 43 expression levels change in lipopolysaccharide-stimulated rat C6 glioma cells. C6 cells were treated with 0.05, 0.25, 0.5, 1,2.5 and 5 IJg/mL lipopolysaccharide for 24 hours. The nitrite estimation-detected nitric oxide release level was elevated substantially after lipopolysaccharide stimulation. To test the transcriptional level changes of inducible nitric oxide synthase, tumor necrosis factor-a and connexin 43 mRNA, C6 cells were treated with 5 pg/mL lipopolysaccharide for 3 48 hours. Reverse transcription-PCR showed that the expression of inducible nitric oxide synthase and tumor necrosis factor-a mRNA increased over time, but connexin 43 mRNA levels increased in lipopolysaccharide-stimulated C6 cells at 3 and 6 hours, and then decreased from 12 to 48 hours. Connexin 43 protein expression was detected by immunofluorescence staining, and the protein levels matched the mRNA expression levels. These results suggest that connexin 43 expression is biphasic in lipopo^ysacchadde-induced neuroinflammation in C6 cells, which may be correlated with the connexin 43 compensatory mechanism.

Stable EGFP Gene Expression in C6 Glioma Cell Line after Transduction with HIV-1-based Lentiviral Vector

Objective： To establish a stable C6/EGFP glioma cell transfected with the human immunodeficiency virus line for studies on glioma. Methods： The C6 glioma cell line was type I （HIV-1） based lentivirus vector containing two enhancer-promoters CMV and EF1α. Enhanced green fluorescent protein （EGFP）-positive C6 cells were sorted out by fluorescence-activated cell sort. Expression of EGFP was observed by fluorescent microscopy. EGFP gene in C6 genome was assessed by Polymerase chain reaction （PCR） and DNA sequencing. Original and transfected cells were compared biologically and cytomorphologically. Results： Lentivirus vector transfection produced up to 40% EGFP-positive cells. After fluorescence-activated cell sort selection, a pure cell line C6/EGFP was established. PCR and DNA sequencing revealed integration of EGFP gene in C6 cell genome. Analysis of cell characteristics revealed no difference between transfected and original cells. Conclusion： A C6/EGFP cell line expressing EGFP as a marker is established, in which the EGFP gene is integrated into the genome. This cell line can be served as a promising tool for further basic research and gene therapy studies.

Effects of selective cyclooxygenase-2 inhibitor on C6 glioma cell proliferation and apoptosis

BACKGROUND： Studies have shown that cyclooxygenase-2 is associated with proliferation and apoptosis of glioma cells. OBJECTIVE： To investigate the effects of selective cyclooxygenase-2 inhibitor celecoxib on proliferation and apoptosis of C6 glioma cells in vitro. DESIGN, TIME AND SETTING： A cellular, molecular, controlled study was performed at the Central Laboratory and Room of Electron Microscope, Medical School, Xi＇an Jiaotong University, China from March 2007 to March 2008. MATERIALS： C6 glioma cells during in vitro log phase were assigned to control and experimental groups. Celecoxib （Pfizer, USA）, dimethyl sulfoxide （Sigma, USA）, and MTT （Sigma, USA） were used for this study. METHODS： The control group was subdivided into blank control and dimethyl sulfoxide control groups. C6 glioma cells in the blank control and dimethyl sulfoxide control groups were incubated in Dulbecco＇s modified Eagle＇s medium supplemented with 10% calf serum and 0.3% dimethyl sulfoxide respectively. C6 glioma cells in the experimental group were separately treated with 60, 80 and 100 μmol/L celecoxib. MAIN OUTCOME MEASURES： Activity of C6 glioma cells was examined by MTT assay. C6 glioma cell cycle and apoptosis were determined by annexin V-fluorescein isothiocyanate/propidium iodide double-staining, followed by flow cytometry. Morphology and ultrastructure of C6 glioma cells were observed with an inverted microscope and a transmission electron microscope, respectively. RESULTS： Compared with the blank control group, cell density was reduced, adherence ability weakened, and irregular nuclei were visible, with the presence of chromatin condensation, margination, and some apoptotic bodies in the experimental group. Activity of C6 glioma cells was significantly decreased （P 〈 0.05）, cell number was reduced during S phase, cell number was significantly increased during G2/M phase （P 〈 0.01 ）, and the apoptotic rate was significantly increased （P 〈 0.05） in the experimental group. These results were displayed in a dose- and time-dependent fashion. The outcomes were obvious in the 100 IJmol/L celecoxib group following 72 hours of treatment. CONCLUSION： Celecoxib blocked proliferation and induced apoptosis of C6 glioma cells in a dose- and time-dependent fashion.

Induction of apoptosis and inhibition of proliferation of C6 glioma cells in vitro by tamoxifen

Objective To investigate the anti-tumor effect and mechanism of tamoxifen on rat C6 glioma cells. Methods C6 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 3% fetal calf serum (FCS), and treated with tamoxifen of different concentrations, i.e. group A (1.25μmol/L), group B (2.50 μmol/L), group C (5.00 μmol/L), group D (10.00 μmol/L), group E (20.00 μmol/L) and control group (0.00 μmol/L). Morphological changes, MTT assay and 5-bromo-2’-deoxyuriding labeling ratio were assessed. Apoptosis was observed by flow cytometry. Results C6 cells treated with different doses of tamoxifen for 24, 48, and 72 hours became irregular in shape, while cells treated with vehicle grew normally. MTT assay showed that tamoxifen did not suppress C6 cell growth until 72 hours after treatment. Seventy-two hours after treatment, there were significant differences in cell viable rate between group A versus groups C, D and E; so did group B versus group D as well as group E (P<0.05). BrdU incorporation assay indicated significant difference of BrdU labbled index (BrdU LI) among groups A, C, E and control group 48 hours after treatment (P<0.05). And the BrdU LI decreased with the increased concentration of tamoxifen. Flow cytometry (FCM) showed significant difference between treated group and control group at 24, 48, and 72 hours after treatment (P<0.05). Conclusion Tamoxifen significantly suppresses the growth of C6 glioma cells in a time-and dose-dependent manner. The mechanism of tamoxifen suppressing C6 glioma cells may be inhibiting proliferation and inducing apoptosis. Therefore, tamoxifen can be a candidate as a chemotherapy agent for glioma.

Intramedullary Spinal Cord Glioma Following Microinjection of Glioblastoma Cell Line C6 in Rats

Background: This paper describes the establishment of a rat intramedullary spinal cord tumor (IMSCT) model and histopathological characterization of the tumor model. Methods: Fourteen male Wistar rats were randomized into two groups. The rats in group 1 (control group, n = 7) received a 5 μl intramedullary injection of serum physiologic (SF). Those in group 2 (experimental group, n= 7) received a 5 μl intramedullary implantation of media containing 5 × 105 C6 glioma cells. The animals were sacrificed for histopathological examination at 21 days. Results: The control group showed normal functional and histopathological findings. The group 2 rats implanted with C6 glioblastoma cells developed hind-limb paraplegia. Pathological sections confirmed intramedullary C6 glioblastoma invading the spinal cord. Conclusions: A rat C6 IMSCT model was successfully established. This model may be useful in increasing understanding of intramedullary spinal cord gliomas in humans.

RNA interference affects tumorigenicity and expression of insulin-like growth factor-1,insulin-like growth factor-1 receptor,and basic fibroblast growth factor-2 in rat C6 glioma cells

BACKGROUND： Human gliomas are more likely to express basic fibroblast growth factor-2 （FGF-2） insulin-like growth factor-1（IGF-1）, and IGF-1 receptor （IGF-1R） than normal brain tissue. These factors activate signal transduction systems of Ras/MAPK and PI3K/Akl, which promote glioma growth. OBJECTIVE： To utilize RNA interference （RNAi） technique to down-regulate FGF-2, IGF-1, and IGF-1R gene expression, and to investigate the effects of these genes on rat C6 glioma cells, as well as the feasibility of RNAi for treating glioma. DESIGN, TIME AND SETTING： This neurooncological, randomized, controlled, in vivo and in vitro experiment, which used RNAi methodology, was performed at the Laboratory of Molecular Biology, Institute of Biochemistry, Chinese Academy of Sciences between August 2005 and February 2008. MATERIALS： Rat C6 cell lines were purchased from Shanghai Institute of Cellular Biology Affiliated to Chinese Academy of Sciences. Small interfering RNA （siRNA） was synthesized by Shanghai GenePharma. Anti-IGF-1, anti-IGF-1R, anti-FGF-2, anti-mouse and anti-rabbit IgG G1-HRP antibodies were provided by Santa Cruz Biotechnology, USA. Four to six week-old BALB/c nude mice were purchased from the Laboratory Animal Center, Chinese Academy of Sciences. METHODS： C6 glioma cells were transfected with siRNA, which was chemically synthesized in vitro to correspond to endogenous FGF-2, IGF-1, and IGF-1R genes. The inhibition ratio of targeting mRNA expression was detected by semiquantitative RT-PCR, and protein expression was determined by Western blot analysis. C6 glioma cell proliferation was observed using a growth curve C6 glioma cell apoptosis rate and cell cycle were detected by flow cytometry. C6 glioma cell growth regression was observed by transwell migration assay. In addition, nude mouse subcutaneous tumor models were used in this study. For studying the anti-tumor effects of IGF-1 and IGF-1R siRNA, two blank control groups, with six mice each, were set up： A （2.5 μg siRNA was injected one week after C6 cells were inoculated, Le., when tumor volume reached 8 mm × 8 mm） and B （siRNA was injected at the same time with C6 cells were inoculated. To study the effects of FGF-2 siRNA, the groups consisted of a blank control group, negative control group, 2.6 μg siRNA group, 4 μg siRNA group, and 5.3 μg siRNA group, with six mice each. MAIN OUTCOME MEASURES： mRNA and protein inhibition ratio of FGF-2, IGF-1, and IGF-1 R; C6 glioma cell proliferation, apoptosis, and cycle growth arrest; C6 glioma cell growth regression and subcutaneous tumorigenicity rates. RESULTS： All siRNA constructs proved to be effective. After 48 hours, transfection of 200 nmol/L siRNA resulted in a FGF-2 or IGF-1R gene inhibition ratio 〉 80% and an IGF-1 gene inhibition ratio of approximately 70%. Protein expression levels for FGF-2, IGF-1, and IGF-1R decreased in a dose-dependent manner following siRNA transfection, with an inhibition rate 〉 85%, 60%, and 50%, respectively. C6 glioma cell proliferation and apoptosis rates increased in proportion to siRNA. The apoptosis rate of C6 glioma cells induced by FGF-2, IGF-1, and IGF-1R siRNA was 39.96%, 15.07% and 22.47%, respectively （P 〈 0.01）. Transfection of 200 nmol/L IGF or IGF-1R siRNA for 48 hours suppressed C6 glioma cell migration. At 30 days after intratumoral injection of 2.6, 4, and 5.3 tJg FGF-2 siRNA, tumor growth regression rate of FGF-2 siRNA was 56%, 67%, and 86%, respectively. The tumor growth regression rate was 71.88% and 45.71%, respectively, when IGF-1 or IGF-1R siRNA was intratumorally injected 1 week after C6 glioma cell transplantation. When IGF-1 or IGF-1 R siRNA was intratumorally injected during C6 glioma cell transplantation, the tumor growth regression rate was 78.13% and 74.29%, respectively. CONCLUSION： siRNA transfection downregulated gene expression of FGF-2, IGF-1, and IGF-1R In addition, siRNA treatment markedly suppressed glioma cell proliferation, growth, and migration, and concomitantly reduced subcutaneous tumorigenicity.

Heat shock induction of a 65 kDa ATP-binding proteinase in rat C6 glioma cells

The 45, 55, 65 and 100 kDa ATP-binding proteinases (ATP-BPases) of the heat-shocked (44 ℃ for 30 min, recovery for 12h) rat C6 glioma cells were purified by DEAE-ionexchange and ATP-affinity chromatography. Their molecular masses, isoelectric points (pI), pH-optima and other properties were analyzed by native proteinase gels.It was shown that the 65 kDa ATP-BPase is specifically induced by heat shock and not detectable in control cells.Its N-terminal 1-9 amino acid sequence was determined by Edman degradation, but no homologies to other proteins in the protein data bases were found. 30 and 31 kDa proteinases can be cleaved from the 45, 55 and 65 kDa proteinases to which they are linked. A possible relationship of the heat-induced 65 kDa ATP-BPase with the ATP-dependent proteinases (ATP-DPases) in prokaryotes and eukaryotes is discussed.

Apoptosis in glioma-bearing rats after neural stem cell transplantation

Abnormal activation of the Ras/Raf/Mek/Erk signaling cascade plays an important role in glioma. Inhibition of this aberrant activity could effectively hinder glioma cell proliferation and promote cell apoptosis. To investigate the mechanism of gJioblastoma treatment by neural stem ceiJ trans- plantation with respect to the Ras/Raf/Mek/Erk pathway, C6 glioma cells were prepared in sus- pension and then infused into the rat brain to establish a glioblastoma model. Neural stem cells isolated from fetal rats were then injected into the brain of this glioblastoma model. Results showed that Raf-1, Erk and Bcl-2 protein expression significantly increased, while Caspase-3 protein expression decreased. After transplantation of neural stem cells, Raf-1, Erk and Bcl-2 protein expression significantly decreased, while Caspase-3 protein expression significantly in-creased. Our findings indicate that transplantation of neural stem cells may promote apoptosis of glioma cells by inhibiting Ras/Raf/Mek/Erk signaling, and thus may represent a novel treatment approach for glioblastoma.

Comparative study on the stem cell phenotypes of C6 cells under different culture conditions

Background Glioma stem cell （GSC） hypothesis posits that a subpopulation of cells within gliomas have true clonogenic and tumorigenic potential. Significantly, a more controversial correlate to GSC is that cells in different culture conditions might display distinct stem cell properties. Considering these possibilities, we applied an approach comparing stem cell characteristics of C6 glioma cells under different culture conditions.Methods C6 cells were cultured under three different growth conditions, i.e., adherent growth in conventional 10% serum medium, non-adherent spheres growth in serum-free medium, as well as adherent growth on laminin-coated flask in serum-free medium. Growth characteristics were detected contrastively through neurosphere formation assay and cell cycle analysis. Markers were determined by immunofluorescence, relative-quantitative reverse transcription （RT）-PCR,Western blotting and flow cytometry. Side population cells were analyzed via flow cytometry. Tumor models were detected by magnetic resonance imaging and hematoxylin ＆ eosin staining. Data analyses were performed with SPSS software （17.0）.Results C6 cells （C6-Adh, C6-SC-Sph and C6-SC-Adh） showed distinctive growth patterns and proliferation capacity.Compared to suspending C6-SC-Sph, adherent C6-Adh and C6-SC-Adh displayed higher growth ratio. C6-SC-Sph and C6-SC-Adh showed enhanced capability of neurosphere formation and self-renewal. High side population ratio was detected in C6-SC-Sph and C6-SC-Adh. CD133 was not detected in all three kinds of cells. Conversely, Nestin and β-Ⅲ-tubulin were demonstrated positive, nonetheless with no statistical significance （P 〉0.05）. Interestingly, lower expression of glial fibrillary acidic protein was demonstrated in C6-SC-Sph and C6-SC-Adh. C6-Adh, C6-SC-Sph and C6-SC-Adh were all displayed in situ oncogenicity, while statistical difference of survival time was not confirmed.Conclusions C6 glioma cell line is endowed with some GSC phenotypes that can be moderately enriched in vitro when transferred into stem cell culture condition. The resultant tumor-spheres may be not a prerequisite or sound source of GSCs and adherent culture in stem cell medium is not a growth condition in favor of GSCs expanding in vivo.

Ethanol Extract of Lycopodium serratum Thunb. Attenuates Lipopolysaccharide-Induced C6 Glioma Cells Migration via Matrix Metalloproteinase-9 Expression

Objectives： To elucidate how ethanol extract of L. serratum（ELS） could exert anti-migratory effects on glioma with the suppression of nuclear factor kappa B（NF-κB） downstream pathway. Methods： Cell viability of ELS on C6 glioma was detected by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT） assay. Nitric oxide（NO） assay and 2＇,7＇-dichlorofluorescin diacetate（DCFH-DA） assay were applied to measure NO production and reactive oxygen species（ROS） generation on lipopolysaccharide（LPS）-induced C6 glioma cells. NF-κB, mitogen-activated protein kinase（MAPK）, inducible nictric oxide synthase（i NOS） and cyclooxygenase-2（COX-2） protein were determined by Western blot. Wound healing assay was used to investigate the inhibitory effect of ELS on fetal bovine serum（FBS）-induced migration and matrix metalloproteinase（MMP）-9 and-2 activity was examined by zymography. Results： ELS suppressed LPS-induced phosphorylation of extracellular signal-regulated kinase（ERK）, c-Jun N-terminal kinase（JNK）, and p38 through inhibiting the expression of chemokine CCL2（or monocyte chemoattractant protein-1, MCP-1）. In addition, ELS inhibited the expression of i NOS, COX-2, and the production of NO by LPS in C6 glioma cells. ELS also significantly decreased serum-induced migration of C6 glioma cells in scratch wound healing in a dose-dependent manner（P〈0.01）. The activity of MMP-9 and-2 were also significantly attenuated by ELS with LPS treatment（P〈0.01）. Conclusion： Our results suggest that downregulation of MMP-9 gene expression might be involved in the anti-migration effect of ELS against LPS-induced C6 glioma cells.

Preparation, Release-control and Cell Apoptosis of C_6 Glioma Cells in PEG-PLGA-Rg3 Nanoparticles

With biodegradable material poly（ethylene glycol）-poly（lactide-co-glycolide） （PEG-PLGA） as substrate, the size distribution of Rg3-NPs was approved by the scanning electron microscopy. MTT assay was used to detect the effects of Rg3-NPs on the growth rate of C6 cells at various concentrations and flow cytometry（FCM） was applied to assay the cell cycle and cell apoptosis of C6 glioma cells. Western blot analysis was used to measure the protein level of PCNA. The results show that Rg3-NPs are slick and uniformity, the average diameter of the nanoparticles is about 75-90 nm, entrapment efficiency is （89.7±1.7）%. MTT assay shows the growth of C6 Glioma Cells can be significantly inhibited by Rg3-NPs in a dose-dependence manner. FCM and Western blot analysis show Rg3 can be released from the conjugated nanoparticles to function in the cell nuclei so as to lead to the changes in the growth cycle of the cells, which results in the arrest of G0-G1 cell cycle and induces the apoptosis of C6 cells. Therefore, Rg3-NPs may be used for the auxiliary therapy of brain glioma.

发酵乳杆菌C6全细胞催化剂的制备及产D-塔格糖催化条件优化

以L-阿拉伯糖异构酶产生菌发酵乳杆菌(Lactobacillus fermentum)C6为全细胞催化剂,对其发酵制备工艺以及生物转化D-塔格糖的催化反应条件和细胞透性化学处理方式进行优化。结果表明,菌株C6以最优培养基配方(葡萄糖10 g/L,酵母浸粉20 g/L,蛋白胨20 g/L,无水乙酸钠10 g/L,MgSO_(4)·7H_(2)O 0.4 g/L,MnSO_(4)·2H_(2)O 0.05 g/L,K_(2)HPO_(4)0.4 g/L,L-阿拉伯糖3 g/L,ZnSO_(4)·7H_(2)O 0.04 g/L,生物素100μg/L,焦磷酸硫胺素400μg/L)在最优发酵条件(发酵温度37℃、初始pH值7.5、装液量70 mL/150 mL、接种量1%)下培养24 h,生物量(OD_(600 nm)值=1.25)和L-阿拉伯糖异构酶酶活(107.81 U/mL)较优化前分别提高了92.31%和116.44%;全细胞催化剂在优化的催化反应条件(反应温度60℃、反应pH值7.5、Mn^(2+)10 mmol/L、底物浓度0.5 mol/L、底物加入体积2 mL,反应时间24 h)及细胞透性处理(20%丙酮处理60 min)方式下,D-塔格糖产率达45.07%。

注射后留置时间对大鼠C6脑胶质瘤模型的影响

目的:采用不同的留置时间建立大鼠C6胶质瘤模型,比较不同的留置时间对于模型成功率的影响,提高肿瘤种植成功率。方法:36只SD大鼠按留置时间3min、5min、10min分为3组,C6细胞体积10μl细胞悬液、注射时间5min。第一次未种植成功者再次种植。术后用骨蜡封骨窗并清创缝合。造模术后2周和3周分别行MRI平扫及增强证实模型肿瘤生长。统计学方法采用Fisher精确检验。结果:36只大鼠,16只第一次种植成功,20只种植失败,改变条件再次种植,2只死于麻醉意外,18只成功。留置时间3min与5min,其0.01<P<0.05,留置时间对于肿瘤种植成功二者差异具有统计学意义,留置5min肿瘤成功率高于3min;留置时间5min与10min,其P<0.01,留置5min与10min两种不同的留置时间对于肿瘤种植成功二者差异具有显著的统计学意义,留置10min,肿瘤成功率明显高于5min。结论:微量注射器的留置时间与肿瘤种植成功率有关,随着留置时间的延长,肿瘤种植成功率也会提高。

槲皮素对大鼠脑胶质瘤C6细胞增殖调控的作用

目的:研究类黄酮化合物槲皮素(quercetin,QUE)对体外培养的大鼠脑胶质瘤C6细胞增殖调控的作用。方法:按QUE浓度分成10、25、50、75及100μmol.L-15个处理组和空白对照组(生理盐水)及溶剂对照组(二甲亚砜),大鼠脑胶质瘤C6细胞在RPMI 1640培养基中生长达1×106.mL-1后,在96孔板中分别加入上述浓度的QUE继续培养,每组设3复孔,作用24、48及72 h,采用MTT比色法检测QUE对大鼠脑胶质瘤C6细胞的增殖抑制情况,流式细胞术(FCM)对50及100μmol.L-1的QUE作用48 h的大鼠脑胶质瘤C6细胞进行周期分析,免疫组化法检测50μmol.L-1的QUE作用48 h的p53和bcl-2基因产物。结果:与空白对照组比较,QUE处理组随药物浓度增加和作用时间的延长,A值减小(P<0.05),对C6细胞的增殖抑制作用增强,使停滞在G0/G1期的细胞增加(P<0.01),而S和G2/M期细胞减少(P<0.05)。P53蛋白表达增加和Bcl-2蛋白表达减少。结论:QUE对C6细胞增殖抑制作用具有浓度、时间依赖性,通过P53蛋白表达增加和Bcl-2蛋白表达减少诱导细胞凋亡来实现。

立体定向建立大鼠C6脑胶质瘤模型

目的 建立大鼠C6脑胶质瘤模型 ,采用影像学方法观察肿瘤的生长规律和时间窗 ,为肿瘤的疗效检测提供可行的方法。方法 16只SD大鼠随机分成两组 ,每组 8只 ,用立体定向技术将C6细胞接种在大鼠的右侧尾状核 ,接种细胞量分别为 1 0× 10 6 个 (第 1组 )和 1 5× 10 6 个 (第 2组 )。观察大鼠生存期 ,测量肿瘤的最大径 ,计算肿瘤体积 ,计算观察时间窗。结果 两组大鼠皆成功接种肿瘤 ,成功率为10 0 %。第 1组大鼠平均生存期为 ( 2 3 5 0± 3 93 )d ,明显大于第 2组 ( 17 75± 2 3 8)d(P <0 0 1)。肿瘤最大直径与瘤龄呈直线关系 ,体积与瘤龄呈指数关系。观察时间窗第 1组为 ( 12 0 0± 3 5 1)d ,大于第 2组 ( 8 88± 1 73 )d(P <0 0 5 )。结论 立体定向脑内定量注射C6细胞制作大鼠脑胶质瘤模型成功率高 ,肿瘤最大径和体积增长有规律性。

核糖核酸酶抑制因子对H_2O_2损伤的大鼠神经胶质瘤细胞系C6的影响

核糖核酸酶抑制因子 (ribonucleaseinhibitor,RI)是广泛存在于哺乳动物细胞浆中的一种酸性糖蛋白 .为了进一步了解RI的功能 ,根据RI分子结构富含巯基的特点 ,研究了RI对过氧化氢(H2 O2 )损伤的大鼠神经胶质瘤细胞 (C6 )的影响 .用不同浓度的H2 O2 分别作用于转染有RIcDNA并且RI过表达的C6细胞和正常C6细胞 ,对比损伤前后 2者的细胞存活率、LDH漏出量、细胞内GSH和MDA含量差别 ,以及细胞内抗氧化酶类GPX、CAT和GST活性的差别 .结果表明 ,与正常C6细胞相比 ,RI过表达的C6细胞在H2 O2 作用下存活率高 ,LDH漏出量、MDA含量明显减少 ,而细胞内GSH较多 ;RI过表达的C6细胞在损伤前后均表现出更强的CAT和GST活性 .提示RI具有抗氧化功能 ,能够减轻H2 O2 所致的细胞过氧化损伤 .

外源性IL-18基因对C6胶质瘤细胞周期及相关基因表达的影响

目的:本研究拟从分子水平探讨IL-18基因转染对C6胶质瘤细胞周期及其相关基因表达的影响。方法:用流式细胞仪检测C6/IL-18细胞周期、细胞增殖指数的变化;以MTT法检测细胞的增殖活性及细胞抑制率,用RT-PCR、Western blot分析C6/IL-18细胞PCNAc、yclin D1c、yclin B1、p21 mRNA及蛋白的表达。结果:与C6/LXSN细胞及亲代C6细胞相比,C6/IL-18细胞周期有所改变,表现为G0/G1期细胞增多、G2/M期细胞减少,细胞增殖指数(PI)与细胞增殖活性降低。C6/IL-18细胞的PCNAc、yclin D1c、yclin B1 mRNA及蛋白表达降低,p21 mRNA及蛋白表达增高。结论:外源性IL-18基因表达可降低C6细胞的增殖活性,下调PCNA,cyclin D1,cyclin B1表达,上调p21基因表达,其作用机制有待进一步研究。