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.

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.

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.

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.

IN VIVO GROWTH OF CEREBRAL C6 GLIOMA CELLS TRANSFECTED AND TREATED WITH CX43 GENE

Objective: To study the role of connexin gene (Cx 43) on the development of glioma and the feasibility of using Cx43cDNA as a target of gene therapy of gliomas. Methods: Parental rat C6 cells and C6 cells transfected with Cx43cDNA were implanted into right caudate nucleus of SD rats as control and transfected group. Rats bearing cerebral C6 gliomas were treated with Cx43cDNA and empty vector as treated group and empty vector group. The general manifestation, survival time, MRI dynamic scanning and histopathological changes of all rats were observed. In situ hybridization and immunohisto- chemistry were used for examination of Cx43mRNA and its protein in gliomas. Average number of AgNOR staining was used for detection of cell proliferation activity, and TUNEL method for determination of cell apoptosis. Results: All rats in control and empty vector group died of cerebral gliomas within 3 weeks after implantation of C6 cells. Six out of nine rats in the transfected group and eight out of ten rats in treated group kept alive beyond 120 days with totally disappearing of the tumor foci, except one treated rat having a little residue of tumor. In gliomas of transfected and treated groups Cx43 gene expression was upregulated, proliferation activity was lowered, However, the apoptotic cells did not increase. Conclusion: The present study indicates that Cx43 gene is of crucial importance in the development of malignant glioma. It can be an effective target for gene therapy of gliomas.

蒿甲醚对SD大鼠原位脑胶质瘤的抑瘤实验

目的探讨蒿甲醚(Artemether)对SD大鼠原位脑胶质瘤的抑瘤作用。方法采用四甲基偶氮唑蓝(MTT)法测定不同浓度蒿甲醚对大鼠C6脑胶质瘤细胞株的生长抑制作用,计算半数抑制浓度(IC50)。立体定位仪对48只(雌、雄各半)SD大鼠建立脑胶质瘤原位模型,随机分为6组:对照组;模型组,蒿甲醚33.3mg/(kg·d)、50.0mg/(kg·d)、66.6mg/(kg·d);联合用药组,蒿甲醚50.0mg/(kg·d)+硫酸亚铁1.5mg/(kg·d);阳性药对照组。采用灌胃给药法连续给予各组SD大鼠用药10天,对照组给予0.9%氯化钠溶液。肿瘤体积按a2bπз6(a为肿瘤的短径,b为肿瘤的长径)计算。全脑标本用4%多聚甲醛固定。肿瘤组织做病理观察。结果蒿甲醚抑制大鼠C6脑胶质瘤细胞增殖,其抑制作用呈剂量和时间依赖性;模型组和联合用药组对SD大鼠原位脑胶质瘤的抑瘤率分别为:54.5%、61.0%、64.5%和69.8%;模型组组间比较,蒿甲醚66.6mg/(kg·d)用药组的抑瘤率明显高于蒿甲醚33.3mg/(kg·d)用药组;各实验组间比较,联合用药组的抑瘤率显著高于模型组33.3mg/(kg·d)。结论口服蒿甲醚对SD大鼠脑部原位接种C6脑胶质瘤有明显的抑瘤作用。

蒿甲醚抗SD大鼠原位脑胶质瘤血管生成的实验

目的探讨蒿甲醚(Artemether)抗SD大鼠原位脑胶质瘤血管生成作用。方法采用四甲基偶氮唑蓝(MTT)法测定不同浓度蒿甲醚对大鼠C6脑胶质瘤细胞株的生长抑制作用,计算半数抑制浓度(IC50)。采用立体定位仪在SD大鼠大脑皮质层接种C6脑胶质瘤细胞(1×106/μl)40只,雌、雄各半;随机分为5组,每组8只。在接种第3天后,各组采用灌胃给药法连续给药10天。于接种后的第20天解剖大鼠,经活体左心室灌注4%多聚甲醛,固定肿瘤的全脑标本。在大鼠脑部接种穿刺点做冠状切口,按垂直和水平方向测量肿瘤大小。肿瘤体积=a2bπ?6(a为肿瘤的短径,b为肿瘤的长径)。全脑标本用4%多聚甲醛固定,肿瘤组织做病理观察,免疫组化方法检测移植瘤组织微血管密度。结果各实验组血管计数分别为Ⅰ组(39±4),Ⅱ组(29±6),Ⅲ组(12±8),Ⅳ组(10±5),生理盐水组为(52±7)。各实验组血管计数均明显少于生理盐水对照组,差异有统计学意义(分别P<0.05,P<0.01)。各实验组SD大鼠原位脑胶质瘤体积较对照组显著减小。结论在一定剂量范围内,蒿甲醚具有明显抑制SD大鼠原位脑胶质瘤血管生成作用;蒿甲醚抑制原位脑胶质瘤生长和转移的机制之一是透过血脑屏障抑制脑胶质瘤血管生成。

蒿甲醚对大鼠原位脑胶质瘤抑瘤及抗血管生成的实验研究

目的探讨蒿甲醚是否在选择性地杀伤SD大鼠原位脑胶质瘤的同时还具有抑制血管生成的作用.方法采用四甲基偶氮唑蓝(MTT)法测定不同浓度蒿甲醚对大鼠C6脑胶质瘤细胞株的生长抑制作用,计算半数抑制浓度(IC5)0;采用立体定位仪在48只SD大鼠大脑皮质层接种C6脑胶质瘤细胞(1×106个/μL),雌、雄各半;随机分为3组,每组8只.分别为空白对照组、阳性对照和实验组.在接种第3天后,实验组各组采用灌胃给药法连续给药10 d;于接种后的第20天解剖大鼠,经活体左心室灌注4%多聚甲醛,固定肿瘤的全脑标本.在大鼠脑部接种穿刺点做冠状切口,按垂直和水平方向测量肿瘤大小.肿瘤体积=a2bπ/6(a为肿瘤的短径,b为肿瘤的长径).采用免疫组化方法检测移植瘤组织微血管密度.结果实验组各组对SD大鼠原位脑胶质瘤的抑瘤率分别为:54.5%、61.0%、64.5%和69.8%,与空白对照组比较,差异有统计学意义(P<0.01);各实验组血管计数均明显少于空白对照组,差异有统计学意义(P<0.05、P<0.01).各实验组SD大鼠原位脑胶质瘤体积较空白对照组显著减小.结论在一定剂量范围内,口服蒿甲醚对SD大鼠脑部原位接种C6脑胶质瘤有明显的抑制作用;蒿甲醚抑制原位脑胶质瘤生长的机制之一可能是透过血脑屏障抑制脑胶质瘤血管生成.

体外柴胡皂苷元d对C_6大鼠神经胶质瘤细胞前列腺素E_2生成的影响

目的 观察柴胡皂苷元d(saikogenind ,SGD)对C6大鼠神经胶质瘤细胞体外前列腺素E2 (PGE2 )生成的影响。方法 用放射性免疫法测定细胞产生的PGE2 ,液体闪烁测量法测定14 C花生四烯酸 (AA)标记细胞释放14 C AA。结果 SGD在 1～ 2 0 μmol·L-1范围内 ,抑制由钙离子载体A2 3187诱发C6大鼠神经胶质瘤细胞前列腺素E2 (prostaglandinE2 ,PGE2 )释放 ,其IC50 为 3μmol·L-1,但对花生四烯酸 (arachi donicacid ,AA)释放无影响。SGD不影响细胞微粒体组分将AA转化为PGE2 。结论 SGD抑制由钙离子载体A2 3187诱发体外C6大鼠神经胶质瘤细胞PGE2 产生 ,但不抑制AA释放和直接抑制环氧脂酶 (cyclooxygenase ,COX)活性。

C_6胶质细胞及其肿瘤模型细胞DNA含量和细胞周期

目的用流式细胞仪对Ｃ６胶质细胞株及其动物模型肿瘤细胞的ＤＮＡ含量及细胞周期进行分析。方法将对数生长期Ｃ６细胞悬液及７例Ｗｉｓｔａｒ大鼠肿瘤模型单细胞悬液用碘化丙啶染色后，用流式细胞仪（ＦＣＭ）进行ＤＮＡ含量和细胞周期测定，所得结果进行统计学分析。结果（１）Ｃ６细胞ＤＮＡ含量呈超二倍体，Ｃ６肿瘤模型细胞７份中２份为二倍体，３份为超二倍体，２份为异倍体，统计学检查验二者有极显著差异（Ｐ＜０００１）。（２）细胞周期各时相细胞百分比示Ｃ６细胞增殖指数（ＰＩ）值为４６０％，Ｃ６肿瘤模型细胞ＰＩ值为４０７％，二者经统计学处理无显著差异（Ｐ＞００５）。结论流式细胞仪分析脑胶质瘤细胞对判定脑胶质瘤恶性程度和预后提供了客观线索和依据。

DL型丁胱亚磺酰亚胺对大鼠C_6神经胶质瘤细胞放射增敏效应研究

目的 研究有氧及乏氧状态下DL型丁胱亚磺酰亚胺(DL BSO)对大鼠C6神经胶质瘤细胞的放射增敏效应。方法 以60 Coγ射线为放射源 ,分有氧和乏氧状态下单纯照射组、加药照射组。采用细胞克隆形成方法观察DL BSO对神经胶质瘤细胞的放射增敏效应。结果 有氧状态下 ,DL BSO的放射增敏作用和药物作用时间有关。 0 1mmol·L-1DL BSO用药 2、6、12h ,未能观察到放射增敏作用 ,放射增敏作用仅在用药后 2 4、4 8h出现。乏氧状态下 ,0 1mmol·L-1DL BSO用药后 ,各时间点 (2～ 4 8h)均可见放射增敏效应。有氧、乏氧状态下不同浓度DL BSO的放射增敏作用和药物浓度有关。结论 DL BSO对有氧及乏氧状态下的大鼠C6神经胶质瘤细胞均有放射增敏作用。离体状态下 ,DL BSO主要表现出乏氧增敏作用 ,并呈现一定的时间、浓度依赖关系。

电针足三里对严重烫伤致大鼠急性肺损伤的影响

目的 探讨电针足三里对严重烫伤致大鼠急性肺损伤的影响.方法 雄性SD大鼠40只,体重200～250 g,随机分为5组（n=8）：对照组、烫伤组、足三里组、非经非穴组和α-银环蛇毒素组（α-BGT组）.对照组尾静脉注射生理盐水1 ml.烫伤组、足三里组、非经非穴组和α-BGT组先制备30%总体表面积Ⅲ度烫伤模型,然后烫伤组尾静脉注射生理盐水1 ml;足三里组于双侧足三里穴垂直进针7 mm,给予脉冲电流（电压3V,电流2ms,频率3 Hz）持续刺激12 mim,间隔8 h刺激1次,持续2 d;非经非穴组于双侧足三里穴旁5mm处给予脉冲刺激,方法同足三里组;α-BGT组尾静脉注射α-BGT 1.0 μg/kg,再于双侧足三里穴给予脉冲刺激,方法同足三里组.各组处理结束后,处死大鼠,取肺组织,光镜下观察病理学结果,电镜下观察超微结构,采用ELISA法测定肺组织高迁移率族蛋白B1（HMGBl）含量,采用免疫组化法测定HMGBl蛋白表达,采用RT-PCR法测定HMGBl mRNA表达.结果 烫伤组肺组织光镜下可见肺泡壁崩解,泡内大量渗出液,间质水肿、肥厚和增生,伴大量炎性细胞浸润;电镜下可见细胞核形态不规则,核膜僵硬,部分凸凹不平和核溶解,胞质内板层小体明显减少,肺组织病理损伤程度较对照组减轻.与对照组比较,烫伤组、非经非穴组和α-BGT组肺组织HMGBl含量升高,HMGBl蛋白及其mRNA的表达上调（P〈0.05）,足三里组各指标差异无统计学意义（P〉0.05）;与烫伤组比较,足三里组肺组织HMGBl含量降低,HMGBl蛋白及其mRNA的表达下调,非经非穴组和α-BGT组肺组织HMGBl mRNA表达下调（P〈0.05）;与足三里组比较,非经非穴组和α-BGT组肺组织HMGBl含量升高,HMGBl蛋白及其mRNA的表达上调（P〈0.05）.结论 电针足三里可减轻严重烫伤致大鼠急性肺损伤,其机制与激活含α7亚基N型胆碱能受体介导的胆碱能抗炎通路,抑制肺组织HMGBl的表达有关.