九节龙皂苷对胶质瘤U373MG的抑制作用及其机制研究

目的 探讨九节龙皂苷对人脑胶质瘤细胞U373MG增殖的抑制作用及其机制.方法 培养U373MG细胞系,随机分为二甲基亚砜对照组(control组)和九节龙皂苷治疗(ADS)组.采用甲基噻唑基四唑法检测不同剂量ADS对人胶质瘤细胞U373MG增殖的影响;流式细胞仪观察细胞凋亡情况.免疫组化检测凋亡蛋白Caspase-3、p-Caspase-3的表达变化.结果 与对照组比较,ADS可显著降低U373MG细胞的生存率;流式细胞仪检测结果显示,随着ADS浓度的增大,U373MG细胞的凋亡率明显上升,免疫组化结果同样证明,ADS可呈剂量依赖性的促进凋亡信号Caspase-3和p-Caspase-3的高表达,不同组间差异有统计学意义(P＜0.05).结论 ADS可呈剂量依赖性的抑制U373MG细胞增殖,可能通过促进Caspase-3、p-Caspase-3的表达,诱发U373 MG细胞大量凋亡,发挥重要的抗肿瘤作用.     

人参皂甙Rh2对人胶质瘤细胞U87MG凋亡的影响

目的 观察人参皂甙Rh2对胶质瘤细胞凋亡的影响并初步探讨其可能机制。方法 将培养的人胶质瘤细胞U87MG随机分为人参皂甙Rh2组、人参皂甙Rh2+尼莫地平组和对照组。人参皂甙Rh2组在常规培养细胞时加入20 μg/ml的人参皂甙Rh2，人参皂甙Rh2+尼莫地平组在人参皂甙Rh2组培养细胞时加入浓度为10 μmol/L的尼莫地平。利用流式细胞仪检测U87MG细胞凋亡，利用激光共聚焦显微镜和流式细胞仪检测U87MG细胞内钙离子浓度。结果 与对照组相比，人参皂甙Rh2促进U87MG细胞凋亡（P<0.05），且增加细胞内游离钙离子浓度（P<0.05）；尼莫地平显著减少人参皂甙Rh2引起的U87MG细胞凋亡（P<0.05）。结论 人参皂甙Rh2可以通过增加细胞内游离钙离子浓度促进U87MG细胞凋亡。     

Tumor necrosis factor-alpha-induced cell death in U373 cells overexpressing alpha-synuclein

Intracellular alpha-synuclein inclusion formation in glial cells is frequently seen in Parkinson's disease and multiple system atrophy. Microglial activation in these neurodegenerative disorders suggests that neuroinflammatory responses might interact with alpha-synuclein and contribute to the pathogenesis of these disorders. To study the role of tumor necrosis factor-alpha (TNF-alpha), an important proinflammatory cytokine produced by microglia, on cells overexpressing alpha-synuclein we have used the astrocytoma cell line U373 engineered to express C-terminally truncated alpha-synuclein as a fusion protein with red or green fluorescent proteins. We demonstrate that alpha-synuclein overexpression augmented TNF-alpha-induced apoptotic cell death in U373 cells by induction of caspase activation. Furthermore, TNF-alpha exposure was associated with significant cytoskeletal changes characterized by altered inclusion composition with loss of cytoskeletal proteins and elevation of high-molecular-weight alpha-synuclein species. We conclude that alpha-synuclein overexpression significantly increases the vulnerability of U373 cells to apoptosis through TNF-alpha-mediated pathways.     

下调磷酸甘油酸激酶1增强胶质瘤U251细胞的放射敏感性

目的研究磷酸甘油酸激酶1(PGK1)对胶质瘤U251细胞放射敏感性的影响。方法建立放射抵抗的U251(RR-U251)细胞;LipofectamineTM2000方法将抑制PGK1表达的shRNA-PGK1质粒或对照shRNANC转染至U251细胞;荧光定量PCR及Western Blot检测PGK1的表达;MTT检测细胞相对存活率,划痕实验检测细胞迁移能力,Transwell小室实验检测细胞侵袭能力。结果与正常U251细胞相比,RR-U251细胞中PGK1的表达明显增高;放射处理后,RR-U251细胞的相对存活率升高,迁移能力及侵袭能力增强。转染shRNA-PGK1的细胞接受放射处理后,与对照组相比,细胞的相对存活率降低,迁移能力以及侵袭能力减弱。结论下调U251细胞中PGK1的表达可增加其放射敏感性,提示PGK1可能成为增强放射敏感性的调控靶点。     

The role of cannabinoids and leptin in neurological diseases

Cannabinoids exert a neuroprotective influence on some neurological diseases, including Alzheimer's, Parkinson's, Huntington's, multiple sclerosis and epilepsy. Synthetic cannabinoid receptor agonists/antagonists or compounds can provide symptom relief or control the progression of neurological diseases. However, the molecular mechanism and the effectiveness of these agents in controlling the progression of most of these diseases remain unclear. Cannabinoids may exert effects via a number of mechanisms and interactions with neurotransmitters, neurotropic factors and neuropeptides. Leptin is a peptide hormone involved in the regulation of food intake and energy balance via its actions on specific hypothalamic nuclei. Leptin receptors are widely expressed throughout the brain, especially in the hippocampus, basal ganglia, cortex and cerebellum. Leptin has also shown neuroprotective properties in a number of neurological disorders, such as Parkinson's and Alzheimer's. Therefore, cannabinoid and leptin hold therapeutic potential for neurological diseases. Further elucidation of the molecular mechanisms underlying the effects on these agents may lead to the development of new therapeutic strategies for the treatment of neurological disorders.     

白介素-18基因修饰人脑胶质瘤U87／hIL-18细胞的建立

目的构建人白介素18（hIL-18）基因真核表达载体质粒,建立hIL-18基因修饰并星稳定表达的人脑胶质瘤U87／hIL-18细胞。方法从人淋巴细胞cDNA文库钓取hIL-18基因后行PCR扩增,利用pcDNA3．1（＋）质粒为载体,构建hIL—18真核表达质粒pcDNA3．1／hIL-18。将构建的重组质粒转染人胶质瘤细胞U87,采用G418筛选单克隆细胞株,ELISA检测培养上清中的hIL-18蛋白含量,提取细胞RNA,RT—PCR方法检测hIL-18基因在U87／hIL-18细胞中的表达。结果所得hIL-18 cDNA序列与GeneBank比对一致,构建的质粒经PCR、酶切及测序鉴定正确。质粒转染U87细胞后,经加压筛选获得hIL-18基因稳定、高效表达的U87／hIL-18单克隆细胞株,细胞培养72h的上清中,hIL—18蛋白含量达131．2pg／ml,且RT—PCR结果显示细胞中hIL-18基因表达呈阳性。结论成功构建hIL-18真核表达质粒pcDNA3．1/hIL-18,并建立U87／hIL-18单克隆细胞株,为hIL-18基因修饰的胶质瘤疫苗研究打下了基础。     

白头翁皂苷D对胶质瘤细胞转移和凋亡的影响及机制研究

目的 探究白头翁皂苷D （PSD）对胶质瘤细胞U251MG转移和凋亡的影响及可能机制。方法 采用CCK-8试剂盒检测正常星形胶质细胞和U251MG细胞活力;选用0.0、1.0、2.0及5.0 μmol/L PSD处理U251MG,将细胞分为4组：PSD 0.0 μmol/L组、PSD 1.0 μmol/L组、PSD 2.0 μmol/L组和PSD 5.0 μmol/L组。划痕实验检测细胞迁移能力;Transwell检测细胞侵袭能力;Hoechst 33258染色检测细胞凋亡状况;RT-PCR检测mRNA表达水平;蛋白免疫印迹检测基质金属蛋白酶-2（MMP-2）、尿激酶型纤溶酶原激活物（uPA）、纤溶酶原激活物抑制剂-1（PAI-1）、血管内皮生长因子（VEGF）、cleaved caspase-3、cleaved caspase-9、肝细胞生长因子受体（c-MET）蛋白表达水平。结果 PSD抑制正常星形胶质细胞和胶质瘤细胞U251MG细胞活力。与PSD 0.0 μmol/L组相比较,PSD 1.0、2.0及5.0 μmol/L组细胞迁移率降低（P<0.01）,MMP-2、uPA、PAI-1、VEGF、c-MET蛋白水平降低（P<0.05）,PSD 1.0 μmol/L组细胞侵袭数降低（P<0.05）,PSD 2.0及5.0 μmol/L组细胞侵袭数降低（P<0.01）,细胞凋亡率升高（P<0.05）,cleaved caspase-3、cleaved caspase-9蛋白水平升高（P<0.05）。结论 PSD通过靶向c-MET抑制胶质瘤细胞U251MG迁移和侵袭,并诱导细胞凋亡。     

Mobilization of intracellular calcium by substance p in a human astrocytoma cell line (U-373 MG)

Variations in intracellular free calcium concentration (Δ[Ca²⁺]_i) were measured in intact and isolated human astrocytoma cells (U373 MG) loaded with fura-2 acetoxymethylester. Microperfusion of 50 nM substance P (SP), applied for 1 s, increased [Ca²⁺]_i by 351 nM from a stable basal level of [Ca²⁺]_i of 26 nM. The peak Δ[Ca²⁺]_i induced by SP was dose dependent with a threshold of 10_-3 nM, an ED₅₀ of 1.3 nM and a maximal effect for concentrations of SP greater than 100 nM. The NKI receptor agonist, [Sar⁹Met(O₂)¹¹]SP, mimicked the effect of SP, while the NK₂ and NK₃ selective receptor agonists, [N1¹⁰]NKA(4-10) and senktide, respectively, had no effect. The Δ[Ca²⁺]_i induced by SP was unaffected by 100 μM cadmium or by removal of extracellular calcium ions. Caffeine up to 30 mM had no effect on [Ca²⁺]_i. In contrast, thapsigargin increased resting [Ca²⁺]_i by 92 nM and reduced the Δ[Ca²⁺]_i induced by SP. A pertussis treatment (500 ng/ml-24 h) did not modify the Δ[Ca²⁺]_i induced by SP. We conclude that SP, acting on a NK1 receptor, mobilizes cytosolic calcium from an intracellular calcium pool which can be partially depleted by thapsigargin. © 1994 Wiley-Liss, Inc.     

ZnPcS4-BSA对光动力杀伤胶质瘤细胞效应的影响及其机制研究

目的 研究新型光敏剂ZnPcS4-BSA对光动力杀伤人神经胶质瘤细胞U251效应的影响,并对机制进行初步探讨. 方法 紫外光谱法观察U251细胞对ZnPcS4-BSA的摄取规律,确定最佳孵育时间;CCK-8法测定不同浓度ZnPcS4-BSA、不同剂量激光辐射对U251的细胞毒性和ZnPcS4-BSA介导光动力疗法(PDT)的最佳浓度和最佳激光剂量;应用20 μmol/L ZnPcS4-BSA作用U251细胞4h后给予100 J/cm2的激光辐射,以同样条件培养而未作PDT治疗的细胞作为对照,流式细胞术、RT-PCR分别检测细胞凋亡率和VEGF基因表达的变化. 结果 紫外光谱法扫描发现ZnPcS4-BSA作用4h后U251细胞摄取ZnPcS4-BSA的量达到峰值.不同浓度ZnPcS4-BSA处理后细胞生存率的差异无统计学意义(P＞0.05).400J/cm2激光辐射后细胞生存率低于0、25、50、100、200J/cm2激光辐射组,差异有统计学意义(P＜0.05).30 μmol/L ZnPcS4-BSA作用U251细胞4h后给予25、50、100、200 J/cm2激光照射,随着激光剂量的增加,细胞存活率降低,差异有统计学意义(P＜0.05).20、40、60、80、100 μmol/L ZnPcS4-BSA作用U251细胞4h后,给予200J/cm2激光辐射,随着ZnPcS4-BSA浓度的增加,细胞的抑制率增加,差异有统计学意义(P＜0.05).与对照组比较,经20μmol/L ZnPcS4-BSA作用,100 J/cm2的激光辐射后U251细胞凋亡率、VEGF基因的表达升高,差异均有统计学意义(P＜0.05). 结论 新型光敏剂ZnPcS4-BSA具有良好的增强光动力效能,其介导的PDT能诱导细胞凋亡,其机制可能与VEGF基因有关.     

盐酸小檗碱诱导人脑胶质瘤细胞U251凋亡及其与HERG钾离子通道的表达相关研究

目的探讨盐酸小檗碱(ber)诱导人脑胶质瘤细胞U251凋亡及其与HERG钾离子通道表达相关性。方法使用不同浓度的ber处理人胶质瘤细胞U251,采用四甲基偶氮唑盐(MTT)染色法测定细胞增值率,Hoechst33258荧光染色法观察细胞形态学改变,流式细胞术测定细胞凋亡率,分别采用实时荧光定量PCR与Western Blotting检测HERG钾通道的RNA及蛋白的表达情况。结果 MTT法检测显示细胞抑制率呈浓度与时间依赖性,且随药物浓度增加而增大(P0.05),其24 h的半数抑制浓度(IC50))是(8.78±0.20)μmol/l;Hoechst33258荧光染色法检测表明处理组出现典型的凋亡特征;且流式细胞术检测结果表明随着ber的剂量增加U251细胞的早期凋亡率及晚期凋亡率逐渐增大(P0.05);RT-PCR与Western Bloting检测结果显示ber处理后U251细胞的HERG钾通道的RNA含量及蛋白的表达均上调,且随着药物浓度的增高而增高。结论在体外ber可诱导人胶质瘤细胞系U251的凋亡,并可能通过上调HERG蛋白的表达抑制U251增殖。     

Characterization of NTera2/D1 cells as a model system for the investigation of cannabinoid function in human neurons and astrocytes

The limited availability and potential to culture primary human brain cells means that there is still a need for cell lines that reliably model human neurons and glial cells. The human-derived NTera2/D1 (NT2) cell line is a promising tool from which both neuronal (NT2N) and astrocytic (NT2A) cells can be derived in vitro. Here we have investigated the potential to use this cell model to investigate the endocannabinoid system in the CNS. Through immunocytochemical characterization with a range of neuronal and glial markers, we found that these cell lines differentiate into cells with immature neuronal and astrocytic phenotypes, respectively. By real-time PCR, immunocytochemistry, and functional inhibition of cAMP accumulation, the cannabinoid 1 receptors were identified only on NT2N cells, consistent with high levels of expression of this receptor in neuronal cells of the CNS. No evidence of cannabinoid 2 receptor expression was found on any of the NT2 cell types. Both the precursors and the differentiated NT2N and NT2A cells demonstrated mRNA expression for the key enzymes involved in endocannabinoid synthesis and degradation. This work establishes a cannabinergic phenotype in NT2N and NT2A cells, providing an alternative human derived renewable cell model for investigation of cannabinoid receptor function and endocannabinoid synthesis and metabolism in the CNS.     

白藜芦醇抑制U251人胶质瘤细胞增殖及其相关机制的探讨

目的 探讨白藜芦醇（Res）对U251胶质瘤细胞增殖和侵袭的抑制作用及其机制。方法 将不同浓度Res作用于U251胶质瘤细胞系，相差显微镜下观察其形态的变化，MTT法检测其对细胞生长增殖的抑制作用，流式细胞术（FCM）检测其对细胞周期的影响，用蛋白印迹（Western blot）检测Res处理前后U251细胞表皮生长因子受体（EGFR）、p53、p21、CDK4、CyclinD1、Bcl-2及caspase-3的表达，免疫组化分析MMP9、NFKB、P-AKT及AKT2的表达。结果 U251细胞经Res处理后，细胞形态发生一定变化，U251胶质瘤细胞的增殖被抑制，呈浓度和时间依赖性，细胞周期被阻滞在S期，Western blot检测显示EGFR、CyclinD1、CDK4、Bcl-2的表达降低，p21、p53、caspase-3蛋白表达升高，免疫组化检测显示MMP9，NFKB、P-AKT及AKT2的表达降低。结论 Res有可能通过调节EGFR及p53信号通路而抑制U251胶质瘤细胞的增殖和侵袭，诱导肿瘤细胞凋亡。     

WISP-2siRNA对人脑胶质瘤U251细胞增殖、凋亡的影响

目的 探讨siRNA沉默WISP-2基因表达对人脑胶质瘤细胞U251增殖、凋亡的影响.方法 脂质体介导siRNA转染人脑胶质瘤细胞U251后,检测U251细胞的增殖、凋亡的变化,并用Western blot法检测WISP-2、Bcl-2、Bax蛋白的表达.结果 WISP-2 siRNA能有效抑制U251细胞株的生长,诱导凋亡,siRNA干扰组WISP-2蛋白表达水平为(18.67±1.40)％,明显低于空白对照组(70.18±1.82)％和阴性对照组(69.41±1.77)％,且差异有统计学意义(P＜0.01);siRNA干扰组Bcl-2、Bax蛋白表达水平为(29.67±1.47)％、(31.62±1.32)％,明显低于空白对照组和阴性对照组,且差异有统计学意义(P＜0.01).结论 WISP-2 siRNA抑制WISP-2 mRNA和蛋白表达后,能有效抑制胶质瘤细胞的增殖,增加U251细胞凋亡,可能通过下调Bcl-2/Bax蛋白表达的比值来诱导细胞凋亡.     

二氢杨梅素促进胶质瘤U251细胞凋亡的实验研究

目的 探讨二氢杨梅素(Dihydromyricetin,DHM)对人胶质瘤U251细胞凋亡的促进作用及其机制。方法 将体外培养的胶质瘤U251细胞分为对照组和DHM组(根据DHM水平的不同分为3个亚组),MTT法观察细胞活性,Hoechst 33258核染色法观察细胞凋亡,流式细胞仪检测细胞凋亡率,透射电镜观察细胞形态,Western blot检测Bax和bcl-2蛋白表达水平。结果 Hoechst 33258核染色和流式细胞仪检测显示,随着DHM水平的增高,U251细胞凋亡率呈剂量依赖性增高; 电镜观察显示,对照组胶质瘤U251细胞形态正常,DHM组胶质瘤U251细胞肿胀,细胞器呈碎片样分散,随着DHM水平升高,线粒体、内质网等细胞器结构破坏明显。此外, DHM处理后Bax蛋白表达水平增高,Bcl-2蛋白表达水平降低。结论 DHM可能通过改变Bax及Bcl-2蛋白表达水平来抑制胶质瘤U251细胞增殖并促进其凋亡。     

土贝母苷甲通过上调FADD/caspase-8/caspase-3的表达促进人胶质瘤U251细胞凋亡

目的研究土贝母苷甲(TBMSⅠ)诱导人胶质瘤U251细胞凋亡及其可能的内在机制。方法四甲基偶氮唑蓝比色法(MTT法)检测不同浓度的土贝母苷甲对U251细胞增殖的影响。Hoechst 33258荧光染色观察不同浓度土贝母苷甲处理U251细胞后细胞核形态的变化。流式细胞仪检测细胞的凋亡率。Western blot分析土贝母苷甲对FADD、caspase-8和caspase-3蛋白表达水平的影响。结果土贝母苷甲以剂量依赖方式抑制U251细胞的增殖。土贝母苷甲处理后,U251细胞的细胞核表现出固缩浓染等凋亡特征,且随着药物浓度的增加,细胞数目逐渐减少。与对照组相比较,土贝母处理组(10、20、25μg/ml)的早期和晚期凋亡率均显著增加,差异有统计学意义(P﹤0.05)。10、20、25μg/ml土贝母苷甲处理组上调了FADD、caspase-8和caspase-3蛋白的表达水平,差异有统计学意义(P﹤0.05)。结论土贝母苷甲在体外能够明显的抑制U251细胞的增殖,其机制可能与上调FADD、caspase-8和caspase-3蛋白表达从而诱导细胞凋亡有关。     

土木香内酯对人胶质瘤U251细胞增殖、凋亡的影响

目的 探讨土木香内酯对人胶质瘤U251细胞增殖、凋亡的影响及其作用机制。方法 体外培养人胶质瘤U251细胞,取对数期生长的U251细胞分为空白对照组和土木香内酯（浓度分别为2.5、5.0、10.0 μmol/L）组,每组各10个复孔。使用MTT法检测U251细胞的增殖抑制率,采用流式细胞术检测U251细胞的凋亡率,免疫印迹法检测PDK1/Akt/GSK3β蛋白表达,使用RT-PCR检测c-Myc, Cyclin D1 mRNA的表达水平。结果 随土木香内酯浓度增加,U251细胞增殖抑制率明显增高（P<0.05）,细胞调亡率明显增高（P<0.05）。与空白对照组相比,木香内酯组U251细胞PDK1、Akt和GSK3β蛋白表达水平均无明显差异（P>0.05）,但PDK1、Akt和GSK3β蛋白的磷酸化水平均显著降低（P<0.05）。随着土木香内酯浓度的增加,U251细胞c-Myc、CyclinD1 mRNA的表达水平明显降低（P<0.05）。结论 土木香内酯抑制人胶质瘤U251细胞的增殖并促进其凋亡,其作用机制可能是通过调节PDK1/Akt/GSK3β信号通路有关,且在一定范围内呈浓度依赖性。     

Role of CB1 and CB2 receptors in the inhibitory effects of cannabinoids on lipopolysaccharide-induced nitric oxide release in astrocyte cultures

The purpose of this study was to investigate the role of the central cannabinoid receptor (CB(1)) in mediating the actions of the endogenous cannabinoid agonist anandamide and the synthetic cannabinoid CP-55940. Activation of primary mouse astrocyte cultures by exposure to bacterial lipopolysaccharide (LPS) caused a marked (approximately tenfold) increase in nitric oxide (NO) release. Coincubation with the cannabinoid agonists anandamide or CP-55940 markedly inhibited release of NO (-12% to -55%). This effect was abolished by SR-141716A (1 microM), a CB1 receptor antagonist. SR-141716A alone also significantly increased NO release in response to LPS, suggesting that endogenous cannabinoids modify inflammatory responses. In contrast, coincubation with the CB2 receptor antagonist SR-144528 (1 microM) abolished the inhibitory effects of the endogenous cannabinoid anandamide on LPS-induced NO release, although this may reflect nonspecific effects of this ligand or cannabinoid actions through atypical receptors of anandamide. We also showed that endogenous or synthetic cannabinoids inhibit LPS-induced inducible NO synthase expression (mRNA and protein) in astrocyte cultures. These results indicate that CB1 receptors may promote antiinflammatory responses in astrocytes.     

齐墩果酸通过MAPK/ERK信号传导通路对U87 MG细胞增殖及凋亡的体外实验研究

目的 探讨齐墩果酸(0A)对神经胶质母细胞瘤U87MG细胞增殖及细胞凋亡的影响.方法 以U87MG细胞为研究对象,使用25、50、80、100、150和200μg/ml的OA处理24、48和72h后,采用MTT实验检测OA对体外培养U87MG细胞增殖抑制作用,计算其抑制率;采用划痕法检测OA对U87MG细胞迁移能力的影响;用流式细胞仪分析OA对U87MG细胞周期与凋亡的影响;通过Western blot检测OA对MAPK/ERK信号传导通路活性的影响.结果 50μg/ml以上浓度的OA处理48h后,U87MG细胞的形态发生不同程度的变化,大量细胞脱落.150μg/ml和200μg/ml的OA处理U87MG细胞72h后,生长抑制率可达100％,与对照组相比差异有统计学意义(P＜0.01).25μg/ml的OA处理48 h后,迁移至划痕区的细胞数量相比于对照组明显减少.经50μg/mlOA作用48h后,处于Go/G1期的细胞从64.23％上升至75.03％,说明发生Go/G1期阻滞.同一浓度下,细胞凋亡率达到18.77％.U87MG细胞中MEK和ERK蛋白的磷酸化水平受到明显的抑制.结论 OA可显著抑制U87 MG细胞的生长增殖,呈一定的量效关系;OA可明显抑制U87MG细胞的迁移,并能抑制MAPK/ERK信号传导通路的激活.OA通过抑制MAPK/ERK信号传导通路的激活,可以抑制神经胶质母细胞瘤细胞的增殖生长.