氧自由基与脑胶质瘤瘤周水肿关系的实验研究

目的:探讨自由基和脑胶质瘤瘤周水肿的关系。方法:体外培养大鼠C6脑胶质瘤细胞株,采用立体定向技术将细胞株接种在右侧尾状核,建立大鼠脑胶质瘤模型,共60只,术后5天将荷瘤大鼠随机分为3组(EDA高剂量组,EDA低剂量组和对照组),每组各20只,每组10只用来测定荷瘤大鼠瘤周脑组织含水量、SOD活性及MDA含量,剩余10只用来观察荷瘤大鼠生存时间。结果:EDA干预组荷瘤大鼠瘤周脑组织含水量下降,SOD活性增高,MDA含量下降,以EDA高剂量组更为显著。各组荷瘤大鼠瘤周脑组织含水量与SOD活性呈负相关,而与MDA含量呈正相关。且EDA组荷瘤大鼠生存时间延长。结论:由基参与大鼠脑胶质瘤瘤周水肿的形成,自由基清除剂能够减轻大鼠脑胶质瘤瘤周脑组织水肿。     

白藜芦醇预处理对人鼠脑缺血再灌损伤的保护作用

目的:探讨白藜芦醇预处理对大鼠脑缺血再灌损伤的保护作用及其分子机制.方法:大鼠随机分成假手术组、缺血溶剂组、白藜芦醇预处理组,四动脉阻塞(4-VO)法建立前脑缺血模型,缺血10min/再灌22h,试剂盒检测大鼠海马组织SOD活力及NO、MDA含量变化,RT-PCR法观察GRP78 mRNA的表达.结果:缺血溶剂组海马组织SOD活性明显低于假手术组,NO、MDA含量高于假手术组;缺血前白藜芦醇预处理能显著反转缺血诱导的SOD活力和NO、MDA水平变化,脑缺血能明显上调GRP78 mRNA水平;白藜芦醇预处理能有效抑制缺血诱导的GRP78表达,与缺血组比有显著性差异.结论:白藜芦醇能通过上调SOD活力,减少NO、MDA的生成来抑制缺血后自由基的生成和积累,继而缓解内质网应激、下调GRP78的表达,减轻缺血性脑组织损伤.     

半夏总生物碱对帕金森病大鼠的学习记忆及氧化应激反应的影响

目的 探讨半夏总生物碱(total alkaloids from Pinellia Ternate,TAPT)对帕金森病模型大鼠的防治作用及其抗氧化机制.方法 采用脑内定位注射6-羟基多巴胺(6-OHDA)建立帕金森病大鼠模型,在模型建立成功的同时给予半夏总生物碱预防性治疗.采用Morris水迷宫进行帕金森病大鼠的行为学检测,用化学比色法检测大脑皮质及血清超氧化物歧化酶(SOD)活力、丙二醛(MDA)、过氧化氢(H2O2)和还原型谷胱甘肽(GSH)的含量.结果 与正常组比较,帕金森病模型组大鼠的逃避潜伏期明显延长(P＜0.01),跨越平台次数明显减少(P＜0.01);经半夏总生物碱治疗组,大鼠逃避潜伏期明显缩短(P＜0.05,P＜0.01),跨越平台次数明显增加(P＜0.01).帕金森病模型组大鼠脑皮质及血清中MDA、H2O2的含量增加,SOD活性及GSH的含量降低(P＜0.01);经半夏总生物碱治疗组,脑皮质MDA、H2O2的含量显著减少(P＜0.01),皮质GSH、SOD含量显著增加(P＜0.01);半夏总生物碱给药组中低浓度组、中浓度组血清MDA的含量无统计学意义(P＞0.05),高浓度组血清MDA含量下降(P＜0.01),各治疗组中血清H2O2含量明显下降(P＜0.01),血清GSH含量显著增加(P＜0.01).结论 半夏总生物碱具有改善学习记忆能力,对抗大鼠神经系统的退行性变有一定的作用,可能通过改变帕金森病模型大鼠皮质部分及血清SOD、GSH的含量,而抑制了MDA和H2O2的产生.     

急性酒精中毒合并中度创伤性脑损伤大鼠海马AQP4的表达

目的:探讨大鼠急性酒精中毒合并颅脑外伤后AQP4在海马区表达的变化.方法:健康成年雄性SD大鼠96只,随机分为4组:假手术组(N组)、急性酒精中毒组(A组)、中度创伤性脑损伤组(T组)和急性酒精中毒合并中度创伤性脑损伤(AT组).腹腔注射酒精(2.5g/kg),2h后以重物自由落体击打大鼠头部建立急性酒精中毒合并中度创伤性脑损伤(traumatic brain injury,TBI)动物模型.各组动物分别存活1、3、5、14天.免疫组化方法检测海马CA1区AQP4的表达.结果:AQP4阳性产物分布于胶质纤维和毛细血管壁,各实验组表达均高于N组.术后1天T组比AT组表达显著增高(P＜0.01),术后3天AT组比T组表达增高(P＜0.05),术后14天AT组比T组表达显著增高(P＜0.01).结论:大鼠急性酒精中毒合并颅脑外伤后晚期,海马CA1区AQP4表达增高,可能加重晚期继发性脑水肿,是急性酒精中毒合并颅脑外伤预后不良的原因之一.     

急性酒精中毒合并中度创伤性脑损伤大鼠海马AQP4的表达

目的:探讨大鼠急性酒精中毒合并颅脑外伤后AQP4在海马区表达的变化.方法:健康成年雄性SD大鼠96只,随机分为4组:假手术组(N组)、急性酒精中毒组(A组)、中度创伤性脑损伤组(T组)和急性酒精中毒合并中度创伤性脑损伤(AT组).腹腔注射酒精(2.5g/kg),2h后以重物自由落体击打大鼠头部建立急性酒精中毒合并中度创伤性脑损伤(traumatic brain injury,TBI)动物模型.各组动物分别存活1、3、5、14天.免疫组化方法检测海马CAI区AQP4的表达.结果:AQP4阳性产物分布于胶质纤维和毛细血管壁,各实验组表达均高于N组.术后1天T组比AT组表达显著增高(P＜0.01),术后3天AT组比T组表达增高(P＜0.05),术后14天AT组比T组表达显著增高(P＜0.01).结论:大鼠急性酒精中毒合并颅脑外伤后晚期,海马CAI区AQP4表达增高,可能加重晚期继发性脑水肿,是急性酒精中毒合并颅脑外伤预后不良的原因之一.     

甲亢大鼠肝脏组织中HO-1、Fn及肾脏组织中的抗氧化酶活性的变化分析

为了研究甲亢大鼠肝脏组织中血红素加氧酶1 (HO-1)、铁蛋白(Fn)及肾脏组织中抗氧化酶活性的变化及其意义,本研究选取了36只SD雌性成年大鼠,采用随机数字表法分为甲亢组、对照组。采用优甲乐灌胃造模,通过RT-PCR法检测两组大鼠肝脏组织中HO-1 mRNA、Fn mRNA (含有H和L两种亚基)的表达水平,测定肾脏组织中丙二醛(MDA)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化物歧化酶(SOD)的水平并进行比较。研究结果发现,甲亢组大鼠的血清三碘甲状腺素(T3)、甲状腺素(T4)水平显著的高于对照组,差异具有统计学意义(p0.05);甲亢组大鼠血清促甲状腺激素(TSH)水平显著的低于对照组,差异具有统计学意义(p0.05);甲亢组大鼠的肝脏组织中HO-1 mRNA、Fn-H mRNA表达水平显著的高于对照组,差异具有统计学意义(p0.05);甲亢组大鼠肝脏组织中Fn-L mRNA表达水平显著的低于对照组,差异具有统计学意义(p0.05);甲亢组大鼠肾脏组织中MDA的水平显著的高于对照组,差异具有统计学意义(p0.05);甲亢组大鼠肾脏组织中CAT、GSH-Px、SOD的水平显著的低于对照组,差异具有统计学意义(p0.05)。本研究表明,甲亢会导致大鼠肝脏铁蛋白含量水平的增加,引起HO-1表达升高,同时通过氧化应激对肾脏造成损伤。     

钙敏感受体在大鼠脑缺血再灌注损伤时细胞凋亡中的作用

目的:评价钙敏感受体在大鼠脑缺血再灌注损伤时细胞凋亡中的作用。方法:健康成年雄性Wistar大鼠60只,体重250～300 g,采用随机数字表法分为3组(n=20):假手术组(S组)、脑缺血再灌注组(I/R组)和钙敏感受体拮抗剂组(N组)。I/R组和N组采用线栓法经左侧颈外-颈内动脉插线制备大鼠脑缺血再灌注损伤模型,于脑缺血前10 min尾静脉注射等容量二甲基亚砜和钙敏感受体拮抗剂NPS-89636 1 mg/kg。于再灌注24 h时行神经功能评分,随后处死大鼠取脑组织,测定MDA含量和SOD活性,采用TUNEL法观察神经细胞凋亡情况,计算神经细胞凋亡指数,免疫组化法检测Caspase-3阳性细胞的表达,Western blot法检测Caspase-3蛋白的表达。结果:I/R组和N组MDA含量、神经细胞凋亡指数、Caspase-3阳性细胞和Caspase-3蛋白表达水平高于S组,神经功能评分和SOD活性低于S组,差异有统计学意义(P0.05);N组MDA含量、神经细胞凋亡指数、Caspase-3阳性细胞和Caspase-3蛋白表达水平低于I/R组,神经功能评分和SOD活性高于I/R组,差异有统计学意义(P0.05)。结论:钙敏感受体参与大鼠脑缺血再灌注损伤和细胞凋亡的发生。     

氧自由基与脑胶质瘤瘤周水肿关系的实验研究

目的：探讨自由基和脑胶质瘤瘤周水肿的关系。方法：体外培养大鼠C6脑胶质瘤细胞株,采用立体定向技术将细胞株接种在右侧尾状核,建立大鼠脑胶质瘤模型,共60只,术后5天将荷瘤大鼠随机分为3组（EDA高剂量组,EDA低剂量组和对照组）,每组各20只,每组10只用来测定荷瘤大鼠瘤周脑组织含水量、SOD活性及MDA含量,剩余10只用来观察荷瘤大鼠生存时间。结果：EDA干预组荷瘤大鼠瘤周脑组织含水量下降,SOD活性增高,MDA含量下降,以EDA高剂量组更为显著。各组荷瘤大鼠瘤周脑组织含水量与SOD活性呈负相关,而与MDA含量呈正相关。且EDA组荷瘤大鼠生存时间延长。结论：由基参与大鼠脑胶质瘤瘤周水肿的形成,自由基清除剂能够减轻大鼠脑胶质瘤瘤周脑组织水肿。     

大鼠脑缺血再灌注时水通道蛋白4表达与血脑屏障通透性变化

目的:研究大鼠脑缺血/再灌注时脑组织中水通道蛋4(AQP4)表达与脑水肿、血脑屏障通透性间关系。方法:采用大鼠大脑中动脉线栓缺血模型,免疫组化法、蛋白印迹法测定AQP4表达,干湿重法测定脑水含量以评价脑水肿,伊文氏蓝(EB)法测定血脑屏障通透性。结果:脑缺血后再灌注4～6h,AQP4表达上调,至12h上调显著,48～72h达高峰。脑水含量、EB含量均与此趋势相一致,且AQP4表达与脑水含量、EB含量呈显著正相关(P0.05)。结论:AQP4表达参与了缺血性脑水肿的产生,且与BBB通透性改变呈正相关。     

替米沙坦及吡哆胺对自发性高血压大鼠脑组织氧化应激的影响

目的:探讨替米沙坦及吡哆胺对自发性高血压大鼠脑组织氧化应激的影响。方法:自发性高血压大鼠24只随机分为4组(n=6):高血压对照组(HC组);替米沙坦组(T组);吡哆胺组(P组);联合治疗组(TP组)。同龄WKY大鼠作为正常对照组(NC组)。药物干预16周,测定各组脑组织中丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性及烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶p47phox mRNA表达。结果:与NC组比较,HC组脑组织中MDA含量明显升高、SOD活性明显减低(P<0.05);与HC组比较T组、P组、TP组MDA含量明显减低,SOD活性明显升高(P<0.05);与NC组比较HC组(NADPH)氧化酶p47phox mRNA表达显著上调(P<0.01);与HC组比较T组、TP组NADPH氧化酶p47phox mRNA表达明显下调(P<0.01);HC组与P组比较NADPH氧化酶p47phox mRNA表达无统计学差异(P>0.05)。结论:自发性高血压大鼠脑组织处于氧化应激状态,替米沙坦及吡哆胺可抑制自发性高血压大鼠脑组织的氧化应激水平,联合治疗并不优于替米沙坦单药治疗。     

高压氧疗法对创伤性脑损伤大鼠氧化应激指标及促／抗炎细胞因子水平的影响

目的：研究高压氧（HBO）疗法对创伤性脑损伤（TBI）大鼠氧化应激指标及促／抗炎细胞因子水平的影响方法：SD雄性大鼠18只,随机分为3组（n=6）：假手术组、损伤对照组和HBO治疗组采用Feency法建立大鼠TBI模型,假手术组只开放骨面,不予打击HBO治疗组大鼠于脑损伤后6h采用动物高压舱,以3ATA压力纯氧治疗60min。所有动物于手术后24h处死,分离脑组织,取伤侧脑半球行组织匀浆,分别测定匀浆上清液中超氧化物歧化酶（SOD）、丙二醛（MDA）、NO的含量以及促炎细胞因子TNF-α、IL-6、IL-1β及抗炎细胞因子IL-10的水平,结果：与损伤对照组相比,HBO治疗使SOD、NO以及IL-10水平升高,同时降低脑内MDA及TNF-α、IL-6、IL-1β的含量结论：HBO治疗可抑制TBI后自由基的生成,从而减轻脂质过氧化反应;同时,HBO治疗可减少促炎细胞因子的生成,促进抗炎细胞因子的产生,从而可减轻损伤脑组织的炎症反应,有助于减轻TBI后脑组织的继发性损伤.     

姜黄素对大鼠脑缺氧缺血损伤时脑组织MDA变化、caspase-3表达及细胞凋亡的影响

目的:探讨姜黄素对大鼠脑缺氧缺血损伤时脑组织MDA变化、caspase-3表达及细胞凋亡的影响。方法:健康SD雄性大鼠48只,随机分为假手术对照组(SH组)、脑缺氧缺血组(HI组)、姜黄素组(CU组)、溶剂对照组(SC组);生化方法检测脑组织丙二醛(MDA)含量;免疫组织化学测定大脑皮质caspase-3的表达;电镜观察大脑皮质形态学结构变化。结果:姜黄素可使脑组织MDA含量明显减低,并且抑制caspase-3蛋白的表达;神经元细胞凋亡减轻。结论:细胞凋亡参与了大脑缺氧缺血损伤的发生,姜黄素可能通过减低MDA含量、下调caspase-3的表达抑制细胞凋亡,从而减轻脑缺氧缺血性损伤。     

单核细胞趋化蛋白1及其受体CCR2mRNA在急性酒精中毒大鼠脑组织的表达

目的:观察大鼠急性酒精中毒后脑组织趋化因子单核细胞趋化蛋白1(MCP-1)及其受体CCR2mRNA水平的表达变化.方法:制备急性酒精中毒模型,用SYBR Green I荧光实时定量PCR技术动态定量监测脑组织中MCP-1与CCR2 mRNA在急性酒精中毒后不同时间点表达的变化.结果:息性酒精中毒后6h脑组织MCP-1 ...     

促红细胞生成素对脑缺血/再灌注损伤的保护作用

目的:探讨促红细胞生成素(Epo)对大鼠脑缺血/再灌注损伤的保护作用。方法:32只SD大鼠,采用夹闭双侧颈总动脉30min再灌注24h制作脑缺血/再灌注模型。随机分为4组(n=8):假手术组、脑缺血/再灌注组、Epo组及阳性对照组(尼莫地平),观察缺血/再灌注后血清一氧化氮(NO)和脑组织匀浆中超氧化歧化酶(SOD)活性、丙二醛(MDA)含量及脑组织含水量的变化。结果:Epo组血清NO和脑组织匀浆中MDA含量显著下降,SOD活性显著升高,脑组织含水量显著下降,与缺血/再灌注组相比有显著性差异。结论:大鼠脑缺血/再灌注后,Epo能减轻脑组织的含水量,减少自由基的生成,减轻脂质过氧化反应,对脑缺血/再灌注损伤有保护作用。     

心房颤动患者血清脑钠肽水平变化的临床研究

目的：探讨患者心房颤动（房颤,AF）发作时对血清脑钠肽水平的影响。方法：选择阵发性房颤组、持续性房颤组、对照组（窦性心律）患者各30例,观察各组血清脑钠肽水平;并对阵发性房颤组中心室率≤100 beats/min与心室率〉100 beats/min的患者进行亚组分析;观察阵发性房颤组复律后24 h和30 d血清脑钠肽水平。结果：阵发性房颤组和持续性房颤组血清脑钠肽水平明显高于对照组（P〈0.01）,房颤复律后血清脑钠肽水平很快下降。结论：血清脑钠肽水平在房颤发作时明显升高,血清脑钠肽水平的升高与房颤的发作有关。     

Cell Wall,Cytoskeleton, and Cell Expansion in Higher Plants

To accommodate two seemingly contradictory biological roles in plant physiology, providing both the rigid structural support of plant cells and the adjustable elasticity needed for cell expansion, the composition of the plant cell wall has evolved to become an intricate network of cellulosic, hemicellulosic, and pectic polysaccharides and protein. Due to its complexity, many aspects of the cell wall influence plant cell expansion, and many new and insightful observations and technologies are forthcoming. The biosynthesis of cell wall polymers and the roles of the variety of proteins involved in polysaccharide synthesis continue to be characterized. The interactions within the cell wall polymer network and the modification of these interactions provide insight into how the plant cell wall provides its dual function. The complex cell wall architecture is controlled and organized in part by the dynamic intracellular cytoskeleton and by diverse trafficking pathways of the cell wall polymers and cell wall-related machinery. Meanwhile, the cell wall is continually influenced by hormonal and integrity sensing stimuli that are perceived by the cell. These many processes cooperate to construct, maintain, and manipulate the intricate plant cell wall--an essential structure for the sustaining of the plant stature, growth, and life.     

Dissecting the Heat Stress Response in Chlamydomonas by Pharmaceutical and RNAi Approaches Reveals Conserved and Novel Aspects

To study how conserved fundamental concepts of the heat stress response （HSR） are in photosynthetic eukaryotes, we applied pharmaceutical and antisense/amiRNA approaches to the unicellular green alga Chlamydomonas reinhardtii. The Chlamydomonas HSR appears to be triggered by the accumulation of unfolded proteins, as it was induced at ambient temperatures by feeding cells with the arginine analog canavanine. The protein kinase inhibitor staurosporine strongly retarded the HSR, demonstrating the importance of phosphorylation during activation of the HSR also in Chlamydomonas. While the removal of extracellular calcium by the application of EGTA and BAPTA inhibited the HSR in moss and higher plants, only the addition of BAPTA, but not of EGTA, retarded the HSR and impaired thermotoler- ance in Chlamydomonas. The addition of cycloheximide, an inhibitor of cytosolic protein synthesis, abolished the attenu- ation of the HSR, indicating that protein synthesis is necessary to restore proteostasis. HSP90 inhibitors induced a stress response when added at ambient conditions and retarded attenuation of the HSR at elevated temperatures. In addition, we detected a direct physical interaction between cytosolic HSP90A/HSP70A and heat shock factor 1, but surprisingly this interaction persisted after the onset of stress. Finally, the expression of antisense constructs targeting chloroplast HSP70B resulted in a delay of the cell＇s entire HSR, thus suggesting the existence of a retrograde stress signaling cascade that is desensitized in HSP7OB-antisense strains.     

Redox Regulation of Arabidopsis Mitochondrial Citrate Synthase

Citrate synthase has a key role in the tricarboxylic （TCA） cycle of mitochondria of all organisms, as it cata- lyzes the first committed step which is the fusion of a carbon-carbon bond between oxaloacetate and acetyl CoA. The regulation of TCA cycle function is especially important in plants, since mitochondrial activities have to be coordinated with photosynthesis. The posttranslational regulation of TCA cycle activity in plants is thus far almost entirely unexplored. Although several TCA cycle enzymes have been identified as thioredoxin targets in vitro, the existence of any thioredoxin-dependent regulation as known for the Calvin cycle, yet remains to be demonstrated. Here we have investigated the redox regulation of the Arabidopsis citrate synthase enzyme by site-directed mutagenesis of its six cysteine residues. Our results indicate that oxidation inhibits the enzyme activity by the formation of mixed disulfides, as the partially oxidized citrate synthase enzyme forms large redox-dependent aggregates. Furthermore, we were able to demonstrate that thioredoxin can cleave diverse intraas well as intermolecular disulfide bridges, which strongly enhances the activity of the enzyme. Activity measurements with the cysteine variants of the enzyme revealed important cysteine residues affecting total enzyme activity as well as the redox sensitivity of the enzyme.     

Organelle pH in the Arabidopsis Endomembrane System

The pH of intracellular compartments is essential for the viability of cells. Despite its relevance, little is known about the pH of these compartments. To measure pH in vivo, we have first generated two pH sensors by combining the improved-solubility feature of solubility-modified green fluorescent protein （GFP） （smGFP） with the pH-sensing capabil- ity of the pHluorins and codon optimized for expression in Arabidopsis. PEpHluorin （plant-solubility-modified ecliptic pHluorin） gradually loses fluorescence as pH is lowered with fluorescence vanishing at pH 6.2 and PRpHluorin （plant- solubility-modified ratiomatric pHluorin）, a dual-excitation sensor, allowing for precise measurements. Compartment- specific sensors were generated by further fusing specific sorting signals to PEpHluorin and PRpHluorin. Our results show that the pH of cytosol and nucleus is similar （pH 7.3 and 7.2）, while peroxisomes, mitochondrial matrix, and plastidial stroma have alkaline pH. Compartments of the secretory pathway reveal a gradual acidification, spanning from pH 7.1 in the endoplasmic reticulum （ER） to pH 5.2 in the vacuole. Surprisingly, pH in the trans-Golgi network （TGN） and mul- tivesicular body （MVB） is, with pH 6.3 and 6.2, quite similar. The inhibition of vacuolar-type H＋-ATPase （V-ATPase） with concanamycin A （ConcA） caused drastic increase in pH in TGN and vacuole. Overall, the PEpHluorin and PRpHluorin are excellent pH sensors for visualization and quantification of pH in vivo, respectively.