PI3K/AKT通路介导H_2O_2预处理减轻PC12细胞氧化损伤

目的探讨PI3K/AKT信号通路是否参与H2O2预处理诱导的适应性细胞保护作用。方法体外培养PC12细胞,建立H2O2预处理对抗高浓度H2O2诱导细胞损伤的实验模型。应用甲氮甲唑蓝(MTT)法测定细胞的存活率,比色法测定乳酸脱氢酶(LDH)的活性,碘化丙啶(PI)染色流式细胞术检测细胞凋亡率,免疫印迹法(Westernblot)测定AKT的表达。结果 100μmol H2O2预处理PC12细胞90 min可显著地抑制300μmol H2O2引起的损伤,使细胞存活率从50.2%±4.6%升高至83.8%±3.5%,LDH活性由103%±10.2%下降至68.5%±5.3%,细胞凋亡率由65.5%±4.1%下降至37.1%±2.3%(P<0.01)。100μmol H2O2预处理诱导p-AKT的表达,PI3K抑制剂ly294002阻断了H2O2预处理引起的p-AKT表达。同时ly294002拮抗了H2O2预处理诱导的抗细胞损伤和凋亡作用。结论 H2O2预处理通过PI3K途径引起AKT的活化,PI3K/AKT通路的活化介导了H2O2预处理诱导的适应性细胞保护作用。     

肌肽对高糖诱导的H9c2细胞损伤的拮抗作用

 目的：探讨肌肽对高糖诱导的H9c2心肌细胞损伤的拮抗作用。方法：体外培养大鼠心肌细胞H9c2,细胞分为正常对照组、高糖损伤组和肌肽预保护组。MTT法检测细胞存活率,DCFH-DA荧光探针检测细胞内活性氧(ROS)水平,并利用Western blotting检测半胱氨酸天冬氨酸蛋白酶(caspase-8、caspase-9和caspase-3)活性片段的表达。结果：细胞存活率随着高糖刺激时间和高糖浓度的增加而逐渐降低,而肌肽预保护组细胞的存活率显著高于高糖处理组（P<0.05)。高糖组ROS水平比正常对照组明显增加（P<0.05),而肌肽预保护组与高糖组相比ROS水平降低（P<0.05)。高糖组caspase-8、caspase-9和caspase-3活性片段表达量比正常对照组显著升高(P<0.05),而肌肽预保护组与高糖组相比,caspase-9和caspase-3活性片段表达量显著降低（P<0.05),而活化caspase-8相对含量无明显变化（P>0.05)。结论：肌肽能够拮抗高糖诱导的H9c2心肌细胞氧化应激和凋亡损伤。     

Tpc1808对H2O2诱导的C6细胞氧化应激性损伤的保护作用

目的:研究Tpc1808对H2O2诱导的C6细胞氧化应激性损伤的保护作用。方法:采用细胞培养、基因转染、MTT、LDH、免疫荧光组织化学、Western印迹等技术,以星形胶质瘤C6细胞为研究对象,以H2O2作为氧化应激刺激物,建立细胞损伤模型。结果:当终浓度为100μmol/L的H2O2作用于C6细胞24h后,MTT减小,LDH增加,细胞出现明显凋亡;转染Tpc1808基因的细胞受到保护,grp75表达量增加,与对照组相比有显著差异。结论:Tpc1808基因可以保护H2O2引起的C6细胞氧化应激性损伤,该保护作用可能是通过增加grp75表达实现的。     

H_2O_2诱导的PC12应激损伤中CR及SIRT1参与的调节效应

目的研究H2O2诱导的PC12细胞（又称肾上腺嗜铬细胞瘤细胞）氧化应激损伤中,热量限制（caloric restriction,CR）参与的调控效应及SIRT1的表达变化。方法采用MTT法检测H2O2诱导的氧化应激损伤中CR对PC12细胞活力的影响;TUNEL染色法检测各实验组中PC12细胞的凋亡;免疫荧光检测PC12细胞中SIRT1的表达与定位;RT-PCR及Wester-blot检测各实验组中SIRT1、Caspase-3的表达。结果 60μmol/LH2O2作用6h后,H2O2组细胞活力可维持在70%以上,与正常对照组及CR组比较差异具有统计学意义;而120μmol/LH2O2作用下,H2O2组细胞活力显著下降。本实验选取60μmol/LH2O2浓度作为后续实验使用浓度。CR处理后PC12细胞的凋亡率较H2O2组显著降低。PC12细胞中SIRT1可于细胞浆和细胞核中表达,且主要于细胞浆中表达。H2O2作用6h后,与正常对照组相比SIRT1表达降低,Caspase-3表达上升;在CR＋H2O2组中,与H2O2组比较,Caspase-3降低,SIRT1表达上升。结论 CR具有抗应激损伤及凋亡的效应,可上调PC12细胞中SIRT1的表达,在H2O2诱导的PC12细胞应激性损伤中CR-SIRT1的调控具有保护效应。     

血管生成素-1通过调节PI3K/AKT信号途径抑制H2O2诱导小鼠心肌细胞凋亡

目的 观察血管生成素-1(Ang-1)对H2O2诱导小鼠心肌细胞凋亡的影响及其与磷脂酰肌醇3激酶(PI3K) / 丝氨酸苏氨酸蛋白激酶(Akt)信号通路活化的关系。方法 培养新生小鼠心肌细胞,分为对照组、H2O2诱导组、Ang-1干预组、共同干预组,用Hoechst-33342染色法观察细胞凋亡形态学特征,流式细胞仪检测细胞凋亡率;Westernblot检测蛋白p-AKT、AKT、active Caspase-3的表达。结果 H2O2可以诱导心肌细胞发生凋亡,凋亡的心肌细胞呈现细胞核聚集、皱缩、碎裂等典型的凋亡形态学特征;与H2O2诱导组相比对照组、Ang-1干预组的细胞凋亡率均降低（2.13%±0.61% vs 48.16%±1.37% p<0.01;31.20%±2.01% vs 48.16%±1.37% p<0.01）;共同干预组的细胞凋亡率高于Ang-1干预组（47.42±2.02% vs 31.20±2.01% p<0.01）,与H2O2诱导组没有统计学差异（47.42%±2.02% vs 48.16%±1.37%）;与H2O2诱导组相比Ang-1干预组磷酸化AKT（p-AKT）水平升高、active Caspase-3的表达水平降低,这种差别在共同干预组中不明显。结论 Ang-1通过调节PI3K/AKT信号途径对H2O2诱导的小鼠心肌细胞凋亡起到保护作用。     

雌激素对H_2O_2所致N2a细胞损伤的保护作用

目的探讨雌激素对氧自由基(H_2O_2)所致野生型鼠成神经瘤细胞株(N2a)损伤的保护作用及机制。方法应用H_2O_2制作N2a细胞凋亡的细胞模型,采用细胞计数、酶组织化学、免疫细胞化学方法,检测在雌激素作用下,神经细胞的形态结构、细胞活性和细胞早期凋亡的相关性变化。结果细胞计数结果显示,雌激素组的细胞数量[(0.60±0.12)个/mL(×10~4)]高于其它组,雌激素加H_2O_2组的细胞数量[(0.51±0.11]×104个/mL)明显高于H2O2组[(0.30±0.09)×10~4个/mL],组间差异有统计学意义(P0.05)。免疫细胞化学染色显示,Akt1阳性反应在雌激素组最强,在H2O2组最弱,但雌激素加H_2O_2组与正常对照组Akt1的表达没有明显差别,介于两者之间。Fasl呈相反变化。Caspase3酶活性检测结果显示,H_2O_2组Caspase3酶活性最高,雌激素加H_2O_2组Caspase3酶活性明显降低,其差别有统计学意义(P0.01)。结论雌激素有促进N2a细胞生长的作用,并可保护H_2O_2对N2a细胞的损伤且可以对抗凋亡。     

创伤性脑损伤后核因子E2相关因子2在星形胶质细胞中的表达

目的:探讨创伤性脑损伤后核因子E2相关因子2(Nrf2)在星形胶质细胞中的表达变化。方法:雄性昆明小鼠随机分成假手术组、创伤性脑损伤后1、4、7d组。采用免疫印迹检测脑损伤后Nrf2的表达,免疫荧光染色法检测Nrf2在星形胶质细胞中的定位表达。结果:免疫印迹结果显示脑损伤后Nrf2在4d时表达最高;免疫荧光染色结果显示在假手术组中Nrf2在细胞质中有少量表达,损伤后1d组Nrf2在细胞质和细胞核均有表达,4d时表达显著增高。结论:损伤后Nrf2表达升高并入核可能与对星形胶质细胞的活化有关。     

天麻素保护过氧化氢诱导的神经元氧化损伤的研究

目的:探究天麻素对过氧化氢引起的神经细胞氧化损伤的保护作用及其作用机制。方法:培养SH-SY5Y细胞,并分为3组:对照组、氧化损伤组和天麻素处理组。用流式细胞术检测细胞凋亡率及细胞活性氧(ROS)水平,用ELISA检测细胞内超氧化物歧化酶(SOD)表达水平,用蛋白质免疫印迹实验检测细胞内PI3K、AKT磷酸化水平。结果:过氧化氢处理引起细胞凋亡率升高,胞内ROS平上升,SOD表达下调以及PI3K、AKT磷酸化水平下降;天麻素处理后可以显著抑制过氧化氢引起的细胞凋亡、ROS水平上升及SOD表达下调,并缓解氧化损伤对PI3K/AKT磷酸化的抑制作用。结论:天麻素通过激活PI3K/AKT信号通路保护过氧化氢诱导的神经细胞氧化损伤。     

AKT-糖原合成激酶-3β信号通路对SOD1G93A突变N2a细胞的保护作用

目的探讨AKT-糖原合成激酶-3β(GSK-3β)信号通路对SOD1G93A突变N2a细胞的作用及机制。方法选取小鼠成神经瘤细胞系N2a,分别转染p EGFP-WT-SOD1和p EGFP-G93A-SOD1质粒,应用Western blotting和免疫荧光染色方法检测AKT、GSK-3β和细胞周期蛋白D1(cyclin D1)在细胞模型中的表达变化。应用RT-PCR和Western blotting技术检测siRNA沉默AKT后GSK-3β和cyclin D1在SOD1突变N2a细胞中的表达变化,通过MTS方法,检测细胞增殖和存活的改变。结果与p EGFP-WT-SOD1转染的N2a细胞比较,p EGFP-G93A-SOD1转染的N2a细胞中AKT及GSK-3β总蛋白在转染后24 h和48 h表达均无明显变化,p-AKT(Ser473)、p-GSK-3β(Ser9)和cyclin D1表达均升高。免疫荧光染色结果显示,转染后24 h和48 h,p-AKT(Ser473)、p-GSK-3β(Ser9)和cyclin D1在p EGFP-G93A-SOD1转染的N2a细胞中表达均升高。应用siRNA沉默AKT后与对照组相比,在转染后48 h和72h,AKT、p-AKT(Ser473)、GSK-3β、p-GSK-3β(Ser9)和cyclin D1蛋白均降低。MTS实验结果显示,在AKT沉默后72h、96 h、120 h,N2a细胞增殖和活力降低。结论 SOD1G93A突变影响N2a细胞中AKT、GSK-3β翻译后磷酸化修饰及cyclin D1的表达,AKT可能通过调控GSK-3β和cyclin D1影响SOD1G93A突变N2a细胞的增殖和存活。     

N-乙酰-L-色氨酸减轻H2O2诱导小鼠海马神经元细胞凋亡

 目的 探讨N-乙酰-L-色氨酸(L-NAT)对海马神经元（PHN）缺血低氧损伤的影响。方法 用600μmol/L H2O2诱导PHN制备海马神经元细胞凋亡模型,采用免疫荧光染色检测caspase-3的表达,Rhodamine 123染色检测线粒体膜势能（ΔΨm）的改变,台盼蓝染色检测细胞存活率,比色法检测caspase-3、乳酸脱氢酶（LDH）的活性,Western blot检测caspase-3及凋亡诱导因子（AIF）和细胞色素C（CytC）等线粒体促凋亡因子在胞质蛋白和线粒体蛋白中的表达。结果 L-NAT可减轻H2O2所引起的细胞形态的死亡、存活率的降低、LDH的释放、caspase-3的激活、线粒体膜势能的丧失及AIF和CytC等线粒体促凋亡因子的释放。 结论 L-NAT能通过抑制caspase依赖性和非依赖性的细胞凋亡途径,减轻H2O2诱导的小鼠海马神经元的细胞损伤。     

An apparent KIR2DS2-negative KIR2DL2-positive genotype discloses the novel allele KIR2DS2*00104

KIR2DS2*00104 lacks a distinctive synonymous substitution of KIR2DS2 in nucleotide 418 that affects KIR genotyping.     

CaO-MgO-P_2O_5-SiO_2-CaF_2系统自硬性玻璃的研究

为研究钙离子、镁离子在体内环境中对自硬性玻璃结晶行为的影响,为自硬性生物活性玻璃的临床应用提供依据,本文设计了CaO-P2O5-SiO2-CaF2(Ca-glass)和CaO-MgO-P2O5-SiO2-CaF2(CaMg-glass)系统玻璃并使用模拟体液(simulated body flu id,SBF)进行了研究。首先采用磷酸氢二氨[(NH4)2HPO4]/[NH4H2PO4]硬化液与Ca-glass、CaMg-glass制成硬化体,然后使用X射线衍射(XRD)、扫描电镜(SEM)、失重、力学分析等方法,研究硬化体在SBF中的结晶性、降解性和力学性能。实验结果表明,玻璃粉末与磷酸铵缓冲溶液反应形成了磷酸铵钙[(NH4)2.Ca(HPO4)2.H2O]硬化体。硬化体经过SBF浸泡,Ca-glass系统硬化体中部分磷酸铵钙转化成羟基磷灰石,而CaMg-glass系统硬化体仍然为磷酸铵钙。Ca-glass与CaMg-glass硬化体在SBF中浸泡28天分别降解19.4%和31.3%,抗压强度分别为93.14MPa和64.52MPa。镁离子的歧化作用是导致Ca-glass、CaMg-glass硬化体结晶性能、降解性能以及力学性能差别的主要原因。     

K2O-B2O3-Al2O3-SiO2-MgO-F系统可切削生物微晶玻璃

目的 可切削微晶玻璃的制备温度高达1500 ℃以上,此特性严重制约其产业化发展.本文设计制备了K2O-B2O3-Al2O3-SiO2-MgO-F系统低温云母生物微晶玻璃,并探讨制备工艺对材料结构和性能的影响.方法 采用1300 ℃熔化工艺与600～750 ℃晶化热处理工艺制备微晶玻璃,通过X射线衍射分析方法研究微晶玻璃的晶相组成,利用扫描电子显微镜观察微晶玻璃的形貌,并通过显微硬度分析、高速砂轮切削实验考察微晶玻璃的可切削性能.结果 分别经过600 ℃、650 ℃、700 ℃、750 ℃晶化热处理2 h、4 h、8 h后,玻璃中均形成了主晶相为氟金云母的微晶玻璃,微晶玻璃的显微硬度为3～8 GPa.且随着晶化温度的升高,微晶玻璃层状结构逐渐明晰,但硬度不断下降,其可切削性持续提高.结论 低温下熔化K2O-B2O3-Al2O3-SiO2-MgO系统玻璃工艺降低了可切削微晶玻璃的制备温度和成本,利于产业化生产和推广应用.     

2-Perbromomethyl-2-oxazoline: a novel trifunctional initiator for the ring-opening polymerization of 2-methyl-2-oxazoline

Polymerization of 2-methyl-2-oxazoline was carried out using a trifunctional initiator, 2-perbromomethyl-2-oxazoline. The degree of polymerization (DP) of the resulting polymer was very close to the feed mole ratio of the monomer to initiator. The number-average molecular weight M?_n increased linearly with conversion, indicating the living nature of the propagating chain end. ¹H NMR and end-group analyses results are consistent with the proposal that the polymer possesses a star-shaped structure.     

Cross-Protection in Mice After Immunization with H2N2, H3N2, and Heq2Neq2 Influenza Virus Strains

Mice were vaccinated with the influenza viruses A/Japan/57 (H2N2), A/Hong Kong/68 (H3N2), and A/Equi/Miami/63 (Heq2Neq2) and the hemagglutinin and neuraminidase recombinants derived from these viruses. After infection with the parent viruses, protection was compared with serological findings. It was found that influenza vaccine protects not only against infection with a strain identical or closely related to the vaccine strain, but against heterologous strains as well. Vaccination with Hong Kong/68 and its neuraminidase recombinant resulted in a heterologous neuraminidase inhibition titer against Japan/57 and in a protection against infection with Japan/57. By contrast, after vaccination with Japan/57 and its neuraminidase recombinant, no relevant heterologous neuraminidase inhibition titer against Hong Kong/68 was observed, whereas a protection against infection with Hong Kong/68 did exist. A cross-protection between Hong Kong/68 and Miami/63, but no relationship in the hemagglutination or neuraminidase inhibition tests, was established in the preinfection sera. A one-way antigenic relationship between these viruses was confirmed by the rise of hemagglutinin or neuraminidase antibodies against Hong Kong/68 in the postinfection sera. No cross-protection or serological relationship existed between Miami/63 and Japan/57. Besides the hemagglutinin and neuraminidase, a third factor, the “mouse-protecting antigen,” was considered to contribute to the protection obtained. According to the protection observed, the mouse-protecting antigen of Hong Kong/68 virus is related to that of Japan/57 as well as Miami/63 virus. The mouse-protecting antigens of both Japan/57 and Miami/63 are related to that of Hong Kong/68.     

H2O2 enhances Ca2+ release from osteoblast internal stores

The physiological activity of osteoblasts is known to be closely related to increased intracellular Ca2+ activity ([Ca2+]i) in osteoblasts. The cellular regulation of [Ca2+]i in osteoblasts is mediated by Ca2+ movements associated with Ca2+ release from intracellular Ca2+ stores, and transmembrane Ca2+ influx via Na+-Ca2+ exchanger, and Ca2+ ATPase. Reactive oxygen species, such as H2O2, play an important role in the regulation of cellular functions, and act as signaling molecules or toxins in cells. In this study, we investigated the effects of H2O2 on cellular Ca2+ regulation in osteoblasts by measuring intracellular Ca2+ activities using cellular calcium imaging techniques. Osteoblasts were isolated from the femurs and tibias of neonatal rats, and cultured for 7 days. The cultured osteoblasts were loaded with a Ca2+-sensitive fluorescent dye, Fura-2, and fluorescence images were monitored using a cooled CCD camera, and subsequently analyzed using image analyzing software. The results obtained are as follows: (1) The osteoblasts with lower basal Ca2+ activities yielded a transient Ca2+ increase, a Ca2+ spike, while osteoblasts with higher basal Ca2+ activities showed a continuous increase in [Ca2+]i leading to cell death. (2) Ca2+ spikes, generated after removing Na+ from superfusing solutions, were blocked by H2O2 and this was followed by a sustained increase in Ca2+ activity. (3) ATP- induced Ca2+ spikes were inhibited by pretreating with H2O2 and this was followed by a continuous increase of [Ca2+]i. When cells were pretreated with the exogenous nitric oxide (NO) donor S-Nitroso-N-acetylpenicilance (SNAP, 50 microM), treatments of ATP (1 mM) induced a Ca2+ spike-like increase, but [Ca2+]i did not return to the basal level. (4) The expression of inositol- 1,4,5-triphosphate receptor (IP3R) was enhanced by H2O2. Our results suggest that H2O2 modulates intracellular Ca2+ activity in osteoblasts by increasing Ca2+ release from the intracellular Ca2+ stores.     

KIR2DL2/S2 and KIR2DS5 in alcoholic cirrhotic patients undergoing liver transplantation

IntroductionThe molecular mechanisms underlying alcoholic liver fibrosis and cirrhosis are not completely understood. Hepatic fibrosis involves the interplay of diverse cells and factors, including hepatic stellate cells (HSCs), Kupffer, NK cells, and T-lymphocyte subsets. Killer-cell immunoglobulin-like receptors (KIR) are membrane receptors involved in mediation between NK and activated HSCs, regulating NK cell function through their interaction with HLA-I molecules. The aim of this study was to analyse the genetic association between KIR genes and the susceptibility to or protection from alcoholic cirrhosis (AC) in a cohort of male AC patients undergoing liver transplantation (LT) with and without concomitant viral infections.Material and methodsKIR genotyping was performed in nuclear DNA extracted from 281 AC patients and compared with 319 male controls.ResultsSignificant differences between total AC patients and healthy controls were only found in the case of KIR2DL2 and KIR2DS5. KIR2DL2 was significantly underrepresented in non-viral AC patients (52.6% vs. 63.3%; p = 0.015), while patients heterozygous for KIR2DL2 were also underrepresented in the non-viral AC group compared with controls (p = 0.034). KIR2DS5 was overrepresented in this group compared with healthy controls (p = 0.002). All these observations were only evident in AC patients older than 54 years old.ConclusionsOur data suggest a contrary effect of KIR2DL2 and KIR2DS5 in AC patients older than 54 years, in whom the presence of KIR2DL2 appears to be protective against AC, whereas the presence of KIR2DS5 seems to promote the fibrotic process, particularly in patients with no associated viral infection.     

ExspiratorischepO2-undpCO2-Kurven bei Atmung von N2-, He- und Ar−O2-Gemischen

Zusammenfassung Zur Untersuchung der intrapulmonalen Gasmischung wurden an zehn Versuchspersonen die exspiratorischenpO₂- undpCO₂-Kurven fortlaufend und simultan massenspektrometrisch in Abhängigkeit vom Atemvolumen bei Atmung von Stickstoff-Sauerstoff-, Helium-Sauerstoff- und Argon-Sauerstoff-Gemischen registriert.Im Mischluftanteil wurden für den Abfall despO₂ von 75% auf 25% der Endamplitude im Mittel bei N₂–O₂-Atmung 81,6 ml, bei He–O₂-Atmung 66,1 ml und bei Ar–O₂-Atmung 71,9 ml benötigt. Die entsprechenden Zahlen für den Anstieg despCO₂ sind bei Atmung von N₂–O₂ 84,9 ml, von He–O₂ 68,5 ml und von Ar–O₂ 80.6 ml.DerpO₂ des Alveolarluftanteils sank während der letzten 300 ml Exspirationsvolumen bei Atmung des N₂–O₂-Gemisches im Mittel um 4,7 Torr, bei He–O₂ um 3,4 Torr und bei Ar–O₂ um 6,8 Torr. DerpCO₂ stieg gleichzeitig im Mittel bei Atmung des N₂–O₂-Gemisches um 2,8 Torr, bei He–O₂ um 2,1 Torr und bei Ar–O₂ um 3,7 Torr.Die Ursachen dieser Differenzen werden für den Mischluftanteil auf unterschiedliche Diffusions- und Strömungsbedingungen in den zentralen Lungenabschnitten zurückgeführt. Demgegenüber lassen sich die unterschiedlichen Partialdruckänderungen im Alveolarplateau durch Diffusion in den peripheren Lungenabschnitten und durch die Form der O₂ und CO₂-Bindungskurven erklären.Mit finanzieller Unterstützung der Europäischen Gemeinschaft für Kohle und Stahl durchgeführte Forschungsarbeit.     

13C NMR spectroscopy of 2-formamido-2-methylpropyl acrylate and poly(2-formamido-2-methylpropyl acrylate)

The synthesis and characterization of 2-formamido-2-methylpropyl acrylate (FMPA) is reported. ¹³C NMR spectra of FMPA in CDCl₃, CD₃OD, DMSO-d₆, DMF-d₇, and D₂O exhibit two pairs of lines for all seven carbon atoms at room temperature; the ratio of the two conformers varies moderately with solvent (21 : 79 to 41 : 59). The conformers are believed to involve strong internal hydrogen bonding which is not completely broken even by the addition of trifluoroacetic acid to CDCl₃ (1/1, v/v). However, the pairs of lines coalesce in turn as the temperature is raised to 120°C in DMSO-d₆. FMPA was polymerized at 35°C in DMF and CHCl₃, using a free radical initiator and the polymer was characterized by ¹³C NMR spectroscopy.