雌激素减轻H2O2诱导PC12细胞凋亡

目的观察雌激素对H2O2诱导细胞凋亡的作用,探讨雌激素保护作用机制。方法在PC12细胞建立H2O2诱导细胞凋亡的实验模型。用MTT法检测细胞存活率,比色法测定乳酸脱氢酶(LDH)活性,Hoechst染色检测细胞凋亡,碘化丙啶(PI)染色流式细胞术检测细胞凋亡率,比色法测定caspase-3活性。结果H2O2明显降低PC12细胞的存活率,使LDH释放增加,促进细胞凋亡,并能明显地升高caspase-3的活性。雌激素能显著地减轻上述变化。结论雌激素对抗H2O2诱导的细胞凋亡,抑制caspase-3的激活是其细胞保护机制之一。     

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。     

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。     

管花苷B对抗H202诱导的PCI2细胞凋亡

目的:观察肉苁蓉提取物管花苷B对H:O:诱导的PCI2细胞损伤的影响.方法:用MTr法检测细胞存活率,以激光共聚焦显微镜荧光染色法检测细胞内活性氧的产生和线粒体膜电位的变化,DNA琼脂糖凝胶电泳和流式细胞仪检测细胞凋亡的发生,并用荧光酶标仪测定caspase-3的活性.结果:100 μmol稬-1H2O2处理细胞24 h显著降低细胞的存活率;诱导细胞发生凋亡,凋亡率达48.O％;细胞内活性氧水平及caspase-3的活性显著升高;而线粒体膜电位却明显降低,红／绿荧光强度的比值由正常的5.97降低为0.41左右.而预先给予l、10或100 mg.L-1浓度的管花苷B处理细胞12 h,可显著提高细胞存活率;并可有效抑制DNA ladder的发生;流式细胞仪检测凋亡率分别降低到30.9％、18.3％和6.2％;激光共聚焦显微镜结果显示管花苷B可明显降低细胞内活性氧的水平;并可逐渐恢复线粒体的高能量状态;easpase-3的活性不断降低,并呈现了一定的剂量依赖性.结论:管花苷B能显著地抑制H2O2诱导的PCI2细胞凋亡,其神经细胞保护作用可能与其降低细胞内活性氧水平,维持线粒体膜电位的高能状态和抑制caspase-3的活性有关.     

NOD8对H2O2诱导的人肝细胞凋亡的影响

 目的:探讨NOD8对H2O2诱导的人肝细胞L02凋亡的影响。 方法: pEGFP-C2 及pEGFP-NOD8重组质粒经JetPRIME介导转染L02细胞;用H2O2诱导细胞凋亡。实验分为pEGFP-C2组、pEGFP-C2+H2O2组和pEGFP-NOD8+H2O2组。采用MTT法检测细胞活性,Western blotting检测细胞NOD8的蛋白表达,Hoechst 33342染色检测细胞凋亡情况,流式细胞术检测细胞凋亡率,比色法检测细胞caspase-3活性。 结果: 通过MTT检测不同浓度（0.2～2 mmol/L）H2O2刺激6 h后的细胞活性,确定1 mmol/L H2O2为诱导细胞凋亡的剂量。Western blotting检测结果显示,转染pEGFP-NOD8质粒的细胞NOD8蛋白表达明显增加。Hoechst 33342染色法观察发现,pEGFP-C2+H2O2组有较多细胞出现强蓝色荧光细胞核,细胞凋亡较多,而pEGFP-NOD8+H2O2组细胞凋亡明显减少。流式细胞术分析显示,pEGFP-C2+H2O2组的细胞凋亡率明显升高,pEGFP-NOD8+H2O2组的细胞凋亡率则显著下降。pEGFP-C2+H2O2组细胞的caspase-3活性明显升高,而pEGFP-NOD8+H2O2组细胞的caspase-3活性显著下降。 结论: NOD8可抑制H2O2诱导的L02细胞凋亡,其作用机制可能与NOD8抑制细胞的caspase-3活性有关。     

氧化应激介导糖原合成酶激酶-3β促进肝细胞凋亡

目的探讨糖原合成酶激酶-3β(GSK3β)在氧化应激诱导肝细胞凋亡中的作用。方法以人肝HL-7702细胞为研究对象,H2O2/抗霉素A诱导细胞产生氧化应激,建立肝细胞凋亡模型。SB216763为GSK3β特异性抑制剂,在H2O2/抗霉素A给药前2 h干预。采用钙黄绿素乙酰甲酯/碘化丙啶(PI)双染色观察细胞存活情况,采用annexinⅤ-FITC/PI联合流式细胞术检测细胞凋亡,同时对细胞培养上清进行乳酸脱氢酶(LDH)检测来评价细胞死亡程度;Western blot法检测p-GSK3β、GSK3β、caspase-3、cleaved caspase-3、c-Jun氨基末端激酶(JNK)和细胞色素C(Cyt C)蛋白的表达。结果 H2O2/抗霉素A诱导的氧化应激促进了GSK3β活性增加,抑制GSK3β活性缓解了氧化应激和由氧化应激引起的细胞凋亡。与氧化应激模型组相比,SB216763干预组中PI染色的细胞显著减少,流式细胞术检测细胞凋亡率降低,细胞培养上清中LDH显著降低,Western blot法结果显示干预组中cleaved caspase-3、JNK、Cyt C蛋白表达下降。结论 GSK3β是氧化应激诱导细胞凋亡通路中的一种重要信号分子,抑制其活性可减轻氧化应激而改善肝细胞凋亡。     

HBSP抑制缺氧复氧心肌H9C2细胞Omi/HtrA2胞内转位及细胞凋亡

目的探讨促红细胞生成素衍生肽(HBSP)对缺氧复氧心肌细胞Omi/Htr A2胞内转位及细胞凋亡的影响。方法将乳鼠心肌细胞(H9C2细胞)分为对照(Ctrl)组、缺氧复氧(H/R)组、HBSP组和EPO组。培养结束后MTS法检测细胞存活率,酶标仪检测细胞上清液中LDH释放率,Western blot检测细胞内cleaved caspase-3表达,TUNEL法检测心肌细胞凋亡;分离H9C2细胞胞质及线粒体,Western blot分别检测线粒体及细胞质Omi/Htr A2表达。结果与Ctrl组相比,H/R组细胞存活率下降(P0.05),LDH释放、cleaved caspase-3表达、心肌细胞凋亡及Omi/Htr A2胞内转位均明显上升(P0.05);与H/R组相比,EPO组的细胞存活率升高(P0.05),LDH释放降低、cleaved caspase-3表达减弱、心肌细胞凋亡减少、Omi/Htr A2线粒体转位明显减少(P0.05);随着HBSP浓度的增加,各组细胞存活率逐渐上升,LDH释放、cleaved caspase-3表达、心肌细胞凋亡及Omi/Htr A2胞内转位率逐渐下降(P0.05)。结论 HBSP具有与EPO类似的保护作用,可抑制经H/R诱导的心肌细胞凋亡,其机制可能是通过减少Omi/Htr A2蛋白的胞内转位,抑制caspases通路激活,进而发挥细胞保护作用。     

肝细胞生长因子减轻皮质神经元的缺氧/复氧损伤

目的研究肝细胞生长因子(HGF)对缺氧/复氧诱导大鼠皮质神经元凋亡的保护。方法分离培养SD大鼠皮质神经元,经HGF(15、30和60μg/L)预处理后经缺氧/复氧诱导凋亡,用MTT比色法检测细胞存活率;用Hoechst33258染色法和流式细胞仪检测神经元的凋亡;用比色法检测细胞乳酸脱氢酶(LDH)和caspase-3活性变化。结果与正常对照组相比,缺氧/复氧组神经元的细胞存活率显著下降,细胞凋亡率和caspase-3活性升高(均P<0.05);而HGF预处理12 h可显著逆转缺氧/复氧所致的细胞存活率降低、凋亡率增加、LDH活性上升、caspase-3活性升高的改变(均P<0.05),且这些效应与HGF剂量正相关。此外,HGF的抗凋亡效应可被PI3K/Akt通路抑制剂LY294002阻断。结论HGF减轻缺氧/复氧所诱导的神经元凋亡可能与其激活神经元的PI3K/Akt信号通路、减少caspase-3表达有关。     

瘦素抑制H_2O_2诱导的大鼠心肌细胞凋亡

目的:观察瘦素(leptin)对H2O2诱导的大鼠心肌细胞凋亡的影响并探讨其作用机制。方法:应用脱氧三磷酸尿苷缺口末端标记(TUNEL)法观察瘦素对H2O2诱导的大鼠心肌细胞H9c2凋亡的影响;应用Western blotting法观察瘦素、H2O2对caspase-3、胞外信号调控激酶(ERK)活性的影响。结果:(1)瘦素对H2O2诱导的H9c2细胞凋亡具有显著的抑制作用(与对照组比较P0.01),该作用可被ERK激酶抑制剂PD98059所阻断。(2)H2O2明显抑制ERK活性;而瘦素可激活ERK并部分阻断H2O2诱导的caspase-3激活。结论:瘦素对H2O2诱导的H9c2细胞凋亡具有抑制作用,其机制可能与其激活ERK信号途径有关。     

管花苷B对抗H2O2诱导的PC12细胞凋亡

目的：观察肉苁蓉提取物管花苷B对H2O2诱导的PC12细胞损伤的影响。方法：用MTT法检测细胞存活率，以激光共聚焦显微镜荧光染色法检测细胞内活性氧的产生和线粒体膜电位的变化，DNA琼脂糖凝胶电泳和流式细胞仪检测细胞凋亡的发生，并用荧光酶标仪测定caspase-3的活性。结果：100μmol&#183;L^-1H2O2处理细胞24h显著降低细胞的存活率；诱导细胞发生凋亡，凋亡率达48．0％；细胞内活性氧水平及caspase-3的活性显著升高；而线粒体膜电位却明显降低，红／绿荧光强度的比值由正常的5．97降低为0．41左右。而预先给予1、10或100mg&#183;L^-1浓度的管花苷B处理细胞12h，可显著提高细胞存活率；并可有效抑制DNA ladder的发生；流式细胞仪检测凋亡率分别降低到30．9％、18．3％和6．2％；激光共聚焦显微镜结果显示管花苷B可明显降低细胞内活性氧的水平；并可逐渐恢复线粒体的高能量状态；caspase-3的活性不断降低，并呈现了一定的剂量依赖性。结论：管花苷B能显著地抑制H2O2诱导的PC12细胞凋亡，其神经细胞保护作用可能与其降低细胞内活性氧水平，维持线粒体膜电位的高能状态和抑制caspase-3的活性有关。     

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.