硒酸酯多糖诱导白血病多药耐药细胞凋亡的作用机制

目的:观察硒酸酯多糖(Kappaselenocarrageenan,KSC)对K562ADM耐药细胞的诱导凋亡效应,并探讨其作用机制。方法:应用噻唑蓝(MTT)比色法、WrightGiemsa染色、DNA琼脂糖凝胶电泳和流式细胞术(Flowcytometry,FCM)观察K562ADM细胞凋亡;FCM测定K562ADM细胞Fas、P53和Bcl2蛋白表达水平;RTPCR检测Caspase3mRNA的表达。结果:50～500mg·L-1KSC抑制K562ADM细胞增殖,并诱导K562ADM细胞凋亡,细胞出现呈典型凋亡形态改变,DNA电泳可见DNA梯状条带(DNAladder);FCM分析出现亚G1期细胞群,S期细胞比例增高。Fas蛋白表达上调,Bcl2蛋白表达下调,Caspase3mRNA表达显著增强,但P53蛋白表达无明显改变。结论:KSC通过Fas依赖性Caspase3激活途径诱导K562ADM细胞凋亡。     

青蒿琥酯诱导人早幼粒白血病细胞HL60凋亡的线粒体机制

目的 :探讨青蒿琥酯 (artesunate ,Art)诱导人急性早幼粒白血病细胞HL6 0凋亡的线粒体机制。方法 :用Art处理HL6 0细胞 ,通过MTT比色法检测细胞增殖抑制的效果 ;透射电镜、DNA凝胶电泳技术观察细胞的凋亡 ;流式细胞术 (flowcytometry ,FCM)进行细胞周期分析和线粒体膜电位 (mitochon drialmembranepotential ,MMP)水平的检测 ;比色法检测半胱 天冬氨酸蛋白酶 3(cysteinecontainingas partate,Caspase 3)活性。结果 :Art呈浓度和时间依赖性的抑制HL6 0细胞的增殖 (P <0 .0 5 )。Art诱导后HL6 0细胞出现典型的凋亡形态学变化和生化改变 ,G2 M期细胞比例增高 ,S期减少 ,凋亡率上升 (P <0 .0 5 ) ,线粒体膜电位降低 (P <0 .0 5 ) ,Caspase 3活性增强 (P <0 .0 5 )。结论 :Art可能通过线粒体 半胱 天冬氨酸蛋白酶 3特定途径诱导HL6 0细胞凋亡。     

Cr(Ⅵ)诱导肝细胞凋亡与氧化损伤、p53和半胱氨酸天冬氨酸蛋白酶3表达的关系

目的探讨6价铬(Cr(Ⅵ))在不同暴露水平诱导细胞凋亡的途径有无差异。方法L-02肝细胞暴露于不同浓度Cr(Ⅵ)24h。用琼脂糖凝胶电泳和流式细胞术检测细胞凋亡;化学比色法检测SOD,CAT活性与GSH含量;RT-PCR检测p53,caspase3mRNA表达水平;免疫组化检测p53蛋白含量。结果Cr(Ⅵ)诱导L-02肝细胞凋亡率浓度依赖性增加(r=0.997),6~12μmol·L-1Cr(Ⅵ)处理组与对照组比差异有显著性(P<0.05);琼脂糖凝胶电泳表现DNA特征性梯形条带;SOD与CAT活性和GSH含量均下降,Cr(Ⅵ)处理组GSH含量与CAT活性水平明显低于对照组(P<0.05);3~9μmol·L-1Cr(Ⅵ)处理组细胞p53mRNA,caspase3mRNA,p53蛋白表达水平明显高于对照组(P<0.05)。细胞凋亡率与SOD活性、CAT活性呈负相关(r分别为-0.952和-0.885);p53mRNA表达与caspase3mRNA表达正相关(r=0.890);p53蛋白表达与GSH含量呈负相关(r=-0.929)。结论氧化损伤即细胞内抗氧化系统平衡的破坏在Cr(Ⅵ)诱导肝细胞凋亡中具有重要作用;在低浓度水平Cr(Ⅵ)诱导细胞凋亡具有p53和caspase3依赖性,但在较高Cr(Ⅵ)处理水平,不排除非p53及caspase3途径的存在。     

紫花牡荆素对肝癌细胞凋亡的影响及其机制研究

目的研究紫花牡荆素(casticin)诱导肝癌细胞凋亡的作用及其分子机制。方法体外培养人肝癌PLC/PRF/5和HepG2细胞系。通过碘化丙啶(PI)染色流式细胞术分析细胞凋亡率,ELISA法检测组蛋白/DNA碎片的含量,琼脂糖凝胶电泳观察DNA断裂条带,Western blotting检测细胞内DR4和DR5蛋白表达水平。结果 Casticin以浓度依赖方式增加PLC/PRF/5和HepG2细胞系sub-G1细胞的百分率和组蛋白/DNA片段的含量(P<0.05),casticin(30μmol·L-1)作用细胞24h后可出现DNA梯度条带;casticin可以浓度依赖性方式诱导PLC/PRF/5内DR5蛋白水平的表达,但对DR4蛋白表达无影响;DR5/Fc嵌合蛋白预处理能有效降低casticin诱导的细胞凋亡。结论 Casticin可以浓度依赖性方式诱导肝癌细胞凋亡,其机制可能与上调细胞的DR5蛋白表达有关。     

一种海洋来源的ETP类化合物HDN-1诱导K562细胞凋亡作用及机制初探

目的 探讨南极土壤来源真菌的次级代谢产物HDN-1对人慢性粒细胞白血病K562细胞的增殖抑制,诱导凋亡作用及其机制。方法 采用MTT法检测HDN-1对K562细胞的增殖抑制作用;DNA结合染料Hoechst 33342染色,Western Blotting以及DNA琼脂糖凝胶电泳检测细胞凋亡以及凋亡相关蛋白的变化。结果 HDN-1能显著抑制K562细胞的增殖,并呈剂量-时间依赖性;HDN-1作用后,细胞核形态发生明显变化并且出现了基因组DNA的梯状剪切条带;Caspase-9、8、3逐渐被激活,Caspase3的作用底物PARP被活化裂解出现89KD的剪切条带,并且Caspase蛋白的激活可被特异性抑制剂所阻断;抑凋亡蛋白Bcl-2,Mcl-1的表达降低,促凋亡蛋白Bax的表达量增高。另外,融合蛋白Bcr-Abl的表达被HDN-1抑制并呈剂量依赖性。结论 来源于南极土壤来源真菌的次级代谢产物HDN-1可能是通过抑制Bcr-Abl融合蛋白的表达进而诱导K562细胞凋亡,并且凋亡是通过Caspase依赖的线粒体途径和死亡受体途径共同介导发生的。     

三氧化二砷诱导K562/ADM细胞凋亡的作用机制研究

目的 :观察三氧化二砷 (As2 O3 )对K5 6 2 ADM细胞的诱导凋亡效应 ,探讨其作用机制。方法 :应用噻唑蓝 (MTT)比色法、Wright Giemsa染色、DNA琼脂糖凝胶电泳和流式细胞术 (FCM)观察K5 6 2 ADM细胞凋亡 ;FCM测定K5 6 2 ADM细胞Fas、Bcl 2、P5 3蛋白水平的变化 ;比色法检测Caspase3活性变化。结果 :As2 O3 可抑制K5 6 2 ADM细胞增殖 ;K5 6 2 ADM呈典型凋亡形态改变 ;DNA电泳可见梯状条带出现 ;FCM分析示亚G1期细胞比例增高 ,G2 M期阻滞 ;Fas、P5 3蛋白表达明显上调 ;Caspase3活性明显增强。结论 :As2 O3 可通过Fas依赖性Caspase3激活而诱导K5 6 2 ADM细胞凋亡。     

二乙酰二脱水卫矛醇诱导人白血病HL-60细胞凋亡与Bcl-2家族的关系

目的　探讨抗肿瘤药物二乙酰二脱水卫矛醇 (di acetyldianhydrogalactitol,DADAG )诱导肿瘤细胞凋亡机制。方法　透射电镜、DNA梯形条带、流式细胞术及Westernblot法分别检测凋亡和凋亡相关基因Bcl 2家族成员表达的动态变化。结果　透射电镜可见细胞异染色质固缩、边集及凋亡小体形成 ;DNA琼脂糖凝胶电泳呈典型的“DNAlad der” ;流式细胞仪DNA直方图上出现亚二倍体峰。DADAG4 μmol·L-1处理HL 6 0细胞 6h后 ,Bcl 2蛋白水平开始下降 ,Bad蛋白水平明显上调 ,相反 ,Bcl XL 蛋白水平呈时间依赖性地下降。结论　DADAG诱导白血病HL 6 0细胞凋亡 ,与Bcl 2、Bcl XL 蛋白水平下降 ,Bad蛋白水平明显上调有关     

Survivin在PC12细胞对抗化学性缺氧损伤中的作用

目的探讨存活素(survivin)在PC12细胞对抗氯化钴(CoCl2)诱导损伤中的作用。方法应用不同浓度的CoCl2处理PC12细胞不同时间,建立化学性缺氧诱导PC12细胞损伤的实验模型。应用CCK-8比色法检测细胞存活率;Western-blot法检测CoCl2诱导缺氧与survivin表达间的量效(200～1000μmol·L-1)和时效(0～48h)关系。结果CoCl2可明显抑制PC12细胞的存活率,且呈浓度和时间依赖性。应用不同浓度CoCl2处理PC12细胞24h,在200～600μmol·L-1浓度范围内,呈浓度依赖性地促进survivin表达,600μmol·L-1CoCl2诱导survivin表达达高峰,超过此浓度,则随着CoCl2浓度的增加,survivin表达逐渐下降,CoCl2浓度达1000μmol·L-1时,survivin基本不表达;应用600μmol·L-1CoCl2处理PC12细胞,在0～36h时间范围内,呈时间依赖性地促进PC12细胞survivin的表达,但随着处理时间的延长,survivin的表达逐渐下降;加入2μmol·L-1Hsp90抑制剂17-丙烯胺基-17-去甲氧基格尔德霉素(17-AAG),不仅可以降低600μmol·L-1 CoCl2诱导的survivin高表达,而且加重了600μmol·L-1 CoCl2对PC12细胞的损伤作用,使细胞存活率降低。结论survivin表达上调可能是PC12细胞对抗化学性缺氧损伤的内在防御机制之一。     

蛋白酶体抑制剂MG132诱导HepG2细胞凋亡及其机制研究

目的观察蛋白酶体抑制剂MG132对人肝癌细胞HepG2的致凋亡作用,并从泛素蛋白酶体途径(UPP)相关基因E1、E2和E3及天冬氨酸特异的半胱氨酸蛋白酶3(Caspase3)表达初步探讨MG132的致细胞凋亡机制。方法采用多个浓度(2、5、10μmol·L-1)的蛋白酶体抑制剂MG132处理HepG2细胞;流式细胞术检测细胞周期和细胞凋亡率,DNA琼脂糖凝胶电泳检测细胞凋亡;逆转录聚合酶链反应(RTPCR)检测UPP相关基因E1、E2、E3和凋亡相关基因Caspase3的转录水平;免疫细胞化学检测Caspase3蛋白表达。结果对照组HepG2细胞凋亡率低于5%,在2、5和10μmol·L-1MG132作用下,细胞凋亡率分别为42.9%、66.1%、72.8%,MG132诱导HepG2细胞凋亡具有量效关系;RTPCR检测发现细胞内UPP相关基因E1、E2、E3mRNA表达下降,而凋亡相关基因Caspase3mRNA表达上调;免疫组化检测Caspase3蛋白表达水平升高。结论蛋白酶体抑制剂MG132能够诱导HepG2细胞凋亡,其机制可能与MG132抑制UPP活性,使细胞内Caspase3蛋白降解减少,同时上调Caspase3基因转录,促进细胞凋亡。     

人参皂苷Rg1可能通过CDK4-pRB-E2F1通路减少神经元凋亡

目的　探讨人参皂苷Rg1是否通过CDK4 pRB E2F1通路对抗Aβ1～ 4 0 诱导的神经元凋亡。方法　用TUNEL染色、DNA琼脂糖凝胶电泳方法观察神经元凋亡形态学和生化改变 ;免疫印迹方法检测细胞周期相关的周期依赖性激酶4(Cyclindependentkinase 4,CDK4)、磷酸化的视网膜神经胶质瘤蛋白 (Retinoblastomaprotein ,pRB)水平 ;RT PCR技术检测E2F1mRNA表达水平 ;荧光分光光度计法检测凋亡效应分子Caspase 3活力的变化。结果　人参皂苷Rg1预处理可降低Aβ1～ 4 0 诱导的大鼠皮层神经元的凋亡率 ;凋亡细胞特有的DNA梯形条带消失 ;CDK4、磷酸化pRB水平降低 ;E2F1基因表达下调 ;Caspase 3活力下降。结论　人参皂苷Rg1可通过抑制CDK4 pRB E2F1信号传导通路及Caspase 3的激活 ,减少Aβ1～ 4 0 诱导的大鼠皮层神经元凋亡。     

Synthesis,Cytotoxicity and Antileishmanial Activity of Some N-(2-(indol-3-yl)ethyl)-7-chloroquinolin-4-amines

We report herein the condensation of 4,7-dichloroquinoline (1) with tryptamine (2) and D-tryptophan methyl ester (3) . Hydrolysis of the methyl ester adduct (5) yielded the free acid (6) . The compounds were evaluated in vitro for activity against four different species of Leishmania promastigote forms and for cytotoxic activity against Kb and Vero cells. Compound (5) showed good activity against the Leishmania species tested, while all three compounds displayed moderate activity in both Kb and Vero cells.     

Lung disease and PKCs

The lung offers a rich opportunity for development of therapeutic strategies focused on isozymes of protein kinase C (PKCs). PKCs are important in many cellular responses in the lung, and existing therapies for pulmonary disorders are inadequate. The lung poses unique challenges as it interfaces with air and blood, contains a pulmonary and systemic circulation, and consists of many cell types. Key structures are bronchial and pulmonary vessels, branching airways, and distal air sacs defined by alveolar walls containing capillaries and interstitial space. The cellular composition of each vessel, airway, and alveolar wall is heterogeneous. Injurious environmental stimuli signal through PKCs and cause a variety of disorders. Edema formation and pulmonary hypertension (PHTN) result from derangements in endothelial, smooth muscle (SM), and/or adventitial fibroblast cell phenotype. Asthma, chronic obstructive pulmonary disease (COPD), and lung cancer are characterized by distinctive pathological changes in airway epithelial, SM, and mucous-generating cells. Acute and chronic pneumonitis and fibrosis occur in the alveolar space and interstitium with type 2 pneumocytes and interstitial fibroblasts/myofibroblasts playing a prominent role. At each site, inflammatory, immune, and vascular progenitor cells contribute to the injury and repair process. Many strategies have been used to investigate PKCs in lung injury. Isolated organ preparations and whole animal studies are powerful approaches especially when genetically engineered mice are used. More analysis of PKC isozymes in normal and diseased human lung tissue and cells is needed to complement this work. Since opposing or counter-regulatory effects of selected PKCs in the same cell or tissue have been found, it may be desirable to target more than one PKC isozyme and potentially in different directions. Because multiple signaling pathways contribute to the key cellular responses important in lung biology, therapeutic strategies targeting PKCs may be more effective if combined with inhibitors of other pathways for additive or synergistic effect. Mechanisms that regulate PKC activity, including phosphorylation and interaction with isozyme-specific binding proteins, are also potential therapeutic targets. Key isotypes of PKC involved in lung pathophysiology are summarized and current and evolving therapeutic approaches to target them are identified.     

Gender-related symptoms and correlates of alcohol dependence among men and women with a lifetime diagnosis of alcohol use disorders

This study explored gender-related symptoms and correlates of alcohol dependence in a crosssectional study of 150 men and 150 women with a lifetime diagnosis of alcohol use disorders (AUD). Participants were recruited in equal numbers from treatment settings, correctional centres and the general community. Standardized measures were used to determine participants' use of substances, history of psychiatric disorders and psychosocial stress, their sensation seeking and family history of substance use and mental health disorders. Multivariate analyses were used to detect patterns of variables associated with gender and the lifetime severity of AUD. Men had a longer history of severe AUD than women. Women had similar levels of alcohol dependence and medical and psychological sequelae as men, despite 6 fewer years of AUD. More women than men had a history of severe psychosocial stress, severe dependence on other substances and antecedent mental health problems, especially mood and anxiety disorders. There were differences in family history of alcohol-related problems approximating same-gender aggregation. The severity of a lifetime AUD was predicted by its earlier age at onset and the occurrence of other disorders, especially anxiety, among both men and women. The limitations in the generalizability of these findings due to sample idiosyncrasies are discussed.     

Biochemical and molecular characterisation of cubozoan protein toxins

Class Cubozoa includes several species of box jellyfish that are harmful to humans. The venoms of box jellyfish are stored and discharged by nematocysts and contain a variety of bioactive proteins that are cytolytic, cytotoxic, inflammatory or lethal. Although cubozoan venoms generally share similar biological activities, the diverse range and severity of effects caused by different species indicate that their venoms vary in protein composition, activity and potency. To date, few individual venom proteins have been thoroughly characterised, however, accumulating evidence suggests that cubozoan jellyfish produce at least one group of homologous bioactive proteins that are labile, basic, haemolytic and similar in molecular mass (42-46 kDa). The novel box jellyfish toxins are also potentially lethal and the cause of cutaneous pain, inflammation and necrosis, similar to that observed in envenomed humans. Secondary structure analysis and remote protein homology predictions suggest that the box jellyfish toxins may act as α-pore-forming toxins. However, more research is required to elucidate their structures and investigate their mechanism(s) of action. The biological, biochemical and molecular characteristics of cubozoan venoms and their bioactive protein components are reviewed, with particular focus on cubozoan cytolysins and the newly emerging family of box jellyfish toxins.     

Nachweis einiger Heilmittel in der Kniegelenksynovialflüssigkeit

Zusammenfassung Mittels Gaschromatographie und Dünschichtchromatographie wiesen die Autoren 11 Substanzen nach, welche durch Injektion oder nach Verabreichung per os in die Kniegelenksynovialflüssigkeit eindrangen. In ihrer Aufstellung konnten sie eine direkte Beziehung zwischen Struktur sowie chemischphysikalischen Eigenschaften der Substanz und ihrer Fähigkeit, aus dem Blut in die Kniegelenksynovialflüssigkeit einzudringen, nicht nachweisen, außer der Tatsache, daß Substanzen mit starker Affinität zu Eiweißstoffen erst in höheren Dosen nachweisbar waren.     

Dose tolerability of chronically inhaled voriconazole solution in rodents

Invasive pulmonary aspergillosis (IPA) is a fungal disease of the lung associated with high mortality rates in immunosuppressed patients despite treatment. Targeted drug delivery of aqueous voriconazole solutions has been shown in previous studies to produce high tissue and plasma drug concentrations as well as improved survival in a murine model of IPA. In the present study, rats were exposed to 20 min nebulizations of normal saline (control group) or aerosolized aqueous solutions of voriconazole at 15.625 mg (low dose group) or 31.25 mg (high dose group). Peak voriconazole concentrations in rat lung tissue and plasma after 3 days of twice daily dosing in the high dose group were 0.85 ± 0.63 μg/g wet lung weight and 0.58 ± 0.30 μg/mL, with low dose group lung and plasma concentrations of 0.38 ± 0.01 μg/g wet lung weight and 0.09 ± 0.06 μg/mL, respectively. Trough plasma concentrations were low but demonstrated some drug accumulation over 21 days of inhaled voriconazole administered twice daily. Following multiple inhaled doses, statistically significant but clinically irrelevant abnormalities in laboratory values were observed. Histopathology also revealed an increase in the number of alveolar macrophages but without inflammation or ulceration of the airway, interstitial changes, or edema. Inhaled voriconazole was well tolerated in a rat model of drug inhalation.