三氧化二砷对慢性髓系白血病细胞周期及Survivin表达的影响

目的研究三氧化二砷(As2O3)对慢性髓系白血病细胞(K562)的作用特点及其机制。方法不同浓度As2O3作用于K562细胞后，四唑盐(MTT)比色法分析细胞增殖；流式细胞术检测细胞周期分布、细胞凋亡及Survivin抗原表达；RT-PCR检测Survivin mRNA的表达。结果2—10μmoL／L的As2O3能有效抑制K562细胞增殖，但未能诱导明显的细胞凋亡。周期分析显示，G2／M期细胞比例显著增多；同时G2／M细胞周期依赖性表达的Survivin mRNA和蛋白表达增加。结论As2O3明显抑制K562细胞的生长，其机制是诱导G2／M期细胞周期停滞。Survivin表达上调可能是K562细胞对As2O3诱导凋亡抵抗的机制之一。     

三氧化二砷对慢性髓系白血病细胞周期的影响

目的：探讨三氧化二砷(As2O3)对人类慢性髓系白血病细胞系(K562)细胞周期及其凋亡的影响。方法：将K562细胞与不同浓度的As2O3共同孵育，在不同时间点应用MTT方法检测K562细胞存活率，并用流式细胞仪检测As2O3的致凋亡作用，同时对As2O3作用后的K562细胞进行细胞周期分析及时相性周期蛋白表达检测。结果：As2O3明显抑制K562细胞增殖并表现为时间和剂量依赖性；在2．0--10．0μmol/L梯度浓度As2O3的作用下，细胞于12—24h时段内无明显凋亡现象，但K562细胞明显阻滞于G2／M期时相，同时周期调控蛋白cyclinE表达下调，cyclinB1表达基本不变。结论：As2O3可抑制K562细胞增殖，但其抑制机制不在于通过诱导凋亡，而依靠诱发K562细胞的G2／M期阻滞。     

硼替佐米联合二硫化二砷诱导慢性粒细胞白血病细胞凋亡机制研究

目的:探讨硼替佐米(bortezomib)与二硫化二砷(As2S2)对慢性粒细胞白血病(chronic myeloid leukemia,CML)细胞株K562和对伊马替尼耐药的同源细胞株K562R的增殖抑制、诱导凋亡及其作用机制。方法:应用四甲基偶氮唑盐比色法(MTT)增殖抑制实验、瑞氏染色细胞形态观察法、膜联蛋白Ⅴ(Annexin Ⅴ)流式细胞仪检测硼替佐米和As2S2单独或联合作用于CML细胞株的增殖抑制和凋亡情况;蛋白免疫印迹法(Western blot)检测药物对Bcr-Abl蛋白以及凋亡相关蛋白的影响;反转录PCR(RT-PCR)检测Bcr-Abl mRNA的表达。结果:硼替佐米联合As2S2能有效抑制CML细胞增殖,6nmol/L硼替佐米和3μmol/L As2S2联合作用于K562细胞48h,细胞生长抑制率达(76.4±3.9)%以上,坏死和凋亡细胞比例达54.0%,与单药作用(硼替佐米13.9%;As2S2 8.2%)相比,差异有统计学意义(P<0.05)。药物联合作用通过线粒体途径协同诱导CML细胞凋亡,并使K562和K562R细胞的Bcr-Abl蛋白表达和蛋白磷酸化水平下降。结论:硼替佐米联合As2S2有效抑制K562细胞增殖和诱导凋亡,提高浓度后对K562R细胞也产生类似的作用,有增殖抑制和诱导凋亡作用,两者联合应用可能具有克服伊马替尼耐药的作用。     

亚砷酸诱导肝癌细胞株HepG-2细胞凋亡的实验研究

目的探讨亚砷酸(As2O3)对肝癌HepG-2细胞的增殖抑制和诱导凋亡的作用.方法应用细胞计数、FITC-TUNEL染色后荧光摄象及流式细胞仪技术探讨亚砷酸对肝癌HepG-2细胞的增殖抑制及诱导凋亡作用.结果亚砷酸能明显抑制肝癌HepG-2细胞的生长,5μmol/LAs2O3组抑制程度明显大干1μmol/L As2O3组(P＜0.01),二者与不加药阴性对照组相比均有显著性差异(P＜0.01),亚砷酸对肝癌细胞的生长抑制具有时间依赖性,5μmol/L As2O3组已表现出细胞毒作用;1μmol/L As2O3组作用d 3开始出现凋亡细胞,FITC-TUNEL染色后荧光摄象可见典型的凋亡细胞.流式细胞仪可观察到凋亡细胞具有时间依赖性.结论亚砷酸能够抑制肝癌HepG-2细胞的增殖,且能诱导肝癌细胞的凋亡,具有治疗肝癌的潜在价值.     

淫羊藿提取物IC163对K562白血病细胞增殖抑制和凋亡诱导效应观察

目的:观察淫羊藿提取物IC163对K562白血病细胞是否具有增殖抑制和诱导凋亡作用。方法:用MTT实验观察不同浓度的IC163对K562细胞增殖抑制率的影响,用Hochest33258染色法和Annexin V-FITC和PI染色法检测凋亡细胞的百分率,用Western印迹检测药物干预下的K562细胞Caspase-3蛋白表达。结果:IC163以浓度依赖性方式有效地抑制K562细胞增殖,其抑制K562细胞增殖的IC50为18.1μmol/L;IC163以浓度依赖性方式诱导K562细胞凋亡并伴有Caspase-3蛋白表达上调和裂解激活。结论:IC163能够以浓度依赖性方式有效地抑制K562细胞增殖并诱导K562细胞凋亡,其诱导K562细胞的机制可能与Caspase-3凋亡信号启动有关。     

As2O3对肾癌细胞增殖、凋亡的影响及意义

目的观察As2O3对肾癌细胞株786-0增殖、凋亡及细胞内X染色体连锁凋亡抑制蛋白（XIAP）表达的影响,探讨其意义。方法取对数生长期786-0,分别经0．5、1．0、2．0μmol／L的As2O3处理后,采用MTT比色法检测细胞生长情况,流式细胞仪测算细胞凋亡率,蛋白质印迹法及RT—PCR法检测细胞中的XIAP及其mRNA。结果0．5、1．0、2．0μmol／LAs2O3均可抑制786-0增殖。随着作用时间的延长,细胞生长抑制率升高（P均〈0．01）;随着As2O3浓度的增加,细胞凋亡率升高（P均〈0．01）、细胞内XIAP及其mRNA表达明显下调（P均〈0．05）。结论As2O3可抑制786-0增殖,并诱导其凋亡。这一作用与As2O3抑制786-0中XIAP及其mRNA表达有关。     

伊马替尼联合槲皮素对K562细胞增殖和凋亡协同作用的机制研究

目的:探讨槲皮素联合伊马替尼对K562细胞增殖的影响以及诱导凋亡的机制。方法:将不同浓度的槲皮素、伊马替尼单药和联合用药作用于K562细胞,绘制协同曲线。将0.25μmol/L伊马替尼与25μmol/L的槲皮素联合作用于K562细胞,通过细胞计数检测细胞增殖,流式细胞术检测细胞周期、线粒体跨膜电位的变化,蛋白质印迹法检测相关蛋白的表达。结果:0.25μmol/L伊马替尼联合25μmol/L槲皮素对K562细胞有明显的协同抑制生长和诱导凋亡作用。两药联合处理能降低K562细胞线粒体跨膜电位,使胱天蛋白酶(caspase)9和胱天蛋白酶3发生剪切,并明显下调B细胞淋巴瘤(Bcl)2样蛋白1(Bcl-xl)和髓样细胞白血病-1(Mcl-1)蛋白的表达。结论:伊马替尼与槲皮素协同抑制K562细胞生长并诱导细胞凋亡,其机制主要通过下调Bcl-xl以及Mcl-1蛋白的表达,从而激活线粒体凋亡途径来实现的。     

青蒿琥酯对白血病细胞增殖和凋亡的影响

目的研究青蒿琥酯对髓系白血病细胞株K562的增殖抑制和凋亡作用,分析其抗肿瘤机制。方法体外培养K562细胞应用不同浓度的青蒿琥酯处理细胞,台盼蓝染色分析细胞活力,WST-1还原法检测细胞增殖,流式细胞术分析细胞凋亡和周期,采用免疫印迹技术分析Bax和Bcl-2表达。结果 K562细胞活力随着药物浓度的增加而下降,而FTY720对细胞增殖的抑制作用随药物浓度增加而增加。药物作用72 h,K562细胞的IC50值为95μmol/L;与对照组比较,50μmol/L和100μmol/L青蒿琥酯处理48 h导致S期细胞减少,G_2M期细胞增加;当浓度达到200μmol/L后,G_2M期细胞减少,但死亡细胞增加到39.65%。双染和流式细胞术分析发现,100μmol/L青蒿琥酯作用24 h后,早期凋亡细胞百分率增加;与对照组比较青蒿琥酯导致细胞内Bax表达增加。结论青蒿琥酯可通过诱导细胞凋亡和抑制细胞增殖而发挥抗肿瘤作用,其线粒体途径可能参与细胞凋亡过程。     

氧化砷诱导胰腺癌细胞凋亡的实验研究

目的：观察As2O3对胰腺癌细胞株体外生长和裸鼠腹腔种植腹水生成的影响及其作用机制。方法：0．125－2μmol/L的As2O3与胰腺癌细胞株SW－8902共同孵育，观察不同浓度、不同作用时间对胰腺癌细胞生长的抑制作用、作用后胰腺癌细胞的凋亡特征及Fas Fas-L表达的变化。80只BALB／C－nu/nu裸鼠腹腔内接种胰腺癌细胞株SW－8902，并随机分为4组，然后分别腹腔内注射生理盐水及不同剂量的As2O3，观察各组裸鼠的生存时间。结果：1－2μmol/L的As2O3作用后，胰腺癌细胞呈典型的凋亡特征性改变，流式细胞仪检测在G1期前出现亚二倍体凋亡峰，DNA电泳呈现特征性Ladder, 细胞核内可见染色质浓缩、碎裂和边聚。As2O3也可显抑制荷胰腺癌裸鼠腹水的生成，延长生存期（P＜0．01）,Fas、Fas-L表达在As2O3作用后2d上升，3d达最高，以后表达量下降。结论：As2O3诱导胰腺癌细胞凋亡，抑制裸鼠胰腺癌腹腔种植及腹水的生成，延长生存期。Fas、Fas-L表达上调是As2O3诱导肿瘤细胞凋亡的途径之一。     

三氧化二砷体外抑制人宫颈癌Hela细胞生长机制的研究

采用MTT法观察三氧化二砷（AS2O3）对人宫颈癌Hela细胞生长抑制作用;膜联蛋白V（Annexin V）-异硫氰酸荧光素（FITC）＋碘化丙碇（PI）双参数流式细胞术（FCM）检测细胞凋亡情况,FCM测定细胞周期及增殖细胞核抗原（PCNA）表达。结果 AS2O3显著抑制Hela细胞生长增殖,且剂量—效应关系显著（r=0.98,P〈0.01）,其48 h的IC50值为5.59μmol/L。Hela细胞经AS2O3处理后可发生凋亡。AS2O3显著下调PCNA表达且使细胞周期阻滞于G2/M期（P〈0.01）。认为AS2O3在体外可显著抑制人宫预癌Hela细胞生长并诱导凋亡,其机制可能与阻滞细胞周期及降低PCNA蛋白表达有关。     

Analysis of density and epitopes of D antigen on the surface of erythrocytes from DEL phenotypic individuals carrying the RHD1227A allele

Background

The characteristics of the D antigen are important as they influence the immunogenicity of D variant cells. Several studies on antigenic sites have been reported in normal D positive, weak D and partial D cases, including a comprehensive analysis of DEL types in Caucasians. The aim of this study was to assess D antigen density and epitopes on the erythrocyte surface of Asian type DEL phenotypic individuals carrying the RHD1227A allele in the Chinese population.

Materials and methods

A total of 154 DEL phenotypic individuals carrying the RHD1227A allele were identified through adsorption and elution tests and polymerase chain reaction analysis with sequence-specific primers in the Chinese population. D antigen density on the erythrocyte surface of these individuals was detected using a flow cytometric method. An erythrocyte sample with known D antigen density was used as a standard. Blood samples from D-negative and D-positive individuals were used as controls. In addition, D antigen epitopes on the erythrocyte surface of DEL individuals carrying the RHD1227A allele were investigated with 18 monoclonal anti-D antibodies specific for different D antigen epitopes.

Results

The means of the median fluorescence intensity of D antigen on the erythrocyte membrane surface of D-negative, D-positive and DEL individuals were 2.14±0.25, 193.61±11.43 and 2.45±0.82, respectively. The DEL samples were estimated to have approximately 22 D antigens per cell. The samples from all 154 DEL individuals reacted positively with 18 monoclonal anti-D antibodies specific for different D antigen epitopes.

Discussion

In this study, D antigen density on the erythrocyte surface of DEL individuals carrying the RHD1227A allele was extremely low, there being only very few antigenic molecules per cell, but the D antigen epitopes were grossly complete.     

风湿性心脏炎HLA—DR分子表达量的改变

目的　了解风湿性心脏炎 (rheumaticcarditis,RC)外周血淋巴细胞表面HLA DR分子的表达量 ,以探讨HLA DR分子在外周血淋巴细胞表面的表达量在RC发病机制中的意义 ,为RC的诊断和防治提供新的途径。方法　选取门诊及住院确诊风湿性心脏炎者 33例 ,单纯风湿性关节炎 2 1例 ,风心病静止期组 36例 ,链球菌感染后状态组 16例 ,正常对照组 43例。分别予A组 β型溶血性链球菌 (GAS)膜抗原及GM CSF刺激其淋巴细胞 ,另设空白对照 ,参照RandallEllisMorris的cell ELISA方法 ,检测淋巴细胞表面HLA DR的表达情况 ,以OD/cell为单位表示。HLA DR表达量以均数±标数差表示 ,用方差分析其差异 ,P <0 0 5为统计学显著标准。结果　①心脏炎组HLA DR分子表达量明显高于其他各组 (P <0 0 5 ) ,而关节炎组较非活动期组亦有明显增高 (P<0 0 5 )。②加入膜抗原及GM CSF后各样本的HLA DR的表达量均有增加。加入膜抗原刺激者 ,心脏炎组的增加量较其他各组明显大 ,而关节炎组和风心病静止期组增加量与正常对照组及链球菌感染后状态组相比亦有显著差别 (P <0 0 5 )。结论　①对HLA DR分子表达量的检测有助于对风湿热不同型别及不同病期的诊断和监测风湿性心脏炎的活动情况。②膜抗原与HLA DR分子对风湿性心脏炎发病及发展起了重要     

Mapping the HLA-DO/HLA-DM complex by FRET and mutagenesis

HLA-DO (DO) is a nonclassic class II heterodimer that inhibits the action of the class II peptide exchange catalyst, HLA-DM (DM), and influences DM localization within late endosomes and exosomes. In addition, DM acts as a chaperone for DO and is required for its egress from the endoplasmic reticulum (ER). These reciprocal functions are based on direct DO/DM binding, but the topology of DO/DM complexes is not known, in part, because of technical limitations stemming from DO instability. We generated two variants of recombinant soluble DO with increased stability [zippered DOαP11A (szDOv) and chimeric sDO-Fc] and confirmed their conformational integrity and ability to inhibit DM. Notably, we found that our constructs, as well as wild-type sDO, are inhibitory in the full pH range where DM is active (4.7 to ～6.0). To probe the nature of DO/DM complexes, we used intermolecular fluorescence resonance energy transfer (FRET) and mutagenesis and identified a lateral surface spanning the α1 and α2 domains of szDO as the apparent binding site for sDM. We also analyzed several sDM mutants for binding to szDOv and susceptibility to DO inhibition. Results of these assays identified a region of DM important for interaction with DO. Collectively, our data define a putative binding surface and an overall orientation of the szDOv/sDM complex and have implications for the mechanism of DO inhibition of DM.     

Prostate-specific antigen kallikrein and the heart

Currently, there is growing interest regarding prostate-specific antigen (PSA) and the cardiovascular system. Increased PSA serum levels have been reported after prolonged cardiopulmonary resuscitation, cardiac surgery, extracorporeal cardiopulmonary bypass, acute myocardial infarction (AMI) and coronary artery stenting. The possible role of PSA in cardiac events has been questioned due to the finding of PSA decrease during AMI and by the correlation of variation in PSA levels with coronary lesions and occurrence of major adverse cardiac events. Complexed PSA forms and uncomplexed PSA forms are observed in the bloodstream but the increasing formation of irreversible bound PSA seems to be a crucial finding during AMI. Large studies need to be carried out to confirm these preliminary results and to elucidate unclear aspects. These findings present many potential directions for future research including the role of uncomplexed forms of PSA, the possible distribution of PSA in the heart, the relative expression levels in heart disease states, the mode of expression regulation and other potential specific substrates. The journey of PSA investigation could be longer than initially expected.     

布鲁氏菌病与人类白细胞抗原相关关系的初步探讨

本文报道对51名在工作中因接触带菌羊毛而感染发病的布鲁氏菌病患者,15名同厂健康职工及60名厂外健康者进行了人类白细胞抗原(HLA)分布频率的比较研究。结果表明:病例组与同厂对照组间差异无显著性;病例组与厂外组对照比较可见 A9、A30抗原频率增高。经分析,本文认为遗传性因素对布鲁氏菌病易感性的影响较弱。     

日本血吸虫童虫细胞与非细胞型免疫原在小鼠免疫部位滞留的动态观察

目的观察日本血吸虫(Schistosoma japonicum,Sj)童虫细胞型免疫原和童虫细胞碎片免疫原免疫小鼠后在免疫部位滞留的动态变化。方法昆明鼠实验组A和B经大腿肌肉分别注射107日本血吸虫原代童虫细胞(pJCs)和相当剂量的童虫细胞碎片(JCFs),对照组C注射PBS。分别在注射后1、3、6、9和12 d,各组随机处理4或2只小鼠,从注射部位定量获取肌肉组织,用Western blot法和免疫组化法观察、比较两种类型抗原滞留的动态变化;用组织病理学方法观察发生的炎症反应。结果Western blot显示,A组在注射后各时间点均检测到分子质量单位为75 ku的特异性抗原条带,B组在注射后第6 d该条带即消失,C组未检出该条带。免疫组化检测结果与Western blot相符。组织病理学检查结果显示,A组在免疫后第12 d仍可见肌细胞间隙内大量炎性细胞浸润,B组在免疫后第12 d炎性细胞基本消失,C组未见炎性反应。结论血吸虫童虫细胞型免疫原较童虫细胞碎片免疫原在免疫部位可滞留更长时间,引起更持久的炎性反应,这可能是其诱生高保护性抗血吸虫病免疫的重要机制之一。     

中国汉族人群类风湿性关节炎与HLA-DQ基因多态性和HLA-DR-DQ连锁性的关系

目的 评价中国汉族人群类风湿性关节炎(RA)与HLA-DQ基因多态性和HLA-DR-DQ连锁性的关联情况.方法 以RA组和对照组(正常人)各HLA-DQ、HLA-DR-DQ等位基因频数分布的OR值为统计量.全面检索已发表的有关中国汉族人群RA和HLA-DQ的文献,应用Me-ta分析对基因分型研究结果进行汇总分析.结果 四项研究共入选348例RA患者和162名正常者进行对照,经Meta分析中国汉族人群RA的易感基因有HLA-DQA1*0301(OR=1.820,P=0.01)、DQB1*0401(OR=4.807,P＜0.01).DR4.DQA1*0301(OR=8.437,P=0.024)、DR4-DQB1*0401(OR=3.215,P＜0.01)在中国汉族人群RA中有连锁性并且是RA的危险基因型.HLA.DOA1*0102(OR=0.352,P=0.040)、DQB1*0602(OR=0.404,P=0.01)、DQB1*0604(OR=0,P＜0.05)是中国汉族人群RA的保护基因.结论 DQA1*0301、DQB1*0401是中国汉族人群RA的易感基因,其中DR4-DQA1*0301和DQB1*0401与DR4相连锁是RA的危险基因型,DQA1*0102、DQB1*0602和DQB1*0604是中国汉族人群RA的保护基因.     

二倍体型与三倍体型卫氏并殖吸虫虫体抗原与宿主抗原的观察

用间接免疫荧光抗体法,对鼠体内两种类型卫氏并殖吸虫虫体抗原与宿主抗原做了检测。结果显示两类型童虫与感染同源卫氏并殖吸虫小鼠血清反应后,虫体抗原存在于体表皮层、肠管上皮细胞上;两类型童虫与兔抗鼠红细胞抗体血清反应后,主要在表皮层看到宿主抗原,荧光强度比虫体抗原所示为弱;三倍体型童虫的宿主抗原荧光反应有52.6～60％呈(++),而二倍体型大部只是(+)反应;这种荧光强度的差别在非免疫状态所获的童虫切片上也被看到。     

血清CA19—9、CEA、CA125联合检测诊断食管癌的价值

目的探讨多抗原联合检测诊断食管癌的价值。方法应用全自动化学免疫分析检测254例食管癌患者、40例食管炎患者和100例健康体检者血清CA19-9、CEA、CA125的表达。结果三项指标联合检测诊断食管癌的灵敏度和特异度均明显高于单独检测及任两项联合检测（P均〈0．01）。结论三项指标联合检测可提高诊断食管癌的灵敏度和特异度,有利于早期诊断食管癌。     

Analysis of cytoplasmic and surface antigens in childhood T-cell acute lymphoblastic leukaemias: clinical relevance of cytoplasmic TCRβ chain expression

SUMMARY. Expression of surface and cytoplasmic antigens on the blasts from 42 cases of childhood T-cell acute lymphoblastic leukaemia (T-ALL) were analysed. All with childhood T-ALL, except for one case expressing cytoplasmic TCR δ chain, were classified on the basis of differential expression of cytoplasmic CD3 (cCD3), TCRβ chain (cTCR β) and surface CD3 (sCD3) into the following three groups: group I (cCD3+, cTCRβ-, sCD3-), eight cases (19.5%); group II (cCD3+, cTCRβ+, sCD3-), 23 cases (56.1%); group III (cCD3+, cTCRβ+, sCD3+), 10 cases (24.4%). Each group defines the stepwise maturational stage of the CD3/TCR complex along the intrathymic T-cell differentiation. Group I had the lowest initial WBC count among the three groups ( P < 0.05) and showed significantly ( P < 0.05) a higher event-free survival (0.75) than those of group II (0.33). There was no significant difference in both the initial WBC count and the event-free survival between groups II and III. Thus, the absence of cTCRβ in sCD3-negative T-ALL appears to be a good prognostic factor, suggesting that this classification provides a useful tool to predict the prognosis of childhood T-ALL. This is the first report, to our knowledge, studying the relationship between the expression of cytoplasmic CD3/TCR antigens and the clinical features in T-ALL.