天花粉蛋白抑制HeLa细胞生长及诱导细胞凋亡之机制探讨

目的研究天花粉蛋白（TCS）对人宫颈癌HeLa细胞生长抑制作用及其机制,探讨HeLa细胞凋亡发生的分子机制.方法采用CCK-8的方法,检测TCS对HeLa细胞的抑制作用;流式细胞仪检测TCS对细胞周期的影响;电子显微镜和Annexin法观察TCS对HeLa细胞诱导凋亡的作用;应用Western blotting观察HeLa细胞caspase-3酶原的变化;用caspases活性检测试剂盒,检测caspase-3,8,9的活性.结果1.TCS对HeLa细胞的生长具有明显的抑制作用;2.TCS可以将HeLa细胞阻滞于S期;3.TCS诱导HeLa细胞发生凋亡,HeLa细胞出现典型的凋亡特征--染色质浓缩和凋亡小体,且凋亡率具有时间依赖性;4.caspase-3酶原表达的降低呈现时间和浓度依赖性,而caspase-3活性随时间依赖性的增高,提示在TCS诱导HeLa细胞凋亡的过程中caspase-3被激活;5.caspase-8,9的活性增高,有时间依赖性,且具有时间顺序.100 mg/L TCS处理24h后,caspase-8早于caspase-3被激活,提示活化的caspase-8可能对caspase-3起到激活作用.结论TCS对HeLa细胞的抑制作用是通过将HeLa细胞阻滞于S期和诱导细胞凋亡来实现的.caspase-3的激活是介导TCS诱导HeLa细胞发生凋亡的通路,同时,活化的caspase-8,9可能参与了caspase-3的激活和凋亡的诱导.     

Survivin 抑制剂YM155 对甲状腺癌B-CPAP 细胞凋亡的影响及机制研究

目的:探讨人生存素(Survivin)抑制剂YM155{4,9-二氢-1-(2-甲氧基乙基)-2-甲基-4,9-二氧代鄄3-(2-吡嗪甲基)-1H-萘并[2,3-d]咪唑鎓溴化物}对甲状腺癌B-CPAP 细胞活力和凋亡的影响以及凋亡相关酶半胱氨酸蛋白酶-3(Cysteinyl aspartate specific proteinase-3,Caspase-3),半胱氨酸蛋白酶鄄8(Cysteinyl aspartate specific proteinase-8,Caspase-8)和半胱氨酸蛋白酶-9(Cysteinyl aspartate specific proteinase-9,Caspase-9)表达的变化,探讨其诱导B-CPAP 细胞凋亡的可能机制。方法:体外培养B-CPAP 细胞,分别以0、0.5、1、2、4、8 nmol/ L 浓度的YM155 进行处理24 、48 和72 h,采用CCK-8 检测试剂盒检测YM155对B-CPAP 细胞活力的抑制作用;B-CPAP 细胞随机分为4 组:分别以0、1、2 nmol/ L YM155 和5 μmol/ L 顺铂(Cisplatin,阳性对照组)处理24 h。分别采用TUNEL 染色和流式细胞仪AnnexinV-FITC/ PI 法检测凋亡的情况;采用RT-PCR 检测Survivin mRNA 的表达;采用比色法和Western blot 检测Survivin 和Caspase-3、Caspase-8、Caspase-9 的活性和表达。结果:与0 nmol/ L 组比较,YM155 对B-CPAP 细胞活力具有明显抑制作用,并诱导B-CPAP 细胞凋亡(P<0.05 或P<0.01);与阴性对照组比较,YM155 显著降低B-CPAP 细胞Survivin 的表达,并上调Caspase-3、Caspase-8、Caspase-9 的表达(P<0.05 或P<0.01)。结论:YM155 对B-CPAP 细胞活力具有抑制作用,诱导其凋亡,其机制可能与上调Caspase-3、Caspase-8 和Caspase-9 表达有关。     

荧光内参在校正Caspase-3/7活性检测偏差中的应用

目的 改进Caspase-3/7活性检测方法.方法 在顺铂诱导的HeLa细胞凋亡模型中,分别利用传统的和改良的实验方法检测Caspase-3/7的活性变化,并与Western印迹实验结果进行方法学比较.结果 改良的实验方法显示不同剂量顺铂诱导下,HeLa细胞Caspase-3/7活性有剂量依赖性增高,与Western印迹实验结果相一致,但传统实验方法显示HeLa细胞Caspase-3/7活性呈现先增高后降低的趋势.结论 由于参测细胞数不同,导致这种Caspase-3/7活性检测方法不能真实反映细胞凋亡程度.本研究成功建立了一种新的改良型Caspase-3/7活性检测方式,这种检测方法可以排除不同凋亡诱导方式诱导细胞凋亡时所产生的因参测细胞数不同所造成的Caspase-3/7活性检测的误差.     

创伤弧菌溶细胞素融合蛋白细胞毒活性相关分子机制研究

目的 研究创伤弧菌溶细胞素融合蛋白(rVVC)诱导人ECV304细胞(人脐静脉内皮细胞)凋亡过程中Caspase-3,-8,-9活性变化.方法 应用MTT法、Hochest33342/PI荧光双染、流式细胞术及DNA琼脂糖凝胶电泳分析rVVC对人ECV304细胞诱导凋亡的影响;比色法测定rVVC诱导人ECV304凋亡过程中Caspase-3,-8,-9活性变化.结果 MTT结果显示rVVC具有降低人ECV304细胞的存活率活性;浓度为2.0溶血单位(HU)/ml的rVVC作用人ECV304 12 h后,其诱导凋亡的活性高于对照组和浓度为0.5 HU/ml的 rVVC处理组,具有剂量依赖性;浓度为2.0 HU/ml rVVC处理组加40 μmol/L Caspase全酶抑制剂(Z-VAD-FMK)后凋亡率较2.0 HU/ml rVVC处理组有一定程度降低.浓度为2.0 HU/ml rVVC处理人ECV304细胞0.5 h后Caspase-3活性开始增高,于3 h达高峰,与对照组比较差异有统计学意义(P<0.01),Caspase-8,-9活性无明显变化.结论 rVVC对人ECV304具有凋亡诱导的生物学活性,Caspase-3可能与活性rVVC诱导的人ECV304凋亡有关.     

松针油诱导宫颈癌Hela细胞凋亡及其作用机制研究

目的 研究松针油对人宫颈癌Hela细胞生长的抑制作用，探讨Hela细胞发生凋亡的分子机制。方法 采用水蒸气蒸馏法提取松针油，气相色谱一质谱联用仪分析成分。MTT法测定松针油对Hela细胞增殖的影响。流式细胞仪和Hochest 33258检测药物作用前后细胞凋亡的情况。应用Western blotting观察Hela细胞caspase-3酶原的变化。用caspase活性检测试剂盒检测caspase-3，8，9的活性。结果 MTT结果显示松针油对Hela细胞生长有明显的抑制作用，呈显著的时间和浓度依赖性。流式细胞仪分析显示松针油诱导Hela细胞发生凋亡，细胞表现出典型的凋亡特征，并且可见凋亡小体，且凋亡率具有时间依赖性。松针油诱导Hela细胞凋亡过程中caspase．3被激活。Caspase-8，9活性增高，又有时间依赖性，且有时间顺序。结论 松针油对Hela细胞的生长有明显的抑制作用，其作用机制是松针油可诱导Hela细胞发生凋亡。在诱导细胞凋亡的过程中，caspase-3的激活是介导Hela细胞发生凋亡的共同通路。同时，活化的caspase-8，9可能参与了caspase-3的激活，从而诱导凋亡的发生。     

泰素帝对肺腺癌细胞A549凋亡及Survivin表达和Caspase-3活性的影响

目的探讨泰素帝对肺腺癌细胞A549凋亡,生存素(Survivin)表达和Caspase-3活性的影响。方法体外细胞培养,待细胞生长处于对数期时,加入不同浓度泰素帝,MTY比色法检测细胞活性。参考MTT结果,选取100ng/ml泰素帝处理A549细胞。透射电镜,流式细胞术检测细胞凋亡的发生;RT-PCR检测Survivin mRNA的表达;Western印迹法检测Survivin蛋白质的表达;Caspase-3活性检测试剂盒检测Caspase-3活性。结果泰素帝可抑制A549细胞生长,诱导细胞凋亡,呈时间依赖性降低Survivin的表达,增强Caspase-3酶活性。结论 100ng／ml泰素帝可诱导A549细胞凋亡,Survivin表达降低和Caspase-3活性增强可能是其诱导肺腺癌细胞凋亡的分子途径之一。     

Nutlins类似物NL-86诱导HeLa细胞凋亡的研究

目的 观察新型Nutlins类似物NL-86在体外诱导宫颈癌HeLa细胞凋亡的作用,并初步探讨其作用的分子机制.方法 采用MTT法检测NL-86化合物对HeLa细胞增殖的影响;用 FITC-Annexin V及碘化丙锭(PI)双染法,通过流式细胞仪(FCM)检测NL-86诱导HeLa细胞凋亡情况;用Western 印迹...     

呼肠病毒BYD1株诱导细胞凋亡能力及其穿膜蛋白结构分析

目的观察我国新分离呼肠病毒BYD1株诱导细胞凋亡能力，分析病毒致细胞凋亡与主要穿膜蛋白μl结构的关系。方法用流式细胞仪检测感染细胞凋亡比例，经Student t-test检验判断病毒诱导细胞凋亡的能力。通过Lasergen V6 Megalign软件分析比对BYD1株和呼肠病毒标准株T1L、T2J和T3D的μl蛋白582．675部分的一级结构；用SWISS MODEL数据库同源模建方法分析BYD1株μl蛋白的二级结构和三级结构。结果呼肠病毒BYD1株能够诱导HeLa细胞凋亡，其μl蛋白643—675氨基酸部分的α螺旋与同型标准株T2J存在差异。结论我国新分离呼肠病毒BYDl株具有诱导细胞凋亡的能力，其μl蛋白643—675氨基酸部分的α螺旋在细胞凋亡诱导中起重要作用。     

线粒体凋亡途径在槲皮素诱导U937 细胞凋亡中的作用研究

目的:观察槲皮素对人急性髓系白血病U937 细胞增殖、凋亡、线粒体膜电位(Δφm),、人B 细胞淋巴瘤因子- 2、人Bcl-2 相关X 蛋白、细胞色素c 表达及半胱氨酸蛋白酶鄄3、半胱氨酸蛋白酶鄄9 活性的影响,探讨线粒体凋亡途径在槲皮素 诱导U937 细胞凋亡中的作用。方法:体外培养U937 细胞,分别以0、10、20、40、80、160μmol/ L 浓度的槲皮素处理24、48 和 72 h,采用细胞计数试剂盒(Cell counting kit-8,CCK-8) 检测槲皮素对U937 细胞增殖的抑制作用;实验随机分为对照组 (Control)、槲皮素10、20 组和40 μmol/ L 组。采用流式细胞仪AnnexinV-FITC/ PI 双染法检测U937 细胞凋亡情况;采用荧光染 料3,3‘-二己基含氧碳菁碘代物[3,3‘-dihexyloxacarbocyanine iodide,DiOC6(3)]染色,流式细胞仪检测U937 细胞的变化; 采用Western blot 检测U937 细胞Bcl-2、Bax、Cytc 表达;采用比色法检测U937 细胞Caspase-3、Caspase-9 活性。结果:槲皮素可 抑制U937 细胞的增殖,抑制率显著升高,且呈时间-剂量依赖性。槲皮素亦可显著抑制U937 细胞凋亡率,与对照组比较(P< 0.01)。槲皮素显著降低U937 细胞驻鬃m,与对照组比较(P<0.01)。槲皮素显著下调U937 细胞Bcl-2 表达,上调Bax 表达,下 调Bcl鄄2/ Bax 比值及上调Cyt c 表达,与对照组比较(P<0.01)。槲皮素显著升高U937 细胞Caspase-3、Caspase-9 活性,与对照 组比较(P<0.01)。结论:槲皮素可显著抑制U937 细胞增殖,线粒体凋亡途径激活是槲皮素诱导U937 细胞凋亡的途径之一。     

共固定化细胞因子诱导的HeLa细胞凋亡

利用光化学固定方法,将干扰素-γ(IFN-γ)和肿瘤坏死因子-α(TNF-α)共同偶联到高分子材料聚苯乙烯基板上,合成生物材料.从形态学、流式细胞仪定量、磷脂酰丝氨酸分析、Caspase活性四个方面进行研究.形态学和流式细胞仪研究结果显示,固定化(及游离)细胞因子都可以诱导HeLa细胞凋亡,而且发现游离细胞因子比共固定细胞因子发挥药效更快,但随着时间的增加,共固定化细胞因子的药效比游离的细胞因子更好、更持久有效.磷脂酰丝氨酸定量分析进一步证明了固定化细胞因子的长效活性.在Caspase-3活性研究中发现游离的药物处理后Capase-3活性的提高比共固定化药物处理后更明显,推测共固定细胞因子诱导的HeLa细胞的凋亡不仅存在Caspase依赖的通路,也可能存在caspase非依赖的通路.     

Development and initial evaluation of a real-time RT-PCR assay to detect bluetongue virus genome segment 1

Since 1998, multiple strains of bluetongue virus (BTV), belonging to six different serotypes (types 1, 2, 4, 8, 9 and 16) have caused outbreaks of disease in Europe, causing one of the largest epizootics of bluetongue ever recorded, with the deaths of >1.8 million animals (mainly sheep). The persistence and continuing spread of BTV in Europe and elsewhere highlights the importance of sensitive and reliable diagnostic assay systems that can be used to rapidly identify infected animals, helping to combat spread of the virus and disease. BTV has a genome composed of 10 linear segments of dsRNA. We describe a real-time RT-PCR assay that targets the highly conserved genome segment 1 (encoding the viral polymerase--VP1) that can be used to detect all of the 24 serotypes, as well as geographic variants (different topotypes) within individual serotypes of BTV. After an initial evaluation using 132 BTV samples including representatives of all 24 BTV serotypes, this assay was used by the European Community Reference Laboratory (CRL) at IAH Pirbright to confirm the negative status of 2,255 animals imported to the UK from regions that were considered to be at risk during the 2006 outbreak of BTV-8 in Northern Europe. All of these animals were also negative by competition ELISA to detect BTV specific antibodies and none of them developed clinical signs of infection. These studies have demonstrated the value of the assay for the rapid screening of field samples.     

Dot immunoperoxidase assay using monoclonal antibody for detection of bluetongue virus antigens.

A rapid, simple dot immunoperoxidase assay (DIPA) is described for visual detection and identification of bluetongue virus (BTV) antigens in samples of infected cell culture fluid. The assay was performed using nitrocellulose (NC) paper and 'dipsticks'. Dots of samples were adsorbed to the NC surface and the remaining non-specific binding sites were blocked with skim milk solution. BTV was detected with either of two murine monoclonal antibodies (4H4, 5G12) to the major group specific antigens of BTV, and the complex was reacted with a peroxidase conjugated anti-mouse immunoglobulin G (heavy- and light-chain specific). Positive reactions were easily visualized as brown spots after enzyme degradation of substrate containing H2O2 and diaminobenzidine (DAB). The DIPA was specific in detecting BTV in samples of cell culture fluid from baby hamster kidney (BHK-21) cells infected with U.S.A. isolates of the five BTV serotypes (2, 10, 11, 13 and 17) known to exist in the U.S.A., and South African isolates of 17 BTV serotypes (1-12, 14-16, 18 and 20), but not with two North American isolates of epizootic hemorrhagic disease of deer virus (EHDV) representing serotypes 1 and 2. Attempts to detect BTV directly in infected sheep blood cells and chick embryo tissue suspensions by DIPA were unsuccessful. Of 55 cell culture fluid samples examined from BHK-21 or Vero cell monolayers inoculated with 55 clinical specimens, propagated initially in embryonating chicken egg (ECE) 11 proved positive and 44 were negative by DIPA. The results were in complete agreement with the conventional ECE and tissue culture isolation systems. The DIPA appears to have potential application, especially as a 'dipstick' kit, for rapid and inexpensive laboratory diagnosis of bluetongue virus infection.     

Development and evaluation of an IgM-capture ELISA for detection of recent infection with bluetongue viruses in cattle

An IgM-capture enzyme-linked immunosorbent assay (ELISA) was developed for the detection of recent infection of bluetongue virus (BTV) in cattle. The test is based on the use of biotinylated capture anti-bovine IgM antibodies bound to a streptavidin-coated ELISA plate. The captured IgM antibodies were detected by application of BTV VP7 antigen and a VP7 antigen-specific monoclonal antibody. The IgM-capture ELISA was compared with the competitive ELISA by testing serum samples from groups of calves infected experimentally with five USA and 19 South Africa serotypes of BTV. The IgM-capture ELISA was able to detect bovine anti-VP7 antibodies from all animals infected with the 24 BTV serotypes at 10 days post-infection, whereas the competitive ELISA was not. When the detectable IgM diminished after 40 days post-infection by the IgM-capture ELISA, the IgG anti-VP7 antibodies remained high. The IgM-capture ELISA is sensitive and can be applied for the detection of recent infection of BTV in cattle.     

Rapid identification of bluetongue virus by nucleic acid hybridization in solution

A liquid phase hybridization format has been adapted for the identification of bluetongue virus (BTV) with respect to serogroup and serotype. In this paper we present experiments which demonstrate the identification of bluetongue virus with respect to both serogroup and serotype. BTV serogroup-specific and BTV serotype 17-specific single-stranded RNA probes labeled with ³⁵S-CTP were used to identify BTV viral nucleic acids in infected cell culture lysates. Cell lysates were prepared for testing by rapid solubilization in a guanidine isothiocyanate/EDTA/dithiothreitol medium. Hybridizations were done in 12.5 μl reaction volumes for 2 h or overnight using nanogram quantities of probe. An RNAse solution was added subsequent to hybridization and incubated for 30 min. TCA-precipitable counts were collected and quantitated by scintillation counting. Specific hybridization signals were obtained in as little as 3 h in assays consisting of uninfected controls, cells infected with different BTV serotypes, and cells infected with a strain of epizootic hemorrhagic disease of deer (EHDV-1). By using asymmetric single-stranded RNA probes we were able to distinguish viral mRNA and viral double-stranded RNA components. We propose this technique as an ancillary or replacement procedure for the confirmation and classification of viral isolates using an appropriate battery of nucleic acid probes. Potential applications and methods of enhancing the technique are discussed.     

Bluetongue virus detection by two real-time RT-qPCRs targeting two different genomic segments

The detection of the bluetongue virus (BTV) by conventional methods is especially difficult and labour-intensive. Molecular diagnosis is also complex because of the high genetic diversity between and within the 24 serotypes of BTV. In the present study, two laboratories joined forces to develop and validate two new RT-qPCRs detecting and amplifying BTV segments 1 and 5. The 2 assays detect strains from all 24 serotypes. They both have a detection limit of 0.01 ECE50 and all 114 samples from BTV-free goats, sheep and cattle were negative. The two assays resulted in similar C_t values when testing biological samples collected in sheep infected experimentally with a field strain of BTV from the Mediterranean basin. On average, the C_t values obtained with the 2 methods applied to the 24 serotypes were not significantly different from each other, but some moderate to high differences were seen with a few strains. Therefore these two methods are complementary and could be used in parallel to confirm the diagnosis of a possible new introduction of BTV. An RT-qPCR amplifying a fragment of the beta-actin mRNA was also developed and validated as internal control for the bluetongue specific assays. The three assays described allow a reliable and rapid detection of BTV.     

Genetic characterization of bluetongue virus serotype 9 isolates from India

Recent incursions of bluetongue virus (BTV) into previously naive geographical areas have emphasised the need to better understand virus movement and epidemiology. Several bluetongue virus (BTV) serotypes are known to exist in India, and some serotype viruses have been isolated. However, the complete genome of not a single isolate is available to date. We report the complete genome sequence of one, and partial sequences of three other Indian isolates of BTV-9. Evolutionary relationships with segment-2 and -6 sequences of BTV isolates around the world, deduced using four different phylogenetic analyses and a similarity programme, show that BTV-9 (Eastern), BTV-9 (Western), and BTV-5 form a triad of equidistant, genetically distinct groups of viruses. The Indian BTV-9 isolates were closely related to Mediterranean and European BTV-9 isolates (Eastern topotype) based on segment-2 and -6 sequences. By contrast, segment-5 analyses clustered the Indian BTV-9 isolates with South African BTV-3 reference strain (98% identity), which belongs to one of the Western types. These results have implications on BTV origin and movement, genotyping, serotyping, and vaccine design.     

A duplex RT-PCR assay for detection of genome segment 7 (VP7 gene) from 24 BTV serotypes

Since 1998, six distinct serotypes of Bluetongue virus (BTV) have invaded Southern and Central Europe, persisting in some regions for up to 6 years and resulting in the deaths of >1.8 million sheep. Rapid and reliable methods of virus detection and identification play an essential part in our fight against bluetongue disease (BT). We have therefore developed and evaluated a duplex, one-step RT-PCR assay that detects genome segment 7 (encoding the major serogroup (virus-species) specific antigen and outer-core-protein VP7) from any of the 24 BTV serotypes. Although Seg-7 is highly conserved, there are sequence differences in the near terminal regions that identify two distinct phylogenetic groups. Two sets of primers (targeting Seg-7 terminal regions of viruses from these two groups) were included in a duplex RT-PCR assay system. Assay sensitivity was evaluated using tissue culture derived virus, infected vector insects and clinical samples (blood and other tissues). The assay reliably amplified Seg-7 from any of the BTV strains tested, including isolates of the 24 BTV serotypes and isolates from different geographic origins. No cross-reactions were detected with members of closely related Orbivirus species (African horsesickness virus (AHSV), Epizootic haemorrhagic disease virus (EHDV), Equine encephalosis virus (EEV) and Palyam virus (PALV)).     

Detecting bluetongue virus RNA in cell culture by dot hybridization with a cloned genetic probe

A 70% copy of genome segment 7 of bluetongue virus (BTV)-17 has been cloned into the plasmid pBR-322. This cloned BTV segment when used as a radioactive probe will hybridize to BTV double-stranded RNA extracted from cell cultures and dotted onto nitrocellulose paper. This dot hybridization technique is therefore suitable for detecting and identifying BTV in cell culture. The specificity of cloned probes is discussed in relation to detecting gene sequences specific for either the bluetongue serogroup or different serotypes of BTV.     

端粒酶在流感病毒诱导HeLa细胞凋亡中的作用

目的探讨端粒酶活性变化与流感病毒诱导HeLa细胞凋亡之间的关系。方法用A1亚型、B型及紫外线照射后的流感病毒分别感染HeLa细胞,在电镜下观察细胞形态,并用Annexin V-FITC染色流式细胞仪及PCR-ELISA法分别检测细胞凋亡诱导率与HeLa细胞端粒酶活性。结果B型、A1亚型流感病毒的凋亡诱导率最高达38.65%、23.94%(P<0.01),经紫外线照射后的流感病毒仍然具有一定的凋亡诱导能力;同时,HeLa细胞端粒酶活性均有所下降,其中B型流感病毒诱导后的HeLa细胞端粒酶活性下降比A1亚型更明显(P<0.01)。结论流感病毒降低肿瘤细胞的端粒酶活性,可能是其诱导肿瘤细胞凋亡的又一重要机制。