壳聚糖对人子宫颈癌HeLa细胞生长作用的研究

壳聚糖对人子宫颈癌ＨｅＬａ细胞生长作用的研究（中国医学科学院中国协和医科大学基础医学研究所细胞生物研究室，北京１００００５沈江波林建立张世馥）壳聚糖（ｃｈｉｔｏｓａｎ）是由甲壳素（１，４）－α－乙酰胺基－α－脱氧－β－Ｄ－葡聚糖经脱乙酰化反应而得到的...     

靶向hTERT RNA干涉促进人宫颈癌HeLa细胞凋亡的研究

目的：利用RNA干涉技术抑制肿瘤细胞端粒酶表达，探讨其对肿瘤细胞凋亡的诱导作用。方法：将成功构建的针对hTERT的siRNA(small interferencing RNA)表达载体转染HeLa细胞，通过透射电镜、免疫印迹和流式细胞术(FCM)等方法检测人宫颈癌HeLa细胞的凋亡情况。结果：构建的siRNA表达载体可以诱导HeLa细胞发生凋亡。结论：针对人端粒酶逆转录酶的siRNA表达载体稳定转染HeLa细胞，可诱导HeLa细胞发生凋亡。     

沉默Smo基因对人宫颈癌HeLa细胞活力及凋亡的影响

目的:使用RNA干扰技术沉默Smoothened(Smo)基因,探讨其对宫颈癌He La细胞活力和凋亡的影响。方法:采用Smo shRNA转染宫颈癌He La细胞;采用RT-PCR和Western blot技术检测各组He La细胞Smo和转录因子Gli1的mRNA和蛋白表达;采用MTT比色法测定沉默Smo后细胞生长的情况;流式细胞术检测Smo shRNA对细胞周期和凋亡的影响。结果:与对照组比较,Smo shRNA转染细胞72 h后,Smo和Gli1的mRNA和蛋白表达水平均明显降低(P0.05)。Smo基因沉默后,He La细胞的活力明显降低,细胞明显阻滞于G0/G1期,细胞凋亡率显著升高。结论:沉默Smo基因可有效抑制人宫颈癌He La细胞生长,并诱导其凋亡。     

重构型Caspase-6对HeLa细胞凋亡的诱导作用

目的 将人Caspase-6基因大小亚基顺序颠倒，表达为重构型Caspase-6分子，探讨其对细胞凋亡的促进作用。方法 RT－PCR获取人Caspase-6基因，测序判断正确后，通过重组PCR的方法构建大小亚基顺序颠倒的重构型Caspase-6（RevCasp6)基因。将其克隆入含绿色荧光蛋白（GFP）蛋白的真核表达载体pIRES2-EGFP中，转染人宫颈癌细胞系HeLa，在荧光显微镜下观察细胞形态的变化，用电子显微镜进一步观察细胞的凋亡特征。结果 获得了人Caspase-6基因，并成功地构建了大小亚基顺序颠倒的Rev-Casp6基因。以构建的RevCasp6真核表达载体，转染HeLa细胞后，荧光显微镜观察到细胞生长不良，细胞核结构破坏和细胞死亡。电子显微镜观察显示，转染了RevCasp6基因的细胞呈凋亡的典型特征。结论 RevCasp6基因具有诱导HeLa细胞凋亡的作用。     

人截短型凋亡诱导因子的表达对HeLa细胞的促凋亡作用

目的：观察人截短型AIF基因的表达对HeLa细胞的促凋亡作用。方法：用RT-PCR法分段克隆全长人AIF基因，经改造截去其N-端线粒体定位信号及部分Spacer区域（1-120位氨基酸）的编码序列，从而获得人截短型AIF（AIF△1-120）基因。将其克隆入pIRES2-EGFP绿色荧光蛋白（EGFP）共表达载体，用脂质体法转染HeLa细胞，通过荧光显微镜观察。免疫组织化学检测，间接免疫荧光检测，电镜观察等方法。检测目的基因在转染细胞中的表达，以及对转染细胞的形态及生长状况的影响。结果：成功地构建了人截短型AIF（AIF△1-120)基因的真核表达载体。转染HeLa细胞后，可检测到人截短型AIF分子的表达，随着转染后时间的延长，可观察到表达人截短型AIF分子的HeLa细胞呈现典型的凋亡特征。结论：人截短型AIF基因的表达可诱导HeLa细胞凋亡。     

膜联蛋白A2对人宫颈癌HeLa细胞增殖、迁移和凋亡的影响

 目的：研究膜联蛋白A2（annexin A2, ANXA2）对人宫颈癌HeLa细胞增殖、迁移和凋亡能力的影响。方法：以HeLa细胞为研究对象,构建过表达载体以及ANXA2-siRNA,转染入细胞。将细胞分为正常对照组、scrambled组、ANXA2过表达组及ANXA2-siRNA组。应用real-time PCR法检测ANXA2 mRNA表达水平及Western blotting检测ANXA2蛋白表达水平。分别采用MTT法、Boyden小室法和流式细胞术观察ANXA2对HeLa细胞增殖、迁移及凋亡能力的影响。结果：ANXA2过表达组可以显著促进HeLa细胞的增殖和迁移;ANXA2-siRNA组明显抑制HeLa细胞的增殖和迁移;ANXA2对HeLa细胞凋亡几乎无影响。结论:沉默ANXA2对人宫颈癌细胞的凋亡无显著影响,但可显著抑制其增殖能力和迁移能力。ANXA2可能在宫颈癌的发生发展中具有十分重要的作用,提示它有可能成为宫颈癌治疗的分子靶点。     

天花粉蛋白对HepA-H细胞和HeLa细胞抑癌活性研究

目的：比较分析商品天花粉蛋白(TCS1) 和从鲜药材中提取分离出的天花粉蛋白粗品（TCS2），对HepA-H细胞（腹水型肝癌高转移株细胞）和HeLa细胞（人宫颈癌肿瘤细胞）的杀伤作用，并进一步探讨其抑癌作用机理。方法： MTT法检测药物的细胞毒作用，电镜观察细胞超微结构改变，电泳检测细胞DNA生物化学特征改变。结果： TCS1和TCS2对HepA-H细胞作用不明显（P>0.05），而对HeLa细胞具有显著性作用，呈明显时效、量效关系（r>0.864, P<0.05或P<0.01）。在相同作用时间内，TCS2作用组对细胞生长抑制率均高于TCS1组（P<0.01）。进一步研究发现，HeLa细胞经TCS2作用后，细胞表面微绒毛消失，胞膜发泡，核染色质浓缩边集，并出现凋亡小体，细胞DNA经琼脂糖凝胶电泳呈典型的梯形带。结论： HepA-H细胞对天花粉蛋白不敏感，而HeLa细胞对TCS1和TCS2敏感，其中TCS2抑癌活性明显强于TCS1，细胞生长受抑制作用显著，作用机制与诱导细胞凋亡相关。     

白藜芦醇诱导人宫颈癌细胞凋亡的作用

目的观察白藜芦醇对人宫颈癌Hela细胞凋亡的影响。方法采用TUNEL法检测不同浓度的白藜芦醇诱导体外培养的Hela细胞凋亡情况。结果浓度大于50μmol／L的白藜芦醇处理Hela细胞后,细胞凋亡特征明显可见,并呈时间和剂量依赖关系。结论白藜芦醇对人宫颈癌Hela细胞有明显的促凋亡作用。     

东亚钳蝎毒抗癌多肽对Eca 109细胞和HeLa细胞的毒性作用

目的和方法：采用ＭＴＴ比色法、生长抑制实验、集落形成抑制实验,探讨东亚钳蝎毒抗癌多肽（anticancer polypeqtide from Ｂuthrs Ｍartensii Ｖenom,ＡＰＢＭＶ）对Ｅca１０９细胞和ＨeＬa细胞的细胞毒作用。结果：ＡＰＢＭＶ对Ｅca１０９细胞有明显的毒性作用,且呈显著量在系,处理细胞２４h和７２h的ＩＣ５０分别为０．０２７rg．mＬ＾－１ 和０．０２４ug．mＬ＾－１。ＡＰＢＭＶ对Ｅca１０９细胞的生长抑制作用呈明显的时效     

截短型人凋亡诱导因子AIF1-400对HeLa细胞的促凋亡作用

目的:观察人截短型AIF(AIF△1-400)对HeLa细胞的促凋亡作用。方法:在AIF△1-120基因克隆成功的基础上进一步改造,构建截去AIF基因线粒体定位信号、FAD结合结构域和NADH结合结构域(1～400位氨基酸)编码序列的AIF△1-400基因,将其克隆入pcDNA3真核表达载体,用脂质体法转染HeLa细胞,通过Western blot检测该基因在转染细胞中的表达通过电镜观察截短型分子对肿瘤细胞的具有促凋亡活性。结果:经酶切鉴定与测序证实,AIF△1-400的真核表达载体构建成功。通过Western blot证实截短型基因在HeLa细胞中表达,电镜观察可见转染细胞骨架的破坏和细胞核固缩等凋亡特征。结论:人截短型AIF(AIF△1-400)基因的表达可诱导HeLa细胞凋亡。     

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.     

Analysis of genome segments from six different isolates of bluetongue virus using RNA-RNA hybridisation: a generalised coding assignment for bluetongue viruses

Nucleic acid probes prepared directly from bluetongue virus (BTV) genomic double-stranded RNA (dsRNA) have been used to identify the functionally equivalent genome segments from six distinct isolates of BTV after their separation in both agarose and polyacrylamide gel electrophoresis systems. Variations in the rate, and in one case the order, of migration of the equivalent genome segments from different viruses was detected in the polyacrylamide gel system. However, the genomic dsRNA profiles of eleven BTV isolates were found to be identical when analysed by agarose gel electrophoresis. Functionally equivalent genome segments from the six viruses that were analysed were found to migrate in identical relative positions in this gel system. From these data we propose a modified version of the protein coding assignments published for BTV 1 South Africa (Mertens et al., 1984) in which the identification of the genome segments would be based upon their order of migration in the agarose rather than the polyacrylamide gel system. The modified coding assignments, unlike the original assignments, would be applicable to all of those viruses analysed and appear likely to be valid for all normal BTV isolates.     

蓝舌病毒L3基因的克隆与表达

目的　通过高效表达,研究蓝舌病毒（ＢＴＶ） ＶＰ３ 的功能,为后续ＢＴＶ病毒样颗粒的装配作准备。方法　克隆出完整的ＢＴＶ１３ Ｌ３ 基因,将其插入杆状病毒表达载体进行表达。结果　获得了含有全长Ｌ３ 基因的克隆,ＶＰ３ 在昆虫细胞中得到了高效表达,表达蛋白占细胞总蛋白的１０％ ～１５％ ,ＶＰ３ 与ＶＰ７ 共表达可装配出ＢＴＶ 核心样颗粒。结论　在昆虫细胞中表达ＢＴＶＶＰ３ 蛋白具有生物学活性,可用于ＢＴＶ病毒样颗粒的装配研究     

Deep sequencing as a method of typing bluetongue virus isolates

Bluetongue (BT) is an economically important endemic disease of livestock in tropics and subtropics. In addition, its recent spread to temperate regions like North America and Northern Europe is of serious concern. Rapid serotyping and characterization of BT virus (BTV) is an essential step in the identification of origin of the virus and for controlling the disease. Serotyping of BTV is typically performed by serum neutralization, and of late by nucleotide sequencing. This report describes the near complete genome sequencing and typing of two isolates of BTV using Illumina next generation sequencing platform. Two of the BTV RNAs were multiplexed with ten other unknown samples. Viral RNA was isolated and fragmented, reverse transcribed, the cDNA ends were repaired and ligated with a multiplex oligo. The genome library was amplified using primers complementary to the ligated oligo and subjected to single and paired end sequencing. The raw reads were assembled using a de novo method and reference-based assembly was performed based on the contig data. Near complete sequences of all segments of BTV were obtained with more than 20× coverage, and single read sequencing method was sufficient to identify the genotype and serotype of the virus. The two viruses used in this study were typed as BTV-1 and BTV-9E.     

蓝舌病毒两外壳蛋白VP2和VP5在昆虫细胞中的表达 …

目的 研究蓝舌病毒（ＢＴＶ）ＶＰ２与ＶＰ５的免疫学特性,为ＢＴＶ基因工程疫苗研究和病毒样颗粒装配打下基础。方法 将ＢＴＶ１０ ＶＰ２和ＶＰ５基因分别插入杆状病毒表达载体ｐＦａｓｔＢａｃ１,转染昆虫细胞获得重组杆状病毒。用ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎ ｂｌｏｔ检测重组杆状病毒对ＶＰ２和ＶＰ５的表达,运用组织培养中和试验和直接血凝试验检测表达产物的生物学活性。     

Induction of T cell response by bluetongue virus core-like particles expressing a T cell epitope of the M1 protein of influenza A virus

A CD4⁺ T cell epitope of the influenza virus matrix protein corresponding to the C terminus (QAYQKRMGVQMQRFK) was inserted into the VP7 gene of bluetongue virus (BTV). The chimeric protein was expressed by a dual recombinant Autographa californica polyhedrosis virus (AcNPV), which encodes the two inner capsid proteins VP3 and VP7 of BTV. When Spodoptera frugiperda cells (Sf9 cells) were infected with this recombinant BTV, core-like particles (CLPs) were formed as demonstrated by electron microscopy. To study the immunogenicity of a foreign epitope deprived of its natural flanking sequences in vitro, purified CLPs expressing the T cell epitope were used to stimulate two different MHC class II-restricted CD4⁺ human T cell clones. One of these T cell clones, ALF 3.7 was specific for the inserted epitope, whereas the other T cell clone ALF 4.4 recognized shorter derivates of the given epitope. CLPs with the inserted epitope were presented as efficiently as purified influenza virus matrix protein to the clone ALF 3.7, whereas clone ALF 4.4 showed no proliferative response. Received: 17 February 1998     

A European field strain of bluetongue virus derived from two parental vaccine strains by genome segment reassortment

Since 1998, nine bluetongue virus (BTV) strains from serotypes 1, 2, 4, 8, 9 and 16 have invaded Europe, killing >2 million animals (mainly sheep). Live vaccine strains of BTV-2, 4, 9 and 16 have also been used in the region. The BTV genome is composed of ten linear-segments of dsRNA, and events in Europe have provided opportunities for different strains to exchange/reassort genome segments, generating novel progeny-viruses. Genome segment 2 (Seg-2) encodes outer capsid protein VP2, the primary determinant of virus serotype, while Seg-5 encodes NS1, which forms 'tubules' within the cell-cytoplasm. Seg-2 and Seg-5 from 15 European isolates, and vaccine/reference strains, of BTV-2 and BTV-16, were sequenced. Isolates from the same serotype showed >92% nt identity in Seg-5, but <84% identity between types. However, published data for Seg-5 of BTV-16 from Italy 2002 showed <83% nt identity with other BTV-16 strains, but was identical to the BTV-2 vaccine strain (used in Italy during 2002, and annually in a multivalent vaccine in Israel since 1995) indicating that ITL2002 is a reassortant between the BTV-2 and BTV-16 vaccine strains. This represents the first detection of a reassortant BTV strain within Europe, highlighting concerns about the use of live BTV vaccines in the region.     

Current and next-generation bluetongue vaccines: Requirements,strategies, and prospects for different field situations

Bluetongue virus (BTV) causes the hemorrhagic disease bluetongue (BT) in ruminants. The best way to control outbreaks is vaccination. Currently, conventionally modified-live and inactivated vaccines are commercially available, which have been successfully used to control BT, but nonetheless have their specific shortcomings. Therefore, there is a need for improved BT vaccines.

The ideal BT vaccine is efficacious, safe, affordable, protective against multiple serotypes and enables the differentiation of infected from vaccinated animals. Different field situations require specific vaccine profiles. Single serotype outbreaks in former BT-free areas need rapid onset of protection against viremia of the respective serotype. In contrary, endemic multiple serotype situations require long-lasting protection against all circulating serotypes. The ideal BT vaccine for all field situations does not exist and balancing between vaccine properties is needed.

Many new vaccines candidates, ranging from non-replicating subunits to replicating next-generation reverse genetics based vaccines, have been developed. Some have been tested extensively in large numbers of ruminants, whereas others were developed recently and have only been tested in vitro and in mice models. Most vaccine candidates are promising, but have their specific shortcomings and advantages. In this review, current and next-generation BT vaccines are discussed in the light of prerequisites for different field situations.