卵形鲳鲹源创伤弧菌导致宿主细胞凋亡的作用机制

弧菌是自然界菌群中的一部分,作为一种有害菌不仅可以感染常见的水产品,有些弧菌更是对人体有一定的致病性。随着近些年水产养殖的不断扩大,由创伤弧菌(Vibrio vulnificus)引起的鱼病不胜枚举,不仅给养殖行业造成巨大的损失,同时对人类生命安全也存在着巨大威胁。本文对卵形鲳鲹源创伤弧菌感染宿主细胞的机制进行研究,目的是阐明创伤弧菌对宿主细胞的致死机理,为未来研发抗病菌功能产品提供理论依据。本文通过确定创伤弧菌胞外产物(Extracellular products,ECPs)的蛋白浓度,以不同的蛋白浓度为基准,利用光镜观察和细胞活力CCK检测技术测定创伤弧菌ECPs对细胞的毒性。然后使用Hoechst 33342染核观察及DNA ladder来进一步确定其致死机制。根据细胞毒性结果,卵形鲳鲹源创伤弧菌ECPs对宿主细胞有很强的细胞毒性。Hoechst 33342染核结果发现实验组出现了凋亡小体,这与STS处理的阳性对照组中细胞变化相一致。但是ECPs处理后的宿主细胞未出现明显的DNA ladder。卵形鲳鲹源创伤弧菌对宿主细胞具有明显的毒性,再通过理化性质的检测,可以初步确定创伤弧菌导致卵形鲳鲹细胞死亡是通过凋亡途径发生的。未来将针对凋亡途径的具体通路进行深入研究,为控制病菌的感染提供理论依据。     

鳗鲡源创伤弧菌的间接ELISA快速检测

创伤弧菌(Vibrio vulnificus)血清2型对鳗鲡有专一的感染性,是鳗鲡出血性败血症、烂鳃病和烂尾病的重要病原菌。采用间接ELISA法对制备的兔抗创伤弧菌多克隆抗血清进行效价测定,结果表明其效价高达1∶512 000。从发病鳗鲡中分离出创伤弧菌和37株其他致病菌,将38株病原菌以较低浓度(1×106cfu/m L)包被后通过兔抗创伤弧菌血清进行交叉反应水平检测,结果表明,兔抗创伤弧菌血清稀释到1∶256 000或1∶128 000时,其与37株其他病原菌均未产生明显的ELISA交叉反应。该血清分别经1∶64 000和1∶32 000稀释后,采用ELISA方法检测了经创伤弧菌、鳗鲡爱德华氏菌(Edwardsiella anguillarum)和嗜水气单胞菌(Aeromonas hydrophila)攻毒感染死亡的美洲鳗鲡(Auguilla rostrata)脏器组织悬液(肝脏、肾脏和鳃),结果表明,兔抗创伤弧菌血清经1∶32 000稀释后可特异性检出鳗鲡鳃和肾脏组织中的创伤弧菌。     

双酚A暴露致神经细胞凋亡和炎症性死亡的研究进展

双酚A(BPA)是生产聚碳酸酯和环氧树脂塑料的添加剂,具有内分泌干扰物效应。由于大量研究发现BPA具有神经毒性,其暴露对大脑神经功能及行为变化的影响受到越来越多的关注.研究发现,氧化应激常伴随着BPA诱导的神经细胞凋亡和炎症性死亡(焦亡)反应的发生,导致神经细胞形态和功能改变,是BPA暴露与神经系统疾病相关性的重要连接纽带.研究聚焦BPA暴露诱导神经细胞凋亡和炎症性死亡这一生理现象,对BPA暴露通过线粒体、内质网以及死亡受体通路信号上的细胞凋亡的分子作用机制的详细介绍,探讨了细胞凋亡、细胞焦亡之间的关系以及他们与神经系统疾病的潜在关系,指出BPA暴露诱导的神经细胞凋亡与炎性反应的分子机制尚需要大量的动物、靶向敲除实验以及人群实验加以验证.同时,应关注低剂量BPA暴露参与毒性通路或与其他毒性通路相互作用而加重复合污染物暴露的健康效应,为深入研究BPA暴露的神经毒性机制及其毒性干预提供参考.     

脑创伤后NF-kB的作用及其细胞凋亡的基因调控

目的：探讨核因子kB（NF-kB）在脑创伤后的作用及细胞凋亡的调控基因。方法：对近年来有关NF—kB在脑创伤后的作用及细胞凋亡的调控基因的文献进行总结。结果．NF-kB在脑创伤中的作用为双重性，脑创伤后细胞凋亡的调控基因为bcl-2基因家族和Caspase基因家族。结论：NF-kB在脑创伤中是诱导细胞死亡还是促进细胞死亡，还具有分歧，bcl-2基因族和Caspase基因家族在脑创伤中的细胞凋亡起着重要调节作用。     

低氧、运动及低氧训练对肝细胞凋亡影响的研究进展

低氧与健康的研究是近年来临床医学、环境医学、航空航天医学和运动医学的研究重点问题之一.从肝细胞凋亡的角度研究运动对肝细胞的影响及其与运动性疲劳之间的关系,对科学制定训练计划等具有重要的意义.低氧训练使机体处于更加缺血和缺氧状态,血液的重新分配,使肝脏出现暂时的缺血,运动强度降低后血液重新回流形成血液再灌注,导致肝损伤,诱导细胞凋亡.细胞凋亡的过低或过高都导致疾病的发生.肝细胞凋亡的异常在急、慢性肝损伤的发病机制中起着重要的作用.就有关低氧、运动及低氧训练对肝细胞凋亡的影响,引起凋亡发生的基因调控及机制进行相关综述.     

USP13抑制溶瘤病毒诱导的肝癌细胞凋亡机制

新城疫病毒(Newcastle Disease Virus,NDV)是一种复制能力较强且特异性识别并杀伤肿瘤细胞的禽类病毒.肝癌细胞被NDV感染后,会特异性激活肝癌细胞中的凋亡信号通路,诱导肝癌细胞发生凋亡. USP13(Ubiquitin Specific Protease 13)是去泛素化酶家族的一员,能够去泛素化和稳定PTEN分子.在Huh7和HLCZ01这两种肝癌细胞中感染NDV时,USP13的表达水平均明显降低.在NDV感染的Huh7和HLCZ01细胞中过表达USP13,细胞凋亡均明显受到抑制.反之,敲低细胞中USP13的水平,细胞凋亡明显得到增强.进一步揭示了USP13抑制NDV诱导肝癌细胞凋亡的机制,在两种肝癌细胞中过表达USP13虽然不影响细胞中NDV的复制,但发现USP13能够上调凋亡信号通路中的一个重要分子Bcl-2,进而抑制肝癌细胞的凋亡. USP13在NDV诱导的肝癌细胞凋亡中发挥着重要的作用,同时为肝癌的溶瘤病毒治疗提供了一个新的理论依据.     

钾离子及钾通道在细胞凋亡时线粒体变化中的作用

细胞凋亡在多种生命活动及疾病中均具有重要作用,线粒体是决定细胞凋亡的关键因素.近年研究发现,细胞内高钾可以保护细胞免于凋亡.但也有研究表明,钾离子及钾离子通道可参与线粒体的结构和功能改变,影响细胞凋亡.明确细胞内钾离子对线粒体的影响,可以更好的理解细胞凋亡及其相关的多种生理、病理机制.综述了钾离子及钾离子通道在细胞凋亡时对线粒体的作用.     

创伤弧菌OmpU与嗜水气单胞菌OmpⅡ外膜蛋白二联表达及其初步免疫原性

从发病的养殖鳗鲡中分离出创伤弧菌(Vibrio vulnificus)和嗜水气单胞菌(Aeromona shydrophila),提取其基因组DNA,再通过PCR法克隆创伤弧菌外膜蛋白Omp U和嗜水气单胞菌外膜蛋白OmpⅡ的基因全长。采用融合PCR法体外连接这2个基因表达膜外片段的序列并成功构建了二联表达载体(p GEX-2T-Vibr-Aero-his)。在大肠杆菌(BL21)的吸光度A_(600)为0.6~1.0时,采用1.0 mmol/L IPTG诱导剂,16℃过夜诱导表达。表达产物经离心柱亲和层析纯化后获得分子量为82.2 ku的外膜蛋白。蛋白经透析复性后免疫于鳗鲡以测定其血清抗体效价。结果表明,重组蛋白注射组的鳗鲡血清抗体效价在免疫后第14天和第21天显著(P0.05)高于PBS对照组,第28天和第42天达到极显著(P0.01)水平。     

三株弧菌热休克蛋白70基因的克隆和序列分析

 根据GenBank中同类蛋白序列设计特异PCR引物,从2株创伤弧菌Vibrio vulnificus和1株河弧菌Vibrio fluvialis中扩增出热休克蛋 白70（heat shock protein, hsp70）基因片段。对这3个片段进行克隆、测序和分析的结果表明,3个片段长均为1 911 bp,包含完整的 hsp70 ORF,编码636个氨基酸。它们的氨基酸序列与GenBank中其它物种hsp70的氨基酸序列比较发现,2株创伤弧菌hsp70基因序列 和同种其它菌株的同源性高,达98%以上;而河弧菌的hsp70序列属首次克隆;与多种原核和真核生物的hsp70氨基酸序列一起构建 了系统进化树,结果支持传统的分类结果。     

三氧化二砷与肿瘤细胞凋亡机制的研究进展

综述了细胞凋亡的相关概念及其三氧化二砷(As203)诱导凋亡可能机制的研究进展.查阅近几年国内外有关的文献资料,进行分析、整理和归纳得出,细胞凋亡是细胞与生俱来的一种机制,是最常见的细胞生理性死亡形式,它的异常在肿瘤的发病上具有重要作用.三氧化二砷能够诱导多种肿瘤细胞凋亡,现已用于治疗血液系统肿瘤和实体瘤.其诱导凋亡的机制正在逐渐成为研究的热点,但还远未被研究透彻,随着研究的不断深入,相信不久的将来三氧化二砷很可能为研究肿瘤治疗新策略提供参考.     

A novel type of aberrant recombination in immunoglobulin genes and its implications for V-J joining mechanism

Functional kappa light chain genes are formed during B-lymphocyte differentiation by the joining of initially separate V and J gene segments. It has been suggested that the intervening DNA is deleted, however the recent reports of what appear to be the reciprocal products of V and J recombination (back-to-back conserved V and J flanking sequences, called f-fragments) in DNA from mature lymphocytes make a simple deletion model unlikely. An alternative scheme involving unequal sister chromatid exchange has been proposed, supported by the evidence that the f-fragments seem to have segregated from the chromosome carrying the reciprocal complete kappa light chain gene (this and other schemes are briefly reviewed in ref. 8). We report here the analysis of a mouse myeloma (MOPC 41), in which a productive (kappa+) and a non-productive (kappa-) rearrangement has occured, which may help to clarify the mechanism of V-J joining. The aberrant rearrangement has led to the joining of a J1 gene segment to a sequence unrelated to any V gene (L10), and which in the germ line is flanked by a sequence resembling a V region recombination signal sequence. In this case no segregation of the reciprocal recombination products (kappa-41 and f41), which is a required step in sister chromatid exchange models, has taken place. An inversion model provides the simplest explanation of this J rearrangement.     

环境污染物PFOS可致人脑微血管内皮细胞骨架蛋白改变

目的:探讨全氟辛烷磺酰基化合物(PFOS)引起的人脑微血管内皮细胞(HBMEC)骨架蛋白(F-actin)排列变化.方法:培养正常的人脑毛细血管内皮细胞,不同浓度PFOS作用30分钟,利用免疫荧光染色的方法检测细胞骨架蛋白F-actin的变化.结果:PFOS可使细胞骨架蛋白F-actin发生重排,这与PFOS的浓度有关.     

The structure, rearrangement and expression of D beta gene segments of the murine T-cell antigen receptor

It has been postulated that the variable region of the beta-polypeptide of the murine T-cell antigen receptor is encoded by three distinct germ-line gene segments--variable (V beta), diversity (D beta) and joining (J beta)--that are rearranged to generate a V beta gene. Germ-line V beta and J beta gene segments have been isolated previously. Here we report the isolation and characterization of two germ-line D beta gene segments that have recognition signals for DNA rearrangement strikingly similar to those found in the three immunoglobulin gene families and in V beta and J beta gene segments. The D beta and J beta segments can join in the absence of V beta gene segment rearrangement and these rearranged sequences are transcribed in some T cells.     

Aberrant rearrangement of the kappa light-chain locus involving the heavy-chain locus and chromosome 15 in a mouse plasmacytoma

The creation of a functional antibody gene requires the precise recombination of gene segments initially separated on the chromosome. Frequently errors occur in the process, resulting in the formation of a non-functional gene. The non-functional genes can be generated by incomplete rearrangements, frameshifts, or the use of pseudo V or J joining segments. It is likely that these aberrant rearrangements arise by the same mechanism as is used in generating functional genes, a process which we have suggested may involve unequal sister chromatid exchange. Aberrant rearrangements of immunoglobulin genes occur in normal lymphocytes and play a major part in allelic exclusion. However, it has recently been suggested that aberrant rearrangements involving immunoglobulin and non-immunoglobulin genes may be involved in tumorigenesis. This suggestion has been stimulated by the frequent occurrence of translocations involving chromosomes known to carry immunoglobulin genes in B-cell malignancies. The rearrangement of non-immunoglobulin DNA to the heavy-chain locus has recently been reported. Some aberrant rearrangements of the kappa locus appear to be due to rearrangements to sites that do not include the conventional sequence for V gene segment joining. Here we describe an aberrant kappa rearrangement that has led to the joining of DNA from chromosomes 15, 6 and 12, and so appears to be the result of chromosomal translocations or transpositions. As 15/6 or 15/12 translocations have frequently been found in mouse plasmacytomas (as have analogous translocations in human lymphocyte tumours) this aberrant kappa rearrangement may be unique to the plasmacytoma from which it was isolated.     

Functional V region formation during in vitro culture of a murine immature B precursor cell line

B lymphocytes originate from pluripotential haematopoietic stem cells and differentiate into immunoglobulin (Ig)-producing cells. B-cell lineage differentiation is accompanied by two types of immunoglobulin gene rearrangements--rearrangement of V, D and J gene segments to create a functional V gene and rearrangement of CH genes for heavy-chain switching. These results, however, have been obtained mainly by analysis of immunoglobulin gene organization of myeloma cells. Baltimore and his colleagues have established Abelson murine leukaemia virus (A-MuLV)-transformed cell lines and found a few lines capable of carrying out kappa-gene rearrangement or undergoing isotype switching during in vitro culture. To study early B-cell lineage differentiation events, we have now also established A-MuLV-transformed cell lines which are capable of differentiating from mu- to mu+ and of undergoing continuing rearrangement of heavy-chain genes in culture. Analysis of immunoglobulin gene organization of these transformed cells revealed that mu- cells have already undergone DNA rearrangements involving JH segments but an additional rearrangement of JH segments is required for initiation of mu-chain synthesis. Southern blot analysis of the DNA and two-dimensional gel electrophoresis of intracytoplasmic mu-chain show that mu-chain diversity with respect to antigen specificity may be generated during this second rearrangement process. As no rearrangement of light-chain genes takes place in these cells, this implies that light-chain gene rearrangement requires some further change, or a different enzyme.     

Identical V beta T-cell receptor genes used in alloreactive cytotoxic and antigen plus I-A specific helper T cells

T lymphocytes involved in the cellular immune response carry cell-surface receptors responsible for antigen and self recognition. This T-cell receptor molecule is a heterodimeric protein consisting of disulphide-linked alpha- and beta-chains with variable (V) and constant (C) regions. Several complementary DNA and genomic DNA clones have been isolated and characterized. These analyses showed that the genomic arrangement and rearrangement of T-cell receptor genes using VT, diversity (DT), joining (JT) and CT gene segments is very similar to the structure of the known immunoglobulin genes. We have isolated two cDNA clones from an allospecific cytotoxic T cell, one of which shows a productive V beta-J beta-C beta 1 rearrangement without an intervening D beta segment. This V beta gene segment is identical to the V beta gene expressed in a helper T-cell clone specific for chicken red blood cells and H-21. The other clone carries the C beta 2 gene of the T-cell receptor, but the C beta 2 sequence is preceded by a DNA sequence that does not show any similarity to V beta or J beta sequences.     

V-J joining of immunoglobulin kappa genes only occurs on one homologous chromosome

In general, heterozygous animal cells express both alleles at a particular locus. The only exceptions are cells of XX genotype after inactivation of one X chromosome, and immunoglobulin-producing cells; in each case only one of the two alleles is expressed in differentiated cells and their progeny. This phenomenon, termed allelic exclusion, has been described for several mammalian species including man and mouse. It has been shown that the variable (V) and constant (C) region genes of immunoglobulins undergo a rearrangement during ontogeny. We wished to test whether allelic exclusion in B cells could be the consequence of V- and C-region rearrangement on one of the two homologous chromosomes only. For that reason we chose to analyse the rearrangement of immunoglobulin light chain genes in normal B lymphocytes isolated on the fluorescence-activated cell sorter. We now present evidence that during normal B-lymphocyte differentiation V-C rearrangement occurs only on one chromosome.     

Allograft theory: transmission of devil facial-tumour disease

The Tasmanian devil, a large carnivorous Australian marsupial, is under threat from a widespread fatal disease in which a malignant oral-facial tumour obstructs the animal's ability to feed. Here we show that the chromosomes in these tumours have undergone a complex rearrangement that is identical for every animal studied. In light of this remarkable finding and of the known fighting behaviour of the devils, we propose that the disease is transmitted by allograft, whereby an infectious cell line is passed directly between the animals through bites they inflict on one another.