TassC结合过氧化物酶体对早期受染巨噬细胞内的鼠伤寒沙门菌的影响

探讨鼠伤寒沙门菌在感染鼠巨噬细胞早期与细胞器的相互作用。用pTassC-GFP质粒转染鼠巨噬细胞RAW264.7,结合多抗的溶酶体标志物溶酶体相关膜蛋白-1用键合了Alexa594的羊抗鼠二抗显色,以观察标记了绿色荧光蛋白的TassC与溶酶体的关系;用pTassC-GFP和pDsRed2-Perxi质粒共转染RAW264.7细胞,以观察TassC-GFP与过氧化物酶体的关系;用SYTO42标记鼠伤寒沙门菌,感染用pTassC-GFP和pDsRed2-Perxi质粒共转染的RAW264.7细胞,以观察细菌与TassC和过氧化物酶体的关系。免疫荧光显示TassC-GFP不与鼠巨噬细胞RAW264.7中的溶酶体结合,但与标记了红色荧光的过氧化物酶体共定位;感染1 h的RAW264.7胞内SYTO42标记的鼠伤寒沙门菌吞噬泡可招募TassC-GFP和过氧化物酶体。这些发现提示在鼠伤寒沙门菌感染早期过氧化物酶体携带杀菌成分通过TassC介导可参与发挥一定的杀菌作用。     

丝裂原活化的细胞外信号调节激酶在金黄色葡萄球菌诱导的U937细胞凋亡中的作用

目的探讨细胞外信号调节激酶（ERK1／2）在金黄色葡萄球菌（简称金葡菌）诱导的人巨噬细胞系U937细胞凋亡中的作用。方法采用AnnexinVFITc／PI双染流式细胞仪检测U937细胞凋亡,用Westernblotting方法分析不同作用时间MEK和ERK1／2的磷酸化水平。预先用不同浓度的PD98059（ERK途径抑制剂）处理U937细胞1h,观察金葡菌感染30min后U937细胞的凋亡情况。结果U937细胞经过金葡菌处理后,发生凋亡,细胞凋亡率呈时间依赖性升高;随着感染时间的延长,MEK和ERK1／2的磷酸化水平逐渐增加,尤以ERK2比较明显。U937细胞的凋亡可被PD98059抑制。结论金葡菌以时间依赖的方式诱导U937细胞凋亡;金葡菌诱导U937细胞凋亡的效应与激活ERK1／2信号转导通路有关。     

自噬对鼠伤寒沙门菌所致的巨噬细胞凋亡的影响

为探讨鼠伤寒沙门菌与巨噬细胞共作用时细胞自噬对凋亡的影响,用加入自噬诱导剂雷帕霉素(Rapamycin,RAPA)和未加RAPA的RPMI1640过夜培养小鼠腹腔巨噬细胞J774A.1,以携带一分子量为100kb毒力质粒的鼠伤寒沙门菌标准毒株SR-11为受试菌。首先测定RAPA对菌量及细胞活性的影响,然后建立细胞感染模型,在细菌与细胞共作用后动态观察24h,不同时间点检测细胞超微结构变化、自噬泡的形成、Beclin-1和Bcl-2的表达、细胞存活率和胞内活菌计数以及细胞凋亡情况。结果显示,RAPA单独作用于细菌或细胞时菌量及细胞活性均无变化;而对细胞感染模型而言,RAPA作用与否细胞内的细菌数及细胞存活率均有显著改变,RAPA可明显降低细胞内活菌数及其所致的巨噬细胞凋亡率(P0.05);RAPA干预组在细菌与细胞共作用早期,部分细菌可被双层膜包裹形成自噬泡,细胞超微结构正常;Beclin-1的表达量增加,而Bcl-2的表达量降低;后期细胞破坏程度明显轻于未用RAPA组。以上结果提示,通过调控细胞自噬水平以减轻宿主细胞凋亡,可作为防治某些感染性疾病的新途径。     

金葡菌通过线粒体途径诱导U937细胞凋亡

目的探讨线粒体凋亡途径在金黄色葡萄球菌（简称金葡菌）诱导人巨噬细胞系U937细胞凋亡中的作用。方法当细胞：细菌为1∶20时分别培养0 min,15 min,30 min,60 min和90 min,采用Western blot法检测胞质细胞色素C的表达及细胞内Bcl-2、Bax、caspase-9和caspase-3的表达。结果随着金葡菌感染时间的延长,胞质细胞色素C和Bax的表达逐渐增加;Bcl-2蛋白的表达逐渐降低;caspase-9和caspase-3的表达逐渐增加。结论金葡菌可通过抑制Bcl-2表达和促进Bax表达引起线粒体细胞色素C释放入胞质,激活caspase-9和caspase-3,促进U937细胞凋亡。     

猪圆环病毒2型通过外泌体miR-125a-5p靶向Bcl-2诱导淋巴细胞凋亡

为研究miR-125a-5p在猪圆环病毒2型(porcine circovirus type 2,PCV2)诱导淋巴细胞凋亡中的作用及其作用机制,以PCV2感染PK-15细胞外泌体孵育的淋巴细胞为研究对象,采用流式细胞术、蛋白质免疫印迹试验(Western blotting)和实时荧光定量PCR,检测淋巴细胞凋亡率及凋亡相关miRNA表达;合成miR-125a-5p模拟物和抑制物转染PK-15细胞,检测miR-125a-5p过表达或抑制表达后细胞凋亡率;采用生物信息学方法预测miR-125a-5p的靶基因,双荧光素酶报告基因检测miR-125a-5p对靶基因的调控;Western blotting检测外泌体孵育淋巴细胞的线粒体凋亡信号通路相关蛋白Bcl-2、Bax、细胞色素C和caspase-3的表达。结果显示,感染PCV2的PK-15细胞分泌的外泌体极显著提高淋巴细胞凋亡率,在一定浓度范围内呈剂量依赖性;与PCV2诱导细胞凋亡相关的miRNA中,miR-125a-5p表达量极显著升高,miR-125a-5p模拟物转染细胞后极显著提高细胞凋亡率;利用TargetScan预测发现,miR-125a-5p与Bcl-2 3''UTR区有结合位点,miR-125a-5p模拟物极显著抑制pmir-Bcl-2 3''UTR-WT荧光素酶活性,对pmir-Bcl-2 3''UTR-MuT的荧光素酶活性无明显改变;外泌体孵育的淋巴细胞Bcl-2表达量显著降低,Bax、细胞色素C的释放和caspase-3表达量显著升高,Bcl-2/Bax的比值极显著降低。这表明,PCV2通过外泌体诱导淋巴细胞上调miR-125a-5p的表达,进而抑制Bcl-2 mRNA和蛋白表达,激活淋巴细胞线粒体凋亡通路诱导细胞凋亡。     

鼠伤寒沙门菌rtsB基因缺失株的构建及其生物学特性

【背景】鼠伤寒沙门菌(Salmonella typhimurium)是一种重要的人畜共患病原菌,严重危害养殖业及人类健康。调控蛋白在病原菌的生存及感染过程中发挥重要作用。【目的】构建鼠伤寒沙门菌调控基因rtsB缺失株和互补株,分析调控蛋白RstB对鼠伤寒沙门菌生物学特性和致病性的影响。【方法】利用Red同源重组的方法构建鼠伤寒沙门菌SAT52的rtsB基因缺失株,并利用互补质粒构建互补株。然后比较分析野生株SAT52、缺失株?rtsB和互补株C?rtsB的生长特性、运动性、生物被膜形成能力、黏附入侵能力、胞内存活能力及致病性的差异。【结果】缺失rtsB基因不影响SAT52的生长速度,但导致运动能力增强,生物被膜形成能力减弱。细胞感染试验结果表明,rtsB基因有助于鼠伤寒沙门菌对Hela细胞的黏附入侵及RAW264.7细胞内的存活。动物试验结果表明rtsB基因缺失显著降低鼠伤寒沙门菌的致病力。【结论】rtsB基因在鼠伤寒沙门菌感染过程中发挥重要作用,可为阐释鼠伤寒沙门菌的致病机制提供参考。     

菌群失调小鼠感染鼠伤寒沙门菌后Th1和Th2细胞因子的动态研究

目的研究鼠伤寒沙门菌感染小鼠在菌群失调下Th细胞因子的动态变化,以探讨菌群失调对沙门菌感染的免疫机制。方法分别建立菌群失调、感染和空白对照的小鼠模型。各组动物在感染不同时点处死,观察小肠、肝脏和脾脏病理改变。采用流式细胞仪检测脾脏细胞中IFN-γ和IL-4表达,以此代表Th1和Th2细胞。结果菌群失调组病理损害最重,Th1明显增加,表达水平高,Th2变化不大,Th1/Th2比值升高。结论菌群失调后,能够加重小鼠感染鼠伤寒沙门菌引起的的Th1型反应,产生炎症性损伤。     

沙门菌- 斑马鱼感染模型用于Th1/Th2 免疫应答的研究

目的:通过构建斑马鱼成鱼感染模型,研究鼠伤寒沙门菌感染对机体Th1/Th2免疫应答的影响。方法:用不同剂量细菌口饲感染8月龄的斑马鱼成鱼,绘制3周生存率曲线。观察各剂量下对成鱼的感染情况,并用实时荧光定量聚合酶链式反应检测肝脏Th1、Th2型细胞相关基因和细胞因子的mRNA转录水平,计算Th2/Th1相对表达量比值。结果:用105 CFU感染2周斑马鱼全部存活,第15天开始出现死亡,且在3周后死亡率达到50%;感染后3周解剖发现,肝脏、脾脏和肠道有明显红肿和糜烂;肝脏Th1、Th2型细胞相关基因和细胞因子mRNA转录水平明显向Th2偏移。结论:用105 CFU鼠伤寒沙门菌口饲感染斑马鱼成鱼构建的模型,能反映机体感染和免疫功能变化,可用于研究体内Th1/Th2免疫应答,为进一步研究鼠伤寒沙门菌感染与免疫机制提供了很好的实验工具。     

鼠伤寒沙门菌spvB基因缺陷突变株的构建

高度保守的spvB基因,其编码产物具有ADP核糖基转移酶活性,是导致受染后细胞发生凋亡的重要毒力因子.为进一步研究该基因致病机制,构建了鼠伤寒沙门菌spvB基因缺陷突变株.根据GenBank鼠伤寒沙门菌spvB基因序列,用Primer Premier 5.0设计PCR特异性引物,获得spvB基因缺陷性核苷酸片段后连接至自杀质粒PGMB151.将构建的自杀载体导入含有spvB基因的鼠伤寒沙门菌标准株SR-11中进行同源重组,PCR筛选缺陷突变株.经PCR和DNA序列测定,成功获得了spvB基因缺陷的鼠伤寒沙门菌突变株.     

M16添加硫氧还蛋白过氧化物酶Ⅱ无法克服昆明小鼠早胚体外发育2-细胞阻滞

目的观察硫氧还蛋白过氧化物酶Ⅱ(Peroxiredoxin Ⅱ,PrxII)是否可以克服昆明(Kunming)小鼠早胚体外发育2-细胞阻滞。方法取昆明小鼠1-细胞胚置于含PrxII蛋白的M16培养液中培养,观察PrxII对昆明小鼠早胚发育潜能和2-细胞胚内活性氧自由基(reactive oxygen species,ROS)水平的影响;同时比较昆明和B6C3F1小鼠1-细胞胚在M16中各自的发育情况;激光扫描共聚焦显微镜分别检测比较昆明与B6C3F1小鼠体外培养2-细胞胚内ROS水平以及昆明小鼠体外培养与体内发育2-细胞胚内ROS水平。结果M16培养液中添加PrxII蛋白(1nmol/L和100nMol/L)可以明显降低昆明小鼠体外培养2-细胞胚内ROS水平(P<0.05),但不能克服昆明小鼠体外发育2-细胞阻滞;昆明小鼠1-细胞胚在M16中培养存在2-细胞阻滞现象,而B6C3F1小鼠无2-细胞阻滞现象;昆明小鼠体外培养2-细胞胚内ROS水平显著低于体内发育2-细胞胚(P<0.05),亦略低于B6C3F1小鼠体外培养2-细胞胚内ROS水平(P>0.05)。结论M16培养液中添加PrxII可以明显降低2-细胞胚...     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.