细菌及春毒素诱发小鼠胸腺细胞凋亡的机制初探

给ＢＡＬＢ／ｃ小鼠腹腔注射产肠毒素Ｂ金黄色葡萄球菌、绿脓杆菌或葡萄球菌肠毒素,均可引起小鼠腺萎缩,呈现胞腺重量减轻、胸腺细胞数减少、胸腺细胞存活率降低。进一步研究表明,小鼠胸腺细胞凋亡的机制可能与这些细菌或毒素诱导缩生产生ＴＮＦ－α、ＩＦＮｒ和ＩＬ－６等细胞因子有关。     

胸腺树突状细胞对胸腺细胞凋亡的促进作用

小鼠胸腺细胞在体外培养一定时间后自发出现凋亡,在与小鼠胸腺树突状细胞(MTSC4)共育后,其凋亡过程可被明显加速;与此相反,小鼠胸腺上皮细胞(MTECI)有抑制凋亡的作用.与MTSC4共育后的胸腺细胞中不仅CD4⁺CD8⁺双阳性细胞明显减小,而且CD4⁺CD8⁺单阳性细胞也减少.提示胸腺基质细胞可通过其对细胞凋亡的促进作用参与胸腺内的阴性选择,并提示阴性选择可能在胸腺髓质区仍在进行.     

葡萄球菌肠毒素B诱导脐静脉内皮细胞凋亡机制的探讨

目的探讨金黄色葡萄球菌(金葡菌)肠毒素B诱导脐静脉内皮细胞凋亡的机制。方法将不同浓度金葡菌肠毒素B感染脐静脉内皮细胞8 h后,用流式细胞术检测细胞凋亡率,同时用比色法检测TNF-α、caspase-3及caspase-8的产生量,并检测加入TNF-α抗体、caspase-3和caspase-8抑制剂后的细胞凋亡率。结果不同浓度肠毒素B作用脐静脉内皮细胞8 h后均可诱导细胞凋亡,且TNF-α、caspase-3和caspase-8的产生量均高于对照组(P0.01);而加入TNF-α抗体、caspase-3和caspase-8抑制剂后凋亡率明显降低。结论金葡菌肠毒素B可以诱导脐静脉内皮细胞凋亡,其凋亡机制可能是通过TNF-α介导的caspase-8及caspase-3激活的外源性死亡因子受体途径。     

CD3单抗诱导幼龄小鼠胸腺细胞凋亡研究

用抗小鼠ＣＤ３单抗刺激幼龄小鼠胸腺细胞,培养不同时间后,检测小鼠胸腺细胞的凋亡情况。结果表明,胸腺细胞呈现了凋亡的典型形态学改变。流式细胞仪检测可见凋亡细胞特有的ＡＰ峰,ＣＤ３单抗刺激未成熟胸腺细胞可以通过内源性的凋亡途径引起细胞死亡。     

胸腺细胞在胸腺外凋亡的形态学证据

In vitro thymus explants culture designed in this paper can mimic the thymic microenvironment as it were in vivo. Theoretically, thymus explants are cut off free from blood stream. So if some developing or developed thymocytes had the inclination to migrate into the periphery, they would only be accumulated in the blood vessels within thymus explants. After 3-day's culture, under transmission electron microscope we observed the migrating thymocytes accumulated in the blood vessels of C57BL/6 mice thymus explants, and these thymocytes were occurring apoptosis at different stage. To our knowledge, this findings offers the first morphological evidence that thymocytes do not necessarily die inside the thymus in situ, and that having acquired the death signals thymocytes can migrate into the blood stream and die quickly outside the thymus. But this is not to say that we deny the intrathymic death hypothesis. On the contrary, we found the number of thymocytes occurring in situ apoptosis on the surfaces of stromal cells is far more than that of migrating into the blood vessels. So, our proposal is that there are two sites for thymocytes apoptosis, some die inside the thymus and the others die outside the thymus.     

胸腺细胞在胸腺外凋亡的形态学证据

本文采用的胸腺外植块培养法实验既能够模拟胸腺微环境中胸腺细胞的发育周期又能够切断血液流通,理论上造成一种胸腺封闭状态(产生的胸腺细胞不能迁出胸腺外,假如某些胸腺细胞有迁出倾向,它也只能堆积于胸腺血管内)。培养3天后,在透射电子显微镜下观察到胸腺外植块血管内存在有胸腺细胞,并且处于不同的凋亡时期。本文的实验结果,在形态学上首次为胸腺外死亡假说找到了直接证据,说明胸腺细胞不必一定在胸腺原位死亡,它也可以获得死亡信号之后进入血管,随血液循环至外用最终死亡。但是这决不是意味着否定胸腺内死亡假说,因为我们同时观察到在胸腺基质细胞表面原位凋亡的胸腺细胞数量远超过进入血管后凋亡的胸腺细胞(但是在基质细胞表面凋亡的胸腺细胞并没有被基质细胞有效吞噬),所以我们的观点是:胸腺细胞同时存在胸腺内死亡和胸腺外死亡两种方式,一部分胸腺细胞在胸腺原位死亡的,而另一部分胸腺细胞是迁出胸腺后死亡溶解的。     

细胞膜分子在表达Fas配体的小鼠胸腺树突状细胞诱导凋亡中的作用

通过TUNEL方法证明在小鼠胸腺组织中 ,有凋亡的胸腺细胞存在 ,但胸腺微环境对胸腺细胞的凋亡的调控机理一直不明 .研究发现Fas配体 (FasL)阳性细胞主要分布在胸腺的髓质区 .小鼠胸腺树突状细胞系 (MTSC4)表达FasL ,在体外介导34 %的单阳性细胞的消除 ,其中使Fas阳性的胸腺细胞的比率减少 6 0 % .MTSC4促细胞凋亡的作用 ,可被单糖GalNAc完全阻断 ,被抗胸腺基质细胞TSC表面分子的单抗PF1 8- 3和 (D + )半乳糖部分阻断 .这说明胸腺树突状细胞直接参与对胸腺细胞凋亡的调节 ,其作用机理中包括多种膜分子的相互作用     

胸腺B细胞的研究

虽然胸腺是T细胞分化的中央淋巴器官,但也存在少量B细胞,约占全部胸腺细胞总数的1％。表型分析表明:14天小鼠胚胎胸腺就存在B细胞,CD5~+B细胞是胸腺B细胞的主要类群,而人类MG患者胸腺B细胞则表达CD71,CD23,B8.7等B细胞活化标志,并分泌抗-AChR自身抗体。胸腺B细胞可能对T细胞发育的负向选择起重要作用。     

白色念珠菌诱导小鼠胸腺细胞凋亡通路的探究

研究发现白色念珠菌诱导小鼠胸腺细胞凋亡与白色念珠菌上调小鼠血清ＴＮＦ α水平、胸腺细胞ｃａｓｐａｓｅ 3的活性及ｂａｘ ,ｐ5 3基因表达有关。尾静脉注射 4× 10 6白色念珠菌 ,于 2 4ｈ处死小鼠 ,用流式细胞仪检测发现白色念珠菌组小鼠胸腺细胞凋亡百分率 (2 3.6 3± 1.85 ) %显著高于正常对照组 (1.87±0 .81) %,Ｐ <0 .0 1。ＤＮＡ琼脂糖凝胶电泳结果显示 ,白色念珠菌组呈现典型的凋亡“梯状带” ,而正常对照组无“梯状带”出现。ＥＬＩＳＡ方法检测小鼠血清ＴＮＦ α水平结果发现 ,正常对照组小鼠血清ＴＮＦ α小于 2 5ｎｇ/Ｌ ,而…     

补充L-精氨酸对热应激大鼠胸腺细胞凋亡的影响

探导热应激对大鼠胸腺细胞的影响及补充L 精氨酸 (L Arg)对胸腺细胞的作用 ,结果显示 :1.在常温下 ,胸腺细胞结构清晰 ,形态规则 ,自然凋亡率低 ,补充L Arg组自然凋亡率低于灌水对照组 (P <0 .0 1) ;2 .不同温度的热应激均可导致胸腺细胞凋亡 ,电镜和光镜显示其典型的形态学特征 ;3.DNA凝胶电泳呈“梯状”条带 ,FCM检测出现凋亡峰 ;4 .补充L Arg的两组上述结果轻于灌水对照组。热应激后 ,2h便可见到凋亡细胞 ,4 - 8h达高峰。补充L Arg组 2 4h后基本恢复到正常水平 ,而灌水对照组稍差。说明适量补充L Arg对热应激大鼠胸腺具有较好的保护作用     

安徽不同地区奶站牛奶中金黄色葡萄球菌及其肠毒素污染状况评估

目的评定安徽地区各奶站牛奶中金黄色葡萄球菌以及肠毒素的污染情况。方法通过从安徽省不同地区30所奶站采集乳样,进行乳源性金黄色葡萄球菌的分离与生化鉴定,并采用PCR技术对分离出的菌株进行金黄色葡萄球菌肠毒素血清型鉴定。结果安徽省30个奶站中有4个地区奶站的牛奶中污染金黄色葡萄球菌;从污染牛奶中共分离出5株金黄色葡萄球菌,检出率为16.7%。经鉴定,所分离出的金黄色葡萄球菌中2株为肠毒素A型,1株为肠毒素C型,2株为同时产肠毒素A和肠毒素C。结论安徽省不同地区奶站中的牛奶污染的金黄色葡萄球菌产肠毒素类型以肠毒素A为主。     

Evaluation of antibacterial activity induced by Staphylococcus aureus and Ent A in the hemolymph of Spodoptera littoralis

The problem of antibiotic resistance considers one of the most dangerous challenges facing the medical field. So, it is necessary to find substitutions to conventional antibiotics. Antimicrobial peptides (AMPs) are a bio-functional derivative that have been observed as one of the important solutions to such upcoming crisis. Owing to their role as the first line of defense against bacteria, fungi, and viruses. This study was conducted to induce the immune response of Spodoptera littoralis larvae by inoculation of sub lethal doses of Staphylococcus aureus and its enterotoxin. Since Staphylococcal enterotoxin A (SEA) considers the major causative agents of Staphylococcal food poisoning, our study oriented to purify and characterize this toxin to provoke its role in yielding AMPs with broad spectrum antimicrobial activity. A great fluctuation was recorded in the biochemical properties of immunized hemolymph not only in the total protein content but also protein banding pattern. Protein bands of ∼22 kDa (attacin-like) and ∼15 kDa (lysozyme-like) were found to be common between the AMPs induced as a result of both treatments. While protein bands of molecular weight ∼70 kDa (phenoloxidase-like) and ∼14 kDa (gloverin-like) were found specific for SEA treatment. Chromatographic analysis using HPLC for the induced AMPs showed different types of amino acids appeared with differences in their quantities and velocities. These peptides exhibited noticeable antimicrobial activity against certain Gram-positive and Gram-negative bacteria. In conclusion, the antimicrobial potential of the antimicrobial peptides (AMP) induced in the larval hemolymph of S. littoralis will be a promising molecule for the development of new therapeutic alternatives.     

病原菌耐药性与血清PCT水平关系的分析

目的 了解病原菌不同的耐药性对患者血清降钙素原（PCT）水平的影响。方法 运用酶联免疫荧光法检测30例普通耐药铜绿假单胞菌感染患者,30例泛耐药铜绿假单胞菌感染患者,30例普通耐药金黄色葡萄球菌感染患者,30例泛耐药金黄色葡萄球菌感染患者,以及40例健康体检者（对照组）血清中PCT的水平。结果 铜绿假单胞菌感染组患者PCT水平明显高于金黄色葡萄球菌感染组（P<0.01）,且两组均明显高于对照组（P均<0.05）,普通耐药菌组患者PCT水平明显低于泛耐药菌组（P均<0.01）,且两组均明显高于对照组（P均<0.05）。结论 革兰阴性菌感染时,PCT水平显著高于革兰阳性菌感染,且病原菌耐药性越高,PCT水平升高越显著。     

Interaction between granulocytes and antibodies in the enhancement of lung defenses against Staphylococcus aureus after intranasal immunization of mice with live-attenuated bacteria

Abstract Immunization with live-attenuated Staphylococcus aureus induced measurable levels of specific IgG and IgA in the lungs, but the pulmonary clearance of S. aureus in immunized mice did not differ from that of control mice. Aerosol exposure of mice to Pseudomonas aeruginosa induced a significant recruitment of polymorphonuclear leukocytes (PMNL) to the lungs in both immunized and control mice, whereas S. aureus challenge did not. However, challenge with a mixture of P. aeruginosa-S. aureus or exposure to an aerosol of Escherichia coli lipopolysaccharide (LPS) before S. aureus challenge induced PMNL migration and a significant enhancement of pulmonary clearance of S. aureus in immunized mice. The presence of both antibodies and PMNL was required for enhancement of S. aureus pulmonary clearance.     

Structural basis for the neutralization and specificity of Staphylococcal enterotoxin B against its MHC Class II binding site

Staphylococcal enterotoxin (SE) B is among the potent toxins produced by Staphylococcus aureus that cause toxic shock syndrome (TSS), which can result in multi-organ failure and death. Currently, neutralizing antibodies have been shown to be effective immunotherapeutic agents against this toxin, but the structural basis of the neutralizing mechanism is still unknown. In this study, we generated a neutralizing monoclonal antibody, 3E2, against SEB, and analyzed the crystal structure of the SEB-3E2 Fab complex. Crystallographic analysis suggested that the neutralizing epitope overlapped with the MHC II molecule binding site on SEB, and thus 3E2 could inhibit SEB function by preventing interaction with the MHC II molecule. Mutagenesis studies were done on SEB, as well as the related Staphylococcus aureus toxins SEA and SEC. These studies revealed that tyrosine (Y)46 and lysine (K)71 residues of SEB are essential to specific antibody–antigen recognition and neutralization. Substitution of Y at SEA glutamine (Q)49, which corresponds to SEB Y46, increased both 3E2’s binding to SEA in vitro and the neutralization of SEA in vivo. These results suggested that SEB Y46 is responsible for distinguishing SEB from SEA. These findings may be helpful for the development of antibody-based therapy for SEB-induced TSS.     

金黄色葡萄球菌外毒素B特异性适体的筛选及其应用

目的:利用指数富集配基的系统进化(SELEX)技术,筛选能与金黄色葡萄球菌外毒素B(SEB)特异、高亲和力结合的单链DNA(ssDNA)适体,并将该适体应用于患者血清标本的检测。方法:从体外合成的96核苷酸随机ss-DNA文库中,以羧基磁珠作为筛选介质,经逐步PCR扩增、筛选,获得针对SEB的高亲和力、高特异性适体;利用荧光素标记适体测定筛选过程中各轮结合力;利用酶连接适体方法检测适体特异性和结合力。结果:经过13轮筛选,ssDNA文库与SEB的结合百分率从1.1%提高到39.8%,增加了36倍;获得的ssDNA适体(A11)针对SEB的特异性强,与金黄色葡萄球菌表面蛋白A(SPA)结合低,并能初步识别患者血清。结论:利用SELEX技术筛选获得了特异结合SEB的高亲和力的ssDNA适体,为金黄色葡萄球菌的临床诊断与治疗奠定了基础。     

胸腺细胞表面岩藻糖化抗原在细胞凋亡过程中变化的初步研究

细胞表面糖在细胞分化及细胞周期中均有一定的变化,而且还与细胞间的识别与信息传递有关,为了解膜表面糖复合物在细胞凋亡过程中的作用,通过地塞米松诱导小鼠胸腺细胞凋亡为模型,利用对８种抗原结构相关的寡糖特异的单克隆抗体,观察凋亡过程中胸腺细胞表面岩藻糖化糖抗原结构的变化。免疫组化的分析结果表明：正常胸腺细胞表面的糖抗原主要是含有岩藻糖基的Ｈ－２和Ｌｅ￣ｂ．而凋亡的胸腺细胞表面出现ＧｌｃＮＡｃβ１－３Ｇａｌ－,Ｇａｌβ１－４ＧｌｃＮＡｃβ１－３Ｇａｌ－及双岩藻糖化抗原Ｌｅ￣Ｙ,同时Ｌｅ￣ｂ消失。磷脂提取结果表明在给药３ｈ后膜的ＰＳ条带明显增加,通过对诱发细胞凋亡过程中组化分析的时相变化观察发现：凋亡细胞膜表面糖抗原的变化在给药１ｈ（即凋亡发生前）就出现。以上结果说明凋亡过程中胸腺细胞表面岩藻糖化抗原发生了变化,且此变化可能与细胞凋亡的始发有关。     

羧基荧光素脂质体的制备及其与病原菌的响应行为

【背景】由于目前临床上检测细菌感染的方法既耗时又昂贵,所以开发快速简便的鉴别方法势在必行。【目的】通过在脂质体膜材料中添加不同的稳定剂,研究它们对于脂质体稳定性的影响,并探究荧光素脂质体与病原菌之间的响应情况。【方法】以磷脂酰胆碱和胆固醇为主要原料,1,2-棕榈酰磷脂酰甘油[1,2-Dipalmitoyl-sn-glycero-3-phospho-(1?-rac-glycerol),DPPG]、十八胺和10,12-二十三二炔酸(10,12-Tricosadiyonic acid,TCDA)为稳定剂,采用薄膜分散-超声法制备羧基荧光素脂质体。利用病原菌能够分泌毒力因子造成脂质体渗漏的原理,将病原菌菌液和上清液分别与荧光素脂质体孵育,检测从脂质体渗漏的羧基荧光素的荧光强度,反映病原菌的毒性程度。【结果】DPPG和TCDA都能增加脂质体的稳定性,而十八胺的添加则导致脂质体稳定性的降低。与金黄色葡萄球菌ATCC25923和铜绿假单胞菌PAO1菌液和上清液共同孵育的脂质体荧光强度大量增加,与大肠杆菌DH5α和PBS缓冲液共同孵育的脂质体荧光强度几乎不变。【结论】能够分泌外毒素的金黄色葡萄球菌和铜绿假单胞菌都能对脂质体产生响应,而不分泌外毒素的大肠杆菌则不会对脂质体产生响应。