贝类血细胞及其免疫功能研究进展

贝类由于是开放性系统,不存在特异性体液免疫,贝类的宿主免疫防御包括以血细胞为基础的细胞和体液系统。血细胞能够在体内或体外吞噬各种有机和无机颗粒,清除病原生物和自身损伤或死亡细胞,而且血细胞能够产生各种非特异性体液因子来参与宿主的免疫防御过程。所以贝类的血细胞在宿主免疫防御机制中起着重要作用。近年来,随着贝类养殖规模的扩大,贝类病害日益严重,给中国的贝类养殖业造成了重大经济损失[1,2]。所以研究贝类免疫学,特别是贝类血细胞的免疫防御功能和机制,以解决贝类养殖中发生的病害问题已成为研究的热点。本文依据国内外有关文献,就贝类血细胞的分类、结构及免疫功能方面的研究作一综述,为国内外研究提供理论基础及资料。1贝类血细胞结构、分类贝类血细胞的分类一直分歧很大,有人将其分为三类:无颗粒细胞、小颗粒细胞、大颗粒细胞,也有人将其分为四类:大颗粒细胞、小颗粒细胞、透明细胞、浆细胞,还有人将其分为八类,甚至几十类,至今无统一命名[3]。关于血细胞的分类,Moore等[4]根据血细胞超微结构和对染色反应的不同,将紫贻贝(Mytilus galloprovincialis)血细胞分为两大类,无颗粒的嗜碱性细胞和有颗粒的嗜酸性颗粒细胞...     

中华稻蝗血细胞染色方法的比较

通过Giemsa、Wright和Giemsa-Wright混合染色3种方法,对中华稻蝗Oxya chinensis(Thunberg)的血细胞进行了染色和血细胞的形态学观察,其结果是Giemsa染液染色时间较长,对细胞核染色效果较好,细胞质界限清晰,但对细胞质中的颗粒染色较差;Wright染液染色时间较短,对细胞核和细...     

硫代葡萄糖苷及其降解产物异硫代氰酸盐

硫代葡萄糖苷是一种含硫的次级代谢产物,广泛分布于十字花科植物中。不同的栽培种、不同的生理阶段、不同的组织部位以及不同的栽培条件,都会使植物中含有的硫代葡萄糖苷的含量和成分有所变化。当硫代葡萄糖苷经葡萄糖硫苷酶作用时会发生降解,生成异硫代氰酸盐等产物。采后的一系列处理会影响植物中硫代葡萄糖苷的含量。硫代葡萄糖苷的降解产物异硫代氰酸盐作为一种化学预防剂,能抑制阶段Ⅰ酶（phase Ⅰ enzyme）,诱导阶段Ⅱ酶（phaseⅡenzyme）,从而防止癌症的发生。目前对硫代葡萄糖苷的鉴定方法主要是高效液相色谱法,气相色谱法等。     

PAS染色及真菌荧光染色在皮下真菌病诊断中的应用

皮下真菌病(subcutaneous mycoses)是指侵犯真皮、皮下组织和骨骼的真菌感染[1],部分可播及周边组织,如足菌肿等,有些则沿淋巴管扩散,如孢子丝菌病、着色芽生菌病,对人体造成较大危害。这类疾病病程缓慢,早期临床表现不典型,为疾病的早期诊断带来一定困难。真菌培养是其诊断的“金标准”,然而,真菌培养阳性率偏低,重要的是部分早期病例不容易考虑到真菌感染,多在组织病理提示后才考虑到深部真菌感染的可能性。     

草鱼出血病病毒多肽的荧光染色

将草鱼出血病病毒（ＧｒａｓｓＣａｒｐＨｅｍｏｒｒｈａｇｅＶｉｒｕｓ,ＧＣＨＶ）置于还原性的溶液中,然后加入等体积的ＮａＨＣＯ３配制的异硫氰酸荧光索溶液进行多肽的标记,再经ＳＤＳ－ＰＡＧＥ分析,在紫外灯下即可检测到ＧＣＨＶ全部的１１个结构多肽的荧光带。该方法最小检测量为５００ｎｇ,由该方法回收的多肽具有抗原活性,可作为抗原进行免疫学实验。     

一种鉴定小鼠胚胎质量的双重荧光染色方法

尝试改进并建立一种较可靠的、适合小鼠胚胎质量参考鉴定的双重荧光染色(细胞计数)方法.与传统方法相比,本实验中抗羊脾细胞抗血清的最佳稀释度为1∶5,作用时间为30 min.豚鼠补体最佳稀释度为1∶5,两种分染染料H33342和PI的工作浓度均降低至5.3 μg/ml便可着色,染色时间延长至90 min,加大了染料工作液中柠檬酸钠的浓度,并在计数观察前增加压片这一步骤.染色过程中应注意避免血清物质对抗体补体的不良影响以及避光和快速的操作.双重荧光染色后小鼠胚胎内细胞团(ICM)细胞着色为蓝色,滋养层(TE)细胞着色为粉红色,数目清晰可辨.此方法可以作为一种简洁有效的小鼠胚胎质量鉴别参考方法.     

异质性荧光染色的巨噬细胞功能研究──Ⅱ．异质性荧光染色的巨噬细胞过氧化物酶活性

细胞化学研究文献报导,巨噬细胞中过氧化物酶的活性有明显的差异,只有部分巨噬细胞过氧化物酶呈阳性反应。本文用淀粉、白喉类毒素和卡介苗分别诱导和活化小鼠腹腔巨噬细胞,进行过氧化物酶反应,并以７６９０－Ｘｕ荧光染液复染后证明,酶反应阳性细胞呈蓝色荧光,而酶反应阴性细胞为淡蓝绿色和黄色荧光。实验表明,过氧化物酶阳性的巨噬细胞是分化程度低的幼稚细胞,因此,过氧化物酶的活性可作为低分化的巨噬细胞的一种标志酶。同时,本文用免疫荧光单克隆抗体间接染色法观察了三种物质诱导和活化的异质性荧光染色的巨噬细胞的分泌功能。     

用实时荧光PCR方法鉴定转基因玉米T14/T25

本研究以实时荧光PCR技术鉴定商业化种植的转基因玉米T14/T25品系。根据转基因玉米T14/T25转入的外源基因质粒图谱,设计转基因引物和探针进行PCR和实时荧光PCR检测,建立了转基因玉米品系鉴定的实时荧光PCR方法。实验结果表明,用TaqMan探针可检测到T14/T25产生的荧光信号,而对其他玉米品系则检测不到荧光信号,为转基因产品的鉴定检测提供了新方法。Abstract: To identify genetically modified (GM) maize T14/T25 lines, a real-time fluorescent PCR (RTF PCR) assay was performed in this study. Primers and Taqman probes specific for inserted genes in the T14/T25 were used to conduct the real-time fluorenscent (RTF) PCR and PCR assays. The RTF PCR method was established to detect and identify GM maize lines. The results show that the TaqMan probe could identify T14/T25 maize used, while other GM and NO-GM maize didn’t be detected. The RTF PCR could be a new method for detecting other genetically modified organism.     

马铃薯晚疫病菌侵染的Solophenyl Flavine荧光染色方法研究

在植物病害研究中,观察寄主植物被病原菌入侵的过程非常重要。Solophenyl Flavine 7GFE染料可附着于菌丝,在波长为330~380 nm的激发光下被激发出蓝色荧光。为了更好的观察寄主植物中病原真菌的侵染情况,本实验以Solophenyl Flavine 7GFE为染剂,对寄主植物中病原真菌的侵染情况进行了观察研究。结果显示,用0.002%(M/V)Solophenyl Flavine 7GFE溶于0.1 mol/L Tris-HCl(p H 8.5)配制的染色液染色5 min的效果最佳;使用95%乙醇溶液替代0.15%三氯乙酸(W/V)酒精溶液∶氯仿(V/V)(4∶1)对寄主植物叶片脱色的方法操作简便、毒害较低;染色时省略了番红预染步骤。将改良的染色方法用于晚疫病菌入侵的马铃薯叶片观察取得了良好的效果。该技术是一种改良的、快速有效、安全无毒的观察真菌菌丝入侵植物的荧光染色方法,也适用于观察其他真菌入侵寄主植物组织的过程。     

异质性荧光染色的巨噬细胞功能研究I．异质性荧光染色的巨噬细胞吞噬活性

利用淀粉多糖和免疫促进剂（白喉类毒素和卡介苗）诱导和活化小鼠腹腔巨噬细胞,观察了四种异质性荧光染色的巨噬细胞非特异性和特异性吞噬活性。实验证明,深蓝色和淡蓝色荧光的巨噬细胞是分化程度低的幼稚细胞,非特异性吞噬功能较弱,但在特异性吞噬过程中呈现了活跃的吞噬活性,特别是在免疫促进剂的活化下,它们的特异性吞噬功能显著增强、淡蓝绿色荧光的巨噬细胞是分化程度较高、非特异性和特异性吞噬功能最旺盛的巨噬细胞,而黄色荧光的巨噬细胞是分化程度最高、特异性吞噬功能较减退的巨噬细胞。     

Fluorescence detection of cationic amyloid fibrils in human semen

Cationic amyloid fibrils, including the Semen Enhancer of Virus Infection (SEVI), have recently been described in human semen. Simple methods for quantitating these fibrils are needed to improve our understanding of their biological function. We performed high-throughput screening to identify molecules that bind SEVI, and identified a small molecule (8E2), that fluoresced brightly in the presence of SEVI and other cationic fibrils. 8E2 bound SEVI with almost 40-fold greater affinity than thioflavin-T, and could efficiently detect high molecular weight fibrils in human seminal fluid.     

Mechanism of thioflavin T binding to amyloid fibrils

Thioflavin T is a benzothiazole dye that exhibits enhanced fluorescence upon binding to amyloid fibrils and is commonly used to diagnose amyloid fibrils, both ex vivo and in vitro. In aqueous solutions, thioflavin T was found to exist as micelles at concentrations commonly used to monitor fibrils by fluorescence assay ( approximately 10-20 microM). Specific conductivity changes were measured at varying concentration of thioflavin T and the critical micellar concentration was calculated to be 4.0+/-0.5 microM. Interestingly, changes in the fluorescence excitation and emission of thioflavin T were also dependent on the micelle formation. The thioflavin T micelles of 3 nm diameter were directly visualized using atomic force microscopy, and bound thioflavin T micelles were observed along the fibril length for representative fibrils. Increasing concentration of thioflavin T above the critical micellar concentration shows increased numbers of micelles bound along the length of the amyloid fibrils. Thioflavin T micelles were disrupted at low pH as observed by atomic force microscopy and fluorescence enhancement upon binding of thioflavin T to amyloid fibrils also reduced by several-fold upon decreasing the pH to below 3. This suggests that positive charge on the thioflavin T molecule has a role in its micelle formation that then bind the amyloid fibrils. Our data suggests that the micelles of thioflavin T bind amyloid fibrils leading to enhancement of fluorescence emission.     

Kinetics of amyloid aggregation of mammal apomyoglobins and correlation with their amino acid sequences

In protein deposition disorders, a normally soluble protein is deposited as insoluble aggregates, referred to as amyloid. The intrinsic effects of specific mutations on the rates of protein aggregation and amyloid formation of unfolded polypeptide chains can be correlated with changes in hydrophobicity, propensity to convert alpha-helical to beta sheet conformation and charge. In this paper, we report the aggregation rates of buffalo, horse and bovine apomyoglobins. The experimental values were compared with the theoretical ones evaluated considering the amino acid differences among the sequences. Our results show that the mutations which play critical roles in the rate-determining step of apomyoglobin aggregation are those located within the N-terminal region of the molecule.     

Effects of confinement on insulin amyloid fibrils formation

Insulin, a 51-residue protein universally used in diabetes treatment, is known to produce amyloid fibrils at high temperature and acidic conditions. As for other amyloidogenic proteins, the mechanisms leading to nucleation and growth of insulin fibrils are still poorly understood. We here report a study of the fibrillation process for insulin confined in a suitable polymeric hydrogel, with the aim of ascertain the effects of a reduced protein mobility on the various phases of the process. The results indicate that, with respect to standard aqueous solutions, the fibrillation process is considerably slowed down at moderately high concentrations and entirely suppressed at low concentration. Moreover, the analysis of the initial stages of the fibrillation process in aqueous solutions revealed a large spatial heterogeneity, which is completely absent when the fibrillation is carried out in the hydrogel. We attribute this heterogeneity to the diffusion in solution of large amyloidal aggregates, which must be formed very fast compared to the average times for the whole sample. These findings are interpreted in the framework of recently suggested heterogeneous nucleation mechanisms. Moreover, they may be useful for the development of new insulin pharmaceutical formulations, more stable against adverse conditions.     

Fullerene inhibits beta-amyloid peptide aggregation

We report that fullerene inhibits strongly the amyloid peptide aggregation at the early stage. It specifically binds to the central hydrophobic motif, KLVFF, of A beta peptides. The IC(50) value has been measured as 9 microM for both A beta(11-25) and A beta(1-40). On the other hand, a control experiment shows melatonin rather specifically binds to the C-terminus region. The IC(50) value of fullerene appears to be at least four times larger for A beta(1-40), compared with melatonin, and 15 times larger for A beta(11-25). This work shows that fullerene can be a promising candidate in search of AD therapeutics because it has the very high IC(50) value for A beta aggregation.     

Quantification of amyloid fibrils using size exclusion chromatography coupled with online fluorescence and ultraviolet detection

An amyloid fibrils investigation within biofilm samples requires distinguishing the amyloid β-sheet structure of these proteins and quantifying them. In this study, the property of amyloids to incorporate the fluorescent dye Thioflavin T has been exploited to propose a method of quantification. The experimental protocol includes the preparation of amyloids from commercial κ-casein (κCN) and their fractionation through size exclusion chromatography (SEC) to provide calibration curves from fluorescence and absorbance signals. Finally, a bacterial biofilm extract was injected into SEC, and the amyloid fibrils could be expressed as equivalent κCN, representing approximately 21% of the total proteins.     

Thioflavin T fluorescence assay for β-lactoglobulin fibrils hindered by DAPH

The molecule 4,5-dianilinophthalimide was recently found to be an efficient compound in disaggregating amyloid fibrils involved in the Alzheimer’s disease. In this study we have investigated whether the compound 4,5-dianilinophthalimide was able to disaggregate fibrils derived from β-lactoglobulin. In addition to a Thioflavin T fluorescence assay, flow-induced birefringence was used as an independent technique to measure the total length concentration of the fibrils. An additional advantage of the latter technique is that not only the total length concentration, but also the length distribution of the fibrils can be measured. The results from flow-induced birefringence showed that the total amount of fibrils and also the length distribution of the fibrils was not influenced by the addition of 4,5-dianilinophthalimide, even though this was suggested by the results of the Thioflavin T assay. The results of flow-induced birefringence were confirmed by rheological measurements and transmission electron microscopy. Our findings show that the use of a Thioflavin T assay in order to probe the possible disaggregating effect of certain compounds can give misleading results.