DNA稳定的银纳米簇诱导的双酶催化转化检测研究

为了实现对碱性磷酸酶和邻-磷酸-L-酪氨酸的同时检测,本研究利用 DNA 稳定的银纳米簇（AgNCs／DNA）诱导的生物催化转化,基于醌类物质对 DNA 稳定的银纳米簇的荧光的猝灭效应,通过酪氨酸酶能与酪胺、多巴胺以及酪氨酸反应生成醌类物质,间接地达到了对酪胺、多巴胺以及酪氨酸的定量检测。AgNCs／DNA 还被进一步用于双酶催化级联反应,通过碱性磷酸氧化酶（ALP）和酪氨酸酶（TYR）组成的双酶系统,实现了对碱性磷酸酶和邻-磷酸-L-酪氨酸的高灵敏度检测,检测限分别达到2．5×10－5μmol·L－1和0．01μmol·L－1。     

酪胺信号放大-量子点标记银染增强的基因芯片可视化检测方法的建立

目的:建立一种酪胺信号放大-量子点标记银染增强的基因芯片可视化检测方法,提高基因芯片检测的灵敏度。方法:待测靶基因与固定在玻片上的探针杂交,依次加入链霉亲和素标记的辣根过氧化物酶、生物素标记的酪胺及链霉亲和素标记的量子点,37℃孵育,然后加入银增强试剂显色,最后用可视化生物芯片扫描仪扫描并记录结果;以牛布鲁菌210105株为检测对象,以酪胺信号放大-荧光素Cy3(TSA-Cy3)检测法为对照方法,测定酪胺信号放大-量子点标记银染增强(TSA-QDS)检测法的灵敏度。结果:确定了基因芯片量子点标记银染增强可视化检测方法的检测流程,优化了检测条件,并考察了检测灵敏度。优化的检测条件为:酪胺-生物素稀释比例为1∶4000,链酶亲和素标记的量子点稀释比例为1∶50,37℃孵育时间为25~30 min,银染增强时间为6~7 min。检测牛布鲁菌的灵敏度为103CFU/mL。结论:该方法实现了基因芯片高灵敏度可视化检测,其灵敏度与荧光法相当,并且有可视化的优势。     

SARS病毒巨细胞病毒合并感染真菌及其染色方法的应用比较

目的几种染色方法显示星形隐球菌和曲菌的比较。方法Grocott-Gomori六胺银改良法,Gomori氏嗜银法和PAS结果Grocott-Gomori氏六胺银改良法显示上述两种真菌效果最好,该法对真菌的显示颜色鲜艳,图像清晰,真菌的孢子和菌丝被染成深黑色,易与其它成份相区别;Gomori氏嗜银法对大量含有菌丝的组织有一定的染色效果,PAS法也能将上述两种真菌显示出来,但所着染成份较多,较难区别。结论Grocott-Gomori氏六胺银改良法是染真菌的最好方法。     

热休克因子1通过上调蛋白质C减轻脓毒症凝血功能障碍保护小鼠急性肺损伤

目的 探讨热休克因子1 （HSF1）减轻脓毒症凝血功能障碍,保护小鼠急性肺损伤的机制。方法 本研究采用盲肠结扎穿孔术（cecal ligation and puncture,CLP）制备脓毒症小鼠模型,检测凝血相关指标和观察小鼠肺部病理变化,通过酶联免疫吸附实验（ELISA）、q RT-PCR和蛋白质印迹法（Western blot）等方法检测蛋白质C表达水平,通过质粒转染抑制或增强HSF1表达从而观察蛋白质C表达水平的变化,并利用生物信息学、凝胶电泳迁移实验（EMSA）和双荧光素酶报告基因实验探讨HSF1调节蛋白质C转录的机制。结果 在CLP脓毒症小鼠模型中,HSF-/-组小鼠的凝血活性与HSF1^+/+组相比明显增强,肺损伤明显加重。ELISA、qRT-PCR和Western blot检测发现,HSF^-/-脓毒症小鼠血浆和肺组织中的蛋白质C表达水平显著低于野生型小鼠。体外bEnd.3血管内皮细胞的实验结果显示,HSF1抑制脂多糖（LPS）诱导的蛋白质C表达,HSF1过表达则增强蛋白质C表达。生物信息学数据分析提示,蛋白质C启动子区含有HSF...     

改良革兰染色法在显示石蜡切片真菌中的应用

目的建立真菌的病理切片改良革兰染色法。方法选取确诊真菌感染的组织蜡块,切若干空白片,通过苏木素-伊红染色（HE）、高碘酸-无色品红染色（PAS）、六胺银染色（GMS）、改良革兰染色后,比较改良革兰染色法的染色效果。结果改良革兰染色法的染色效果好,该法具有易操作、染色效果稳定等优点。结论改良革兰染色法能够清晰地显示出组织切片中的真菌,并且染色效果稳定,在检测组织中的真菌时可发挥重要的辅助诊断价值,具有广泛的应用前景。     

外源性精胺对缺氧诱导乳鼠心肌细胞凋亡的影响及其机制

目的：观察外源性精胺对缺氧所致的乳鼠心肌细胞凋亡的影响,并探讨其机制。方法：复制原代培养乳鼠心肌细胞缺氧损伤模型（使用pH=6.8的Hank's平衡盐溶液作为细胞培养基,排出氧气,然后在缺氧箱中培养24 h）,细胞随机分为正常对照（Control）组、缺氧（Hypoxia）组和精胺干预（Hypoxia+Sp）组。Western blot检测心肌细胞多胺代谢关键酶（ODC、SSAT）蛋白质表达;CCK-8,Hoechst 33342染色观察细胞凋亡情况;光吸收法检测细胞（或培养液）内T-SOD和Caspase-3/-9活性,MDA、GSH含量;DCFH-DA染色观察细胞内活性氧（ROS）生成。结果：与正常组相比,Hypoxia组SSAT蛋白质表达、细胞凋亡率、MDA含量以及细胞内ROS生成增加,而ODC蛋白质表达、SOD活性、GSH含量降低;与Hypoxia组比较,Sp处理可减轻上述指标的变化。结论：外源性精胺可减轻缺氧引起的乳鼠心肌细胞损伤和凋亡,其机制与恢复多胺稳态和清除活性氧有关。     

中性纤维素酶外切葡聚糖酶CBHⅡ的异源表达

利用聚合酶链式反应（PCR）技术,从本实验室保存的1株特异腐质霉EIM-50上克隆到中性纤维素酶外切葡聚糖酶基因（CBHⅡ）全长序列,大小约为1 586 bp。将其克隆到pPIC9K上,成功构建重组质粒pPIC9K-CBHⅡ,并经电转化引入毕赤酵母（Pichia pastoris）GS115,进行异源表达。SDS-PAGE银染结果表明,该重组质粒在毕赤酵母中获得了异源表达。gel pro Analyser软件分析其表达蛋白的表观分子量约为62.548ku。用BandScan软件分析其蛋白表达量为2.3%,即0.738μg/mL（mg/L）。     

微卫星DNA聚丙烯酰胺凝胶电泳(PAGE)银染法的改良

为了筛选和检测绵羊种群中具有较好表现的微卫星多态性,采用Touch-down PCR方法扩增绵羊基因组上的微卫星DNA序列后,经聚丙烯酰胺凝胶电泳分离,采用改良的银染法对微卫星位点进行多态性检测,染色结果与常规银染法相比,该方法具有灵敏度高、背景浅、污染小、条带清晰等突出特点,能有效地控制染色背景,显著提高检测分辨率,整个染色过程所需时间短,具有广泛的推广价值。     

沙冬青cDNA-AFLP银染和条带回收方法分析

通过比较而获得沙冬青cDNA-AFLP银染和条带回收的最佳方法.银染时采取Bassam法和Sanguinetti法,凝胶条带回收时利用直接回收法、试剂盒法、LiCl高盐法和聚丙烯酰胺凝胶高效回收法.比较不同方法对于开展聚丙烯酰胺凝胶电泳的影响.采用Bassam银染法对获得的扩增产物进行染色后无法得到差异显示条带,而利用Sanguinetti银染法显色后可观察到比较明显的差异表达条带;以直接回收法、PAGE试剂盒法、LiCl高盐法对差异条带进行回收后,二次PCR无法得到扩增产物,采用聚丙烯酰胺凝胶高效回收法后则可获得比较清晰的二次扩增产物.Sanguinetti银染法和聚丙烯酰胺凝胶高效回收法是应用于本研究的最佳方法.     

PI3K/Akt-aPKC_（ι/ζ）-Nrf2调控大鼠气道上皮细胞γ-谷氨酰半胱氨酸合成酶表达

目的：观察香烟烟雾提取物（CSE）是否通过磷酯酰肌醇-3-激酶（PI3K）/Akt-不典型蛋白激酶Cι/ζ（aP-KCι/ζ）-核因子相关因子2（Nrf2）信号通路调控大鼠气道上皮细胞γ-谷氨酰半胱氨酸合成酶（-γGCS）表达。方法：用Western blot法检测-γGCS、Nrf2、p-Akt和p-aPKCι/ζ蛋白质,细胞免疫化学法观察-γGCS蛋白质表达,反转录-聚合酶链反应（RT-PCR）法检测-γGCSmRNA,免疫荧光法检测Nrf2蛋白质,流式分析法检测p-Akt阳性细胞率,双酶法测定-γGCS活性,酶循环分析法测定总还原型谷胱甘肽（GSH）含量。结果：暴露CSE 3 h,GSH含量显著升高,Nrf2胞核蛋白质、p-aPKCι/ζ蛋白质、p-Akt蛋白质及其阳性细胞率、-γGCS蛋白质及其mRNA和活性均显著增强。aPKCι/ζ抑制剂RO813220明显减弱p-aPKCι/ζ蛋白质、-γGCS蛋白质及其mRNA和活性表达,但增强Nrf2胞浆蛋白质表达,对p-Akt无影响。p-Akt抑制剂LY294002及RO813220＋LY294002均降低p-aPKCι/ζ蛋白质、p-Akt蛋白质及阳性细胞率、-γGCS蛋白质及其mRNA和活性表达,增强Nrf2胞浆蛋白质表达。直线相关性分析显示Nrf2与-γGCS、p-Akt及p-aPKCι/ζ呈正相关,p-Akt与Nrf2、p-aPKCι/ζ及-γGCS呈正相关,p-aPKCι/ζ与Nrf2、p-Akt及-γGCS呈正相关（P〈0.05）。结论：CSE可能通过PI3K/Akt-aPKCι/-ζNrf2调节-γGCS表达。     

微量蛋白检测中不同银染方法的比较与分析

随着生物化学技术的不断发展,作为检测SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)中微量蛋白的银染方法也在不断改进和发展.采用4种不同的银染方法检测不同含量的牛血清白蛋白,结果显示单纯的银染过程中如果使用戊二醛固定会使蛋白检出更快速灵敏,而结合考马斯亮蓝的复合银染则较单纯银染灵敏度提高了5～7个数量级.     

A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels.

We have developed a highly sensitive stain for visualizing proteins in polyacrylamide gels. Our modification of the procedure for de Olmos' neural, cupric-silver stain is 100 times more sensitive than the conventional Coomassie blue stain (e.g., detection of 0.38 vs 38 ng/mm² of serum albumin), and is comparable to the sensitivity attained with an autoradiogram of ¹⁴C-methylated proteins following a 5-day exposure. This silver stain will be especially useful for analysis of patterns of proteins from tissue where attainment of the high specific activity of isotope labeling which is necessary to detect minor protein components is expensive, technically difficult or, as in humans, prohibited. In preliminary results with material such as unconcentrated cerebrospinal fluid, the silver stain revealed a complex pattern of proteins not visible with Coomassie blue.     

A rapid sensitive silver stain for polypeptides in polyacrylamide gels

The use of silver to detect polypeptides was originally achieved by modifying tissue stains. By adapting methods of photochemistry we have developed a new silver stain for polypeptides which is nearly as sensitive but much more efficient than these earlier procedures. The new silver stain utilizes only three solutions and allows protein patterns to be visualized within 50 min. Its sensitivity is 100 times that of the Coomassie blue stain.     

Analysis of differences between coomassie blue stain and silver stain procedures in polyacrylamide gels: conditions for the detection of calmodulin and troponin C

It is reported that the conditions used in some silver stain procedures can fail to detect calmodulin, troponin C, and other proteins with similar physical properties. Conditions are described that allow the reproducible detection of these proteins. Two phenomena are described: (1) lack of protein staining when treatment with glutaraldehyde is omitted from the protocol, and (2) loss of small proteins from the gel matrix during prolonged washing procedures. These data directly demonstrate that the use of some silver staining protocols can result in misleading data in biological studies and provide an explanation for at least one class of proteins of how silver staining and Coomassie blue staining of gels can give different results.     

Silver stain for proteins on a cellulose acetate membrane

A rapid and sensitive silver staining method to detect proteins on a cellulose acetate membrane has been established. This method is achieved by modification of the silver-based color staining for detection of proteins in polyacrylamide gels [D. W. Sammons, L. D. Adams, and E. E. Nishizawa, Electrophoresis 2, 135-141 (1981)] and applied to our new type of two-dimensional electrophoresis for analysis of proteins on a cellulose acetate sheet [T. Toda, T. Fujita, and M. Ohashi, Anal. Biochem. 119, 167-176 (1982)]. Maximal sensitivity of silver stain for proteins on a cellulose acetate membrane can be obtained by an optimal balance between deposition of silver on the protein and on the background. Certain kinds of proteins are colored red, orange, or grayish-blue. The silver stain is 20-80 times more sensitive than Coomassie blue and some spots are visualized reproducibly by silver only. Densitometric evaluation of standard proteins stained with silver and Coomassie blue is also demonstrated. The method takes only 50 min to perform and is sensitive, simple, and reproducible.     

Advanced negative detection method comparable to silver stain for SDS-PAGE separated proteins detection

In order to achieve an easy, rapid and sensitive protocol to detect proteins in polyacrylamide gel, an advanced negative detection method comparable to silver stain is described. When a gel was incubated with Phloxine B and followed by the development in acidic solution, the zones where forming protein-dye complex were selectively transparent, unlike opaque gel background. Within 50 min after electrophoresis, down to 0.1–0.4 ng of gel-separated proteins (similar with silver stain) could be observed, without labor-intensive and time-consuming procedure. Comparing with the most common negative stain method, Imidazole-zinc stain, Phloxine B stain has been shown higher sensitivity and distinct contrast between the transparent protein bands/spots and opaque background than those; furthermore, it is no longer necessary to concern about retention time of observation. This technique may provide a sensitive and practical choice for proteomics researches.     

Quantitation of protein and DNA in silver-stained agarose gels

A silver stain for both proteins and DNA in agarose gels is described. Quantitation of proteins with this stain is possible, with individual proteins exhibiting characteristic responses, as observed with other stains. The advantage of the silver stain over Coomassie blue is its increased (50- to 100-fold) sensitivity, which allows samples containing very low protein concentrations to be analyzed without prior concentration. This silver stain, when applied to DNA, is at least as sensitive as ethidium bromide, and gives a linear response for the type of DNA and fragment sizes studied.     

Double staining to increase the sensitivity of protein detection in polyacrylamide gels.

As more and more researchers are examining proteins that are available only in extremely limited quantities, i.e., cellular extracts or genetic engineering products, it is critical to utilize staining methods that maximize sensitivity. The protocol we describe here--double staining of polyacrylamide electrophoresis gels with Pro-Blue (colloidal blue stain) followed by silver staining--yields an extremely sensitive, nonspecific protein stain. On average, this double-staining technique resulted in a 40-fold increase in sensitivity and intensity vs. silver stain alone. This is a tremendous return for a small investment in additional time and materials.     

Silver stain for detecting 10-femtogram quantities of protein after polyacrylamide gel electrophoresis

A rapid and highly sensitive silver stain and color stain were developed for visualizing proteins. The procedure is simple and the bands were clear. This silver stain detects 100 pg quantities of proteins. In order to stain quickly, sensitively, and sharply a protein matrix in a gel, the repeated shrinkage and swelling gel was developed with a hyper- and hypotonic solution to remove the sodium dodecyl sulfate (SDS) from SDS-protein complex and to generate influx of staining solution into the gel. We have found that the silver staining method with the repeated exposure to hyper- and hypotonic solution and a narrow well produced 10 fg order of proteins.     

Histochemical properties of sulfated glycoconjugates in developing enameloid matrix of the fish Polypterus senegalus

I investigated the ultrastructural localization and histochemical properties of sulfated glycoconjugates in developing enameloid matrix of the fish Polypterus senegalus, by use of the high iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining and enzymatic digestion methods. HID-TCH-SP stain deposits were localized in the dental basal lamina and in the whole thickness of developing enameloid matrix, particularly closely associated with enameloid collagen fibrils. Most HID-TCH-SP stain deposits in the enameloid were susceptible to testicular hyaluronidase but some stain deposits survived. HID-TCH-SP stain deposits in the basal lamina resisted the enzymatic digestion, and were regularly localized to the internal and external sites of lamina densa at an early stage of development, subsequently tending to be randomly arranged with the increase in thickness of enameloid matrix layer. Furthermore, enzymatic digestion with heparitinase preferentially removed HID-TCH-SP stain deposits in the region of the basal lamina. Thus, it was confirmed that most HID-TCH-SP stain deposits in developing enameloid matrix are chondroitin 4-sulfate and/or 6-sulfate and that the stain deposits in the basal lamina represent heparan sulfate. The chondroitin sulfates tended to disappear with the advancement of enameloid mineralization.