2种真菌多糖荧光标记及其取代度和稳定性

通过对小刺猴头发酵浸膏多糖和黑木耳发酵浸膏多糖进行荧光标记,得到2种真菌多糖的荧光标记产物,即小刺猴头菌多糖-FITC和黑木耳多糖-FITC。通过紫外分光光度计、酶标仪和荧光分光光度计对标记后的真菌多糖进行扫描,并进行运算,得到该2种真菌多糖的荧光物质的取代度,分别为0.79%和0.71%。同时对标记后多糖的体外稳定性进行研究,结果表明,小刺猴头发酵浸膏多糖标记物和黑木耳发酵浸膏多糖标记物在PBS溶液、血浆及尿液中稳定性良好。     

小刺猴头菌发酵浸膏寡糖对荷瘤小鼠免疫功能的影响

通过建立S180荷瘤小鼠试验动物模型,研究小刺猴头菌发酵浸膏寡糖对S180荷瘤小鼠免疫功能的影响。选择灌胃的方法将不同组别小刺猴头菌发酵浸膏寡糖喂服到荷瘤小鼠体内,观察肿瘤生长情况,对其进行免疫功能的测定。结果表明:小刺猴头菌发酵浸膏寡糖可增加荷瘤小鼠免疫器官脾脏和胸腺质量,提高荷瘤小鼠血清的IL-2和TNF-α含量,抑制肿瘤生长,提高机体免疫功能。     

真菌多糖与花生凝集素的相互作用

通过加入虫草多糖、树舌多糖、灵芝多糖、榆耳多糖、银耳多糖、小刺猴头菌多糖、黑木耳多糖和云芝多糖8种真菌多糖,观察真菌多糖对PNA与血细胞表面受体结合的影响。试验结果表明:小刺猴头菌多糖具有较强的与PNA的结合能力,这可能是由于小刺猴头菌多糖的空间结构或其上的半乳糖的连接方式、空间位置更适合与PNA结合的缘故。     

小刺猴头菌发酵液小分子指纹图谱与多糖含量相关性研究

试验研究了小刺猴头菌发酵液小分子指纹图谱与多糖含量的相关性。在小刺猴头菌摇床培养发酵液整个发酵周期内取得的样品经石油醚、二氯甲烷和乙酸乙酯依次萃取,HPLC检测分析。选择二氯甲烷层的3种小分子物质含量为主要研究对象,通过SAS软件和SIMCA-P 11.5软件对其与发酵液多糖进行相关性分析。结果表明:发酵液二氯甲烷层的3,4,5号峰小分子与发酵液多糖在含量变化上呈极显著相关(P<0.01)。     

刺芹侧耳菌丝体固体发酵多糖在斑马鱼模型上的降血脂活性

高脂血症是诱导脂肪肝、高血压、动脉粥样硬化和心脑血管疾病的一个关键风险因素。本课题组前期研究已经在细胞水平、小鼠和斑马鱼动物模型上证实,刺芹侧耳多糖具有抑制体内脂质积累的生物活性。本文利用农林废弃物玉米芯和麦麸作为主要培养原料,通过固体发酵获得刺芹侧耳菌丝体多糖PESF（polysaccharide from Pleurotus eryngii mycelium solid-state fermentation）;进而,采用斑马鱼幼鱼和成鱼高脂动物模型,研究了刺芹侧耳菌丝体多糖在动物体内的降脂效率并解析了可能的降血脂途径和机理。实验结果证实,剂量400μg/mL的PESF不仅可显著抑制高脂饮食引起的斑马鱼幼鱼体内脂质积累,而且也可以有效抑制高脂饮食下斑马鱼成鱼的肝脏和肠道组织内的脂质积累,证实刺芹侧耳多糖具有显著抑制动物体内脂质积累的活性。这些结果建议,刺芹侧耳多糖降血脂的途径可能是通过降低肠道对脂类物质的吸收,从而减少了脂滴在肝脏中的积累。因此,本研究建议刺芹侧耳多糖具有开发成为降脂食品添加剂或者降脂药物原料的潜力。     

金针菇多糖的提取工艺优化及荧光标记研究

通过响应面法优化金针菇多糖热水浸提的最佳工艺,并用异硫氰酸荧光素(FITC)标记金针菇多糖(FVP),研究标记多糖的体外稳定性和细胞毒性。结果表明:优化的最佳浸提条件为液料比41∶1(m L∶g)、提取温度92℃、提取时间3 h,金针菇多糖的提取率为4.87%;通过还原胺化反应实现了金针菇多糖的FITC标记,FITC的取代度为0.90%,在24 h内标记的金针菇多糖体外稳定性良好,且不影响FVP的抑瘤活性。     

猴头菌丝多糖降血糖作用研究

目的:研究猴头菌丝多糖的降血糖作用。方法:以液体发酵生产的猴头菌丝体为原料,经热水浸提、浓缩、酒精沉淀获得菌丝粗多糖;以常规降糖药物格列本脲为阳性治疗对照,通过四氧嘧啶诱发小鼠糖尿病的预防试验,比较猴头菌丝多糖各剂量与格列本脲的降血糖效果。结果:猴头菌丝多糖得率为7.14%,粗多糖再经Sevage法去除蛋白质,获得猴头菌丝精多糖(HMP),得率为10.92%;猴头多糖高、中、低三个剂量均能有效的对抗四氧嘧啶诱发的高血糖;其中,高剂量的降血糖作用与格列本脲相比,差异极显著。结论:猴头菌丝多糖对四氧嘧啶型高血糖模型小鼠有降血糖作用,作用效果优于格列本脲,对糖尿病小鼠的胰腺具有一定的保护作用。     

荧光标记技术在肿瘤模型研究中的应用

目的利用绿色荧光小鼠和红色荧光蛋白标记肿瘤细胞,建立荧光标记的小鼠肿瘤模型,并建立活体荧光成像和荧光显微镜成像在整体和细胞水平直接观察肿瘤的技术。方法将小鼠B16黑色素瘤细胞接种到绿色荧光蛋白转基因小鼠皮下,建立GFP小鼠肿瘤模型。以红色荧光蛋白作为标记基因导入小鼠黑色素瘤细胞B16细胞,建立稳定表达红色荧光蛋白的细胞株。将表达红色荧光蛋白B16细胞接种到绿色荧光转基因小鼠皮下,建立双荧光小鼠肿瘤模型。用荧光显微镜和活体荧光成像系统检测小鼠肿瘤的发生发展。结果分别建立了GFP小鼠肿瘤模型和双色荧光小鼠肿瘤模型。利用活体荧光影像仪可以观察双色荧光小鼠模型中受体绿色荧光组织和红色荧光移植肿瘤相互融合。利用荧光显微镜,可以观察到肿瘤内绿色荧光标记的来源于受体小鼠的血管和免疫细胞。经香菇多糖刺激的GFP小鼠肿瘤模型的移植瘤组织中,来源于受体小鼠绿色荧光标记的免疫细胞明显多于经生理盐水刺激的对照小鼠。结论利用绿色荧光小鼠和红色荧光RFP标记肿瘤细胞建立荧光标记的小鼠肿瘤模型,采用活体荧光成像仪和荧光显微镜可在整体和细胞水平直接观察肿瘤的生长以及肿瘤与宿主的相互作用。     

一种基于EdU的体外癌细胞与小鼠体内组织细胞标记方法的研究

摘要 目的：比较EdU标记对三种癌细胞和小鼠对细胞增殖的影响,为EdU作为标记开展相关细胞增殖实验和临床研究提供依据。方法：本研究使用不同剂量EdU对人非小细胞肺癌A549细胞、人宫颈癌Hela细胞、人肝癌Huh7进行标记2 h,然后使用荧光显微镜观测EdU在细胞中的标记效率,并使用多波长荧光酶标仪检测这三种癌细胞系标记后的荧光强度;使用流式细胞仪检测小鼠经不同剂量的EdU干预12 h后,体内肺、肝、肾组织标记的荧光强度。结果：与对照组相比,经EdU处理后,A549和Hela细胞系的荧光强度,三个剂量组均有显著性差异（P＜0.01）,Huh7细胞系的荧光强度,50 μmol/L有显著性差异（P＜0.05）;EdU在小鼠体内组织肺、肝、肾组织中均有分布,且在肝组织中分布比肺组织和肾组织高。结论：EdU的体外癌细胞与小鼠体内组织细胞的标记效率各不相同,建立的EdU体外标记癌细胞和小鼠体内组织的方法简单,易操作。     

猴头菌子实体发育过程中多糖结构和活性的变化

猴头菌(Hericium erinaceus)多糖是猴头菌发挥药理作用的主要活性成分,猴头菌生长过程中多糖的合成代谢及其结构的变化目前还鲜有研究报道。该试验研究了猴头菌生长发育过程中多糖的产生及其结构和活性的变化。对猴头菌0605菌株生长发育6个时期的子实体进行了多糖和β-葡聚糖含量的测定,并对提取的粗多糖进行了体外免疫活性的研究。结果表明:在猴头菌的整个生长发育过程中,子实体多糖的含量呈现上升的趋势,成熟期含量最高,达1.54%,分裂期含量最低仅0.89%;β-葡聚糖含量整体上呈上升的趋势,中菌刺期含量最高33.58%,成熟期最高,达到50.67%;体外免疫活性试验表明,现蕾期、分裂期、中菌刺期和成熟期的多糖均能很好地刺激巨噬细胞RAW264.7产生NO,活性均优于无菌刺期和小菌刺期的多糖。分别采用体积分数为30%、50%和70%的乙醇对子实体发育后3个时期的热水浸提物进行分级醇沉,对分级分离多糖进行理化性质研究。结果表明:小菌刺期30%醇沉的粗多糖得率高于中菌刺期和成熟期,其大分子量多糖的比例也较高;3个时期50%和70%醇沉多糖分子量相差不大;分级分离多糖的单糖种类基本相同,都含有岩藻糖、半乳糖、葡萄糖和甘露糖,但摩尔比有差异;3个时期分级分离多糖均能刺激巨噬细胞分泌NO,30%醇沉多糖比50%和70%醇沉多糖的活性好;成熟期多糖的活性优于其他生长阶段。对小菌刺期、中菌刺期和成熟期50%醇沉多糖(H5FP4B、H5FP5B、H5FP6B)进行Sephacryl S300凝胶层析纯化,共得到9个组分:H5FP4B-1、H5FP5B-1、H5FP6B-1、H5FP4B-2、H5FP5B-2、H5FP6B-2、H5FP4B-3、H5FP5B-3、H5FP6B-3。对这9个组分的分子量分布进行研究,发现每个时期的主峰(H5FP4B-3、H5FP5B-3和H5FP6B-3)分子量基本相同,分子量分布差异较大的是3个时期的第1组分,在体外免疫活性上也表现出较大差异。主峰H5FP4B-3的分子量为1.59万,由岩藻糖、半乳糖和葡萄糖组成,摩尔比为5.2∶23.9∶1。经红外光谱、甲基化和核磁共振综合分析,解析出H5FP4B-3均一多糖的结构单元是以α-D-1,6连接的半乳糖和α-D-1,2,6连接的半乳糖构成主链,侧链为α构型的端基岩藻糖。研究结果表明:猴头菌不同发育阶段产生的多糖结构和活性有一定的差异,生长初期,大分子量的多糖较多,后期分子量分布差异变小并趋稳定。多糖主成分变化不大,较稳定;到成熟期,多糖的活性更高。     

Ultrasensitive in situ visualization of active glucocerebrosidase molecules

Deficiency of glucocerebrosidase (GBA) underlies Gaucher disease, a common lysosomal storage disorder. Carriership for Gaucher disease has recently been identified as major risk for parkinsonism. Presently, no method exists to visualize active GBA molecules in situ. We here report the design, synthesis and application of two fluorescent activity-based probes allowing highly specific labeling of active GBA molecules in vitro and in cultured cells and mice in vivo. Detection of in vitro labeled recombinant GBA on slab gels after electrophoresis is in the low attomolar range. Using cell or tissue lysates, we obtained exclusive labeling of GBA molecules. We present evidence from fluorescence-activated cell sorting analysis, fluorescence microscopy and pulse-chase experiments of highly efficient labeling of GBA molecules in intact cells as well as tissues of mice. In addition, we illustrate the use of the fluorescent probes to study inhibitors and tentative chaperones in living cells.     

重组腺相关病毒小鼠骨骼肌中近红外荧光蛋白表达及活体成像

近红外荧光蛋白因激发光和发射光波长位于近红外区,在动物组织中光吸收和光散射最低,更适宜于动物活体组织的深层成像.构建了一种携带近红外荧光蛋白(near-infrared fluorescent protein,iRFP)713基因的重组表达质粒pAAV-iRFP713,将重组表达质粒与辅助质粒共转染AAV-293细胞,包装重组腺相关病毒(recombinant adeno-associated virus,rAAV)rAAV-iRFP713.重组腺相关病毒表达载体感染体外培养的癌细胞,48h后,荧光显微镜检测显示近红外荧光蛋白在癌细胞中高效表达,荧光明亮.重组腺相关病毒表达载体注射小鼠骨骼肌,48h后,用近红外荧光活体成像系统检测证明近红外荧光蛋白在小鼠骨骼肌中表达较强, 活体组织成像清晰.实验结果表明近红外荧光蛋白在体内体外均能很好地表达并荧光成像,为动物活体组织标记和成像的研究提供新方法.     

An HPLC-CAD/fluorescence lipidomics platform using fluorescent fatty acids as metabolic tracers

Fluorescent lipids are important tools for live imaging in cell culture and animal models, yet their metabolism has not been well-characterized. Here we describe a novel combined HPLC and LC-MS/MS method developed to characterize both total lipid profiles and the products of fluorescently labeled lipids. Using this approach, we found that lipids labeled with the fluorescent tags, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY FL), 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene [BODIPY(558/568)], and dipyrrometheneboron difluoride undecanoic acid (TopFluor) are all metabolized into varying arrays of polar and nonpolar fluorescent lipid products when they are fed to larval zebrafish. Quantitative metabolic labeling experiments performed in this system revealed significant effects of total dietary lipid composition on fluorescent lipid partitioning. We provide evidence that cholesterol metabolism in the intestine is important in determining the metabolic fates of dietary FAs. Using this method, we found that inhibitors of dietary cholesterol absorption and esterification both decreased incorporation of dietary fluorescent FAs into cholesterol esters (CEs), suggesting that CE synthesis in enterocytes is primarily responsive to the availability of dietary cholesterol. These results are the first to comprehensively characterize fluorescent FA metabolism and to demonstrate their utility as metabolic labeling reagents, effectively coupling quantitative biochemistry with live imaging studies.     

大鼠卵黄囊的分化及其肿瘤性转化研究

目的研究12d大鼠胚胎脏层卵黄囊(VYS)向多胚层组织分化的潜能和在逆转录病毒感染下的肿瘤性转化特征。方法在不同培养条件、移植位点的条件下,观察VYS体内外分化的改变;另外利用逆转录病毒载体将荧光蛋白基因(GFP)转染12d卵黄囊细胞,对GFP标记的转化细胞进行体内外研究。结果在不同培养条件下,均对体外培养的或体内移植的大鼠卵黄囊向三个胚层分化的进程无特异的导向性。将荧光蛋白标记卵黄囊克隆细胞接种在裸鼠皮下长出了未分化的间质细胞肉瘤。结论12d大鼠胚胎脏层卵黄囊具有向三胚层分化的潜能;逆转录病毒感染导致卵黄囊间质细胞发生肿瘤性转化。     

Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester

This protocol outlines the carboxyfluorescein diacetate succinimidyl ester (CFSE) method for following the proliferation of human lymphocytes in vitro and mouse lymphocytes both in vitro and in vivo. The method relies on the ability of CFSE to covalently label long-lived intracellular molecules with the highly fluorescent dye, carboxyfluorescein. Following each cell division, the equal distribution of these fluorescent molecules to progeny cells results in a halving of the fluorescence of daughter cells. The CFSE labeling protocol described, which typically takes <1 h to perform, allows the detection of up to eight cell divisions before CFSE fluorescence is decreased to the background fluorescence of unlabeled cells. Protocols are outlined for labeling large and small numbers of human and mouse lymphocytes, labeling conditions being identified that minimize CFSE toxicity but maximize the number of cell divisions detected. An important feature of the technique is that division-dependent changes in the expression of cell-surface markers and intracellular proteins are easily quantified by flow cytometry.     

F肌动蛋白作为胞间连丝组分的结构与生理学证据

以蒜 (AlliumsativumL .)瓣鞘外表皮为材料 ,利用荧光特异探针与共焦镜检术 ,结合透射电镜与免疫金标记对表皮细胞间胞间联络的性质、结构进行了系统观察。结果表明 ,加厚壁上的通道是由狭长的管状胞质和初生纹孔场上成束的胞间连丝衔接组成 ,前者实为原生质体的一部分。单个胞间连丝的孔径为 6 0～ 70nm ,属正常胞间连丝范围 ,它们乃相邻细胞间共质联系的所在。荧光探针TRITC_Phalloidin (TRITC_Ph)标记的结果显示 ,整个通道上呈现红色荧光的纤索在接近初生纹孔场处明显变窄 ,与超微结构观察中所见的结构特点相吻合 ,共焦镜下观察到的初生壁上的荧光亮斑乃初生纹孔场中成束胞间连丝被标记的反映 ,从而有效地证实了F肌动蛋白在常态胞间连丝上的存在。免疫金标记实验显示在管状胞质中和胞间连丝上有金颗粒分布 ,这一结果为证实荧光标记物具肌动蛋白性质提供了有说服力的补充。     

Metabolic labeling of proteins for proteomics

Realization of the advantages of stable isotope labeling for proteomics has emerged gradually. However, many stable isotope label approaches rely on labeling in vitro using complex and sometimes expensive reagents. This review discusses strategies for labeling protein in vivo through metabolic incorporation of label into protein. This approach has many advantages, is particularly suited to single cells grown in culture (prokaryotic or eukaryotic), but is nonetheless subject to a number of complicating factors that must be controlled so that meaningful experiments can be conducted. Confounding issues include the metabolic lability of the amino acid precursor, incomplete labeling, and the role of protein turnover in labeling kinetics. All of these are controllable, provided that appropriate precautions are adopted.     

Facile metabolic glycan labeling strategy for exosome tracking

Background

Exosomes are nano-sized vesicles derived from the fusion of multivesicular bodies with the surrounding plasma membrane. Exosomes have various diagnostic and therapeutic potentials in cancer and other diseases, thus tracking exosomes is an important issue.

Carboxyfluorescein diacetate succinimidyl ester fluorescent dye for cell labeling

Our objective was to study the properties of the carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and the methodology of cell labeling using CFDA-SE fluorescent dye. First, we analyzed the kinetics of CFDA-SE fluorescent dye intensity over time. Second, we determined the optimal concentration of CFDA-SE fluorescent dye for cell labeling. Third, we tested the toxicity of CFDA-SE fluorescent dye on labeled cells. Finally, we determined the optimal staining time of CFDA-SE fluorescent dye for cell labeling.The results show that the optimal concentration of CFDA-SE fluorescent dye for cell labeling varies according to different cell types. CFDA-SE fluorescent dye is non-toxic to cells as the cell death rate caused by CFDASE labeling is below 5%. The optimal cell labeling time was determined to be 8 min of incubation with CFDA-SE fluorescent dye. We concluded that the advantages of using CFDA-SE fluorescent dye for cell labeling are as follows: (1) the binding of CFDA-SE fluorescent dye to cells is stable; (2) CFDA-SE fluorescent dye is not toxic and does not modify the viability of labeled cells; and (3) CFDA-SE fluorescent dye is a suitable fluorochrome for cell labeling.     

Metabolic flux analysis in plants using dynamic labeling technique: application to tryptophan biosynthesis in cultured rice cells

The concept and methodology of using dynamic labeling for the MFA of plant metabolic pathways are described, based on a case study to develop a method for the MFA of the tryptophan biosynthetic pathway in cultured rice cells. Dynamic labeling traces the change in the labeling level of a metabolite in a metabolic pathway after the application of a stable isotope-labeled compound. In this study, [1-(13)C] l-serine was fed as a labeling precursor and the labeling level of Trp was determined by using the LC-MS/MS. The value of metabolic flux is determined by fitting a model describing the labeling dynamics of the pathway to the observed labeling data. The biosynthetic flux of Trp in rice suspension cultured cell was determined to be 6.0+/-1.1 nmol (gFWh)(-1). It is also demonstrated that an approximately sixfold increase in the biosynthetic flux of Trp in transgenic rice cells expressing the feedback-insensitive version of anthranilate synthase alpha-subunit gene (OASA1D) resulted in a 45-fold increase in the level of Trp. In this article, the basic workflow for the experiment is introduced and the details of the actual experimental procedures are explained. Future perspectives are also discussed by referring recent advances in the dynamic labeling approach.