蛋白酶体抑制剂MG132诱导K562和HeLa细胞凋亡程度存在明显差异

蛋白酶体抑制剂MG132诱导人白血病细胞K562和宫颈癌细胞HeLa凋亡,用3个不同浓度的蛋白酶体抑制剂MG132处理人白血病细胞K562和宫颈癌细胞HeLa,通过MTT检测、annexin Ⅴ/ PI 双染法、流式细胞术、酶标仪和Western 印迹分别检测MG132对K562细胞和HeLa细胞的生长效应、细胞凋亡率、细胞内活性氧(ROS)水平和caspase-3活性变化的影响.蛋白酶体抑制剂MG132诱导K562细胞凋亡明显,对HeLa细胞诱导凋亡不明显.结果表明,蛋白酶体抑制剂MG132特异性诱导不同肿瘤细胞凋亡的程度存在明显差异.     

表达HIV壳体蛋白转基因枸杞悬浮细胞的培养与鉴定

枸杞是我国珍贵的中药材,利用枸杞作为转基因材料具有易于遗传操作,生物性状稳定的优点。将携有人类免疫缺陷病毒I型（HIV-1）壳体蛋白基因的植物表达载体导入根瘤农杆菌EHA105中,并通过农杆菌侵染枸杞叶片,诱导产生抗性愈伤组织,利用抗性愈伤组织作为材料进行悬浮细胞的培养并对转基因枸杞悬浮细胞鉴定。PCR结果表明已获得遗传转化的转基因枸杞悬浮细胞系。免疫组织化学检测结果表明HIV壳体蛋白已在转基因枸杞悬浮细胞中表达。     

抑制或激活泛素-蛋白酶体途径对大鼠肺泡巨噬细胞内质网应激的影响

目的:研究肺泡巨噬细胞(NR8383)不同蛋白酶体激活程度对内质网应激的影响。方法:构建UbG76V-GFP融合蛋白,将含有UbG76V-GFP的质粒导入NR8383细胞,筛选出可稳定表达UbG76V-GFP的细胞系,通过蛋白酶体抑制剂(MG132)、蛋白酶体激活剂(阿霉素)干预蛋白酶体活性。荧光显微镜观察不同蛋白酶体活性下大鼠肺泡巨噬细胞在缺氧复氧2 h、4 h、6 h时蛋白酶体活性,Western blot及PCR技术检测不同蛋白酶体活性下大鼠肺泡巨噬细胞在缺氧复氧2 h、4 h、6 h时泛素化蛋白及内质网应激相关基因的表达。结果:在缺氧复氧2 h、4 h、6 h这3个时间点,加入MG132组大鼠肺泡巨噬细胞绿色荧光及泛素化蛋白(Ubiquitin)表达明显降低(P0.05),而PCR及Western blot示内质网应激基因BIP(免疫球蛋白结合蛋白)、XBP-1(X-盒结合蛋白)和CHOP(C/EBP同源蛋白)平均扩增量及蛋白表达量明显增加(P0.05);加入阿霉素组大鼠肺泡巨噬细胞在缺氧复氧2 h、4h、6 h表现出相反的实验结果,绿色荧光及Ubiquitin蛋白相对表达均明显增加(P0.05),而PCR及Western blot示内质网应激基因BIP、XBP-1和CHOP平均扩增量及蛋白表达量明显增加(P0.05)。结论:本实验结果表明活细胞泛素-蛋白酶体活性程度与内质网应激存在紧密联系,外源性增强泛素蛋白酶体活性会抑制内质网应激,外源性减弱泛素蛋白酶体活性会增强内质网应激。     

蛋白酶体抑制剂MG132的浓度对人白血病细胞K562凋亡的影响

探讨蛋白酶体抑制剂MG132 在诱导人白血病K562细胞凋亡过程中作用.分别以不同浓度的蛋白酶体抑制剂MG132 处理人白血病细胞K562,通过MTT法检测K562细胞活力,应用Annexin Ⅴ和PI 双染的细胞流式法检测K562细胞凋亡率和细胞内活性氧(ROS) 水平,应用酶标仪法检测K562细胞内Caspase- 3活性变化的情况.结果表明,随着MG132浓度的增加,各个指标与对照组比较差异均有显著性(P<0.05):K562细胞增殖明显受到抑制;细胞凋亡率明显增加,且当MG132浓度为900 nmol/L时,细胞凋亡率达36.5 %;同时,ROS 水平和caspase- 3活性明显升高.因次,蛋白酶体抑制剂MG132可显著抑制人白血病细胞K562增殖并促进其凋亡.     

蛋白酶体抑制剂MG132对青扦花粉萌发和花粉管生长的影响

泛素/蛋白酶体系统(UPP)是真核细胞内蛋白质选择性降解的主要途径,而蛋白酶体是UPP中蛋白质降解的场所。本文应用细胞学、统计学方法以及FTIR技术研究了蛋白酶体抑制剂MG132对青扦(Peceawilsonii)花粉萌发、花粉管生长的影响。结果表明:MG132显著抑制青扦花粉萌发和花粉管生长,并导致花粉管形态异常,主要表现为花粉管亚顶端出现液泡化,并且液泡随着培养时间的延长而扩大到整个花粉管,花粉管濒临死亡;而DMSO以及非蛋白酶体抑制剂E-64不产生类似结果;半薄切片结果表明,MG132处理后不仅花粉管细胞质发生液泡化,生殖细胞也发生液泡化;FTIR分析进一步表明,MG132处理后,花粉管顶端的细胞壁蛋白和果胶质含量大幅度下降。上述结果表明:MG132通过抑制蛋白酶体活性显著影响青扦花粉萌发及花粉管生长;UPP在青扦花粉萌发、花粉管极性生长模式的建立和维持过程中起重要作用;抑制蛋白酶体活性将导致青扦花粉管的程序性死亡。     

人蛋白酶体亚基PSMB1的重组表达、纯化及在蛋白酶体抑制剂体外筛选中的应用

蛋白酶体是真核细胞中的一类多亚基蛋白酶复合物,它在胞内蛋白质降解的泛素-蛋白酶体通路中起关键作用。重组表达蛋白酶体的活性亚基可以用于在体外筛选、寻找具有蛋白酶体抑制剂作用的化合物。将人蛋白酶体催化亚基(PSMB1)cDNA的编码区(全长726 bp)克隆至原核表达载体pET28a(+),构建重组质粒pET28a-PSMB1,转化大肠杆菌BL21(DE3),通过1 mmol/L IPTG,20℃过夜诱导,获得相对分子量约为27 kDa的重组蛋白,采用IMAC亲和层析柱纯化重组蛋白,纯化后的重组蛋白纯度超过95%。重组蛋白酶解后经NanoLC-MS/MS鉴定表明所表达的融合蛋白氨基酸序列完全正确。在体外BIAcore分析中,重组蛋白表现出对不同化合物的选择性结合能力,其中与蛋白酶体抑制剂雷公藤红素的结合较强,10μmol/L的雷公藤红素与重组蛋白的结合达到27 RU,并且具有良好的浓度依赖型。本研究建立了表达、纯化人蛋白酶体催化亚基PSMB1的方法,并应用于具有蛋白酶体抑制活性化合物的体外筛选。     

转基因鸡生物反应器载体的构建及其表达特性分析

以增强型绿色荧光蛋白和萤火虫荧光素酶为报告基因,构建了鸡卵清蛋白启动子表达载体和慢病毒载体,以巨细胞病毒 (Cytomegalovirus,CMV)启动子表达载体为对照,转染或感染鸡原代输卵管上皮细胞、鸡胚成纤维细胞、鼠3T3-L1前脂肪细胞和牛乳腺上皮细胞,通过荧光和酶活性检测,旨在筛选出用于实现转基因鸡生物反应器的高效特异性表达载体。结果发现,鸡卵清蛋白启动子表达载体转染以上4种细胞后2种标记基因均有表达,没有表现出明显的细胞特异性,且荧光素酶检测结果表明其在各细胞组中表达活性都低于CMV启动子表达载体100倍以上;慢病毒载体感染以上4种细胞后2种标记基因均有表达,在鸡输卵管上皮细胞组感染单个细胞的病毒颗粒 (Multiplicity of infection,MOI) 为20时绿色荧光蛋白表达量就可以达到CMV启动子表达载体的水平。上述结果表明,基于卵清蛋白基因调控序列构建的表达载体无法实现外源基因的高效、特异性表达,而慢病毒载体在表达活性和广泛性上可以用于进行鸡输卵管生物反应器的研究。     

一种基于绿色荧光蛋白的蛋白酶体抑制剂细胞筛选模型

为建立基于绿色荧光蛋白(GFP)的药物筛选模型,并用此模型从包括中药提取物在内的化合物中筛选新型蛋白酶体抑制剂,本研究构建了pGC-E1-ZU1-GFP融合蛋白慢病毒表达载体并感染A549细胞,筛选稳定表达细胞株,用已知蛋白酶体抑制剂PS-341处理细胞,荧光显微镜检测处理前后细胞GFP水平变化。结果获得了稳定表达pGC-E1-ZU1-GFP的A549细胞,这些细胞用PS-341处理24h后用荧光显微镜检测,发现细胞绿色荧光强度相对于对照组明显增强。利用这一模型对一些化合物进行筛查,发现了一些新的蛋白酶体抑制剂。     

人H1启动子转录shRNA的细胞种属特异性研究

扬州大学王永娟、王安平、孙顺吕3位从事转基因生物制药研究工作者利用siDirect软件预测绿色荧光蛋白GFP基因特异性小干扰RNA（siRNA）,将人工合成的相应shRNA插入含人H1启动子的pSuper载体,获得表达载体pSuper-shRNA,再将H1-shRNA插入表达GFP基因的peGFP-N1载体,获得表达载体peGFP-H1-shRNA。     

miR-25抑制结肠癌细胞凋亡

为了通过结肠癌细胞实验,探究miR-25对结肠癌细胞凋亡的影响,本研究通过qRT-PCR检测结肠癌细胞中miR-25的表达情况;通过CCK-8法检测miR-25抑制剂转染组细胞活力状态;将miR-25抑制剂转染至结肠癌细胞,通过蛋白免疫印迹法检测结肠癌细胞中Bax和Bcl-2蛋白的表达情况;通过Caspase-3活性检测试剂盒检测结肠癌细胞中Caspase-3表达情况;通过流式细胞仪检测结肠癌细胞中ROS水平;通过Elisa试剂盒检测4-HNE、GSH和MDA含量。结果表明:结肠癌细胞中miR-25表达明显高于正常结肠粘膜组;miR-25抑制剂转染结肠癌细胞后,结肠癌细胞活力明显低于空载体对照组;miR-25抑制剂转染后,结肠癌细胞中Bax蛋白显著高于空载体对照组(p0.05),且Bcl-2蛋白低于空载体对照组(p0.05),Caspase-3活性高于空载体对照组(p0.05);miR-25抑制剂转染后,结肠癌细胞中ROS水平明显高于空载体对照(p0.05);miR-25抑制剂转染后,结肠癌细胞中ROS水平明显高于对照组(p0.05);miR-25抑制剂转染后结肠癌细胞中4-HNE (p0.05)和MDA (p0.05)含量明显增加,GSH (p0.05)含量显著降低。miR-25抑制剂可促进结肠癌细胞凋亡,其机制可能与增加结肠癌细胞中ROS水平导致氧化应激水平增强有关。     

枸杞多糖与黄芪多糖抑菌活性的研究*

目的:研究黄芪多糖和枸杞多糖的抑菌活性并探讨不同pH值对其抑菌活性的影响。方法:采用滤纸片扩散法,分析不同浓度黄芪多糖和枸杞多糖在不同pH值下对几种常见细茵和霉菌(大肠杆菌、沙门氏菌、金黄色葡萄球菌、黑曲霉、产黄青霉)的抑制效果。结果:对于细菌,枸杞多糖8mg/mL时出现抑菌圈,而黄芪多糖0.02 mg/mL时效果最佳;对于霉菌,随着枸杞多糖浓度的增大,抑菌圈的直径增大,而黄芪多糖0.02 mg/mL时效果最佳;当枸杞多糖和黄芪多糖在pH6的条件下,二者抑菌活性均最强。结论:枸杞多糖和黄芪多糖对细菌、霉菌都有一定的抑制效果,pH值可影响枸杞多糖和黄芪多糖的抑菌效果。     

HIV-1衣壳蛋白在转基因枸杞中表达的免疫组织化学定位

目的:研究转基因植物中重组蛋白的细胞定位,有助于进一步了解转基因枸杞中HIV-1衣壳(CA)蛋白融合蛋白的分泌表达途径。方法:利用含有MA4-CA融合基因的农杆菌转化枸杞,转化植株获得再生。采用免疫组织化学方法对转基因枸杞表达的CA融合蛋白进行初步定位。结果:免疫组织化学定位表明,在转基因枸杞愈伤组织中,HIV-1CA主要在细胞浆、细胞壁和细胞间隙中表达。结论:免疫组织化学结果初步证明了CA融合蛋白在转基因枸杞中的表达分布。     

Separation of cathepsin A-like enzyme and the proteasome: evidence that lactacystin/beta-lactone is not a specific inhibitor of the proteasome

Previous studies have described a human platelet cathepsin A-like enzyme with a number of similarities to the "acidic" and "neutral" chymotrypsin-like activities of the proteasome. This includes its strong inhibition by the highly specific proteasome inhibitor Lactacystin/beta-lactone, suggesting that either the Cbz-Phe-Ala-hydrolyzing activity attributed to cathepsin A was due to the chymotrypsin-like activity of the proteasome or that lactacystin was not a specific inhibitor of the proteasome. In the present study we discard the first possibility on the basis of the following findings: (a) human platelet cathepsin A, unlike proteasome, binds to concanavalin A, and does not bind to Heparin-Sepharose at pH 7.4; (b) neither the chymotrypsin-like activity of the proteasome, nor proteasome antigens are detected in the cathepsin A preparation; (c) purified proteasome does not exhibit Cbz-Phe-Ala-hydrolyzing activity; (d) Z-lle-Glu-(Ot-Bu)Ala-leucinal (PSI), a compound that selectively inhibits the chymotrypsin-like activity of the proteasome at a concentration of 10 microM has no inhibitory effect on the carboxypeptidase activity of cathepsin A; (e) cathepsin A, free of the proteasome, is completely inhibited by micromolar concentrations of lactacystin/beta-lactone. It is therefore concluded that lactacystin/beta-lactone is not a specific inhibitor of the proteasome.     

Modulation of the chymotrypsin-like activity of the 20S proteasome by intracellular redox status: effects of glutathione peroxidase-1 overexpression and antioxidant drugs

ATP- and ubiquitin-independent proteolysis by the 20S proteasome is responsible for the selective degradation of oxidized proteins. In vitro, the 20S proteasome shows an increased proteolytic activity toward oxidized polypeptides and the suc-LLVY-MCA peptide specific for its chymotrypsin-like activity. We have analyzed the effect of the intracellular redox status on the chymotrypsin-like activity of the 20S proteasome in human T47D cells overexpressing the detoxifiant enzyme seleno-glutathione peroxidase-1 (GPx-1). We report a 30% decreased activity of the chymotrypsin-like activity in cells overexpressing GPx-1. This phenomenon correlated with a 2-fold increase in IkappaB alpha half-life, a protein whose basal turnover is 20S proteasome-dependent. Following exposure to H2O2, these cells showed a seleno-dependently decreased accumulation of intracellular reactive oxygen species and 20S proteasome chymotrypsin-like activity. Similar results were obtained in HeLa cells transiently overexpressing human GPx-1. Moreover, exposure of HeLa cells to antioxidant compounds reduced the proteasome 20S chymotrypsin-like activity. In contrast, no effects were observed when HeLa cell extracts used to determine proteasome activity were incubated with either reduced or oxidized glutathione. These results suggest that GPx-1 activity or pro-reducing conditions can downregulate basal 20S proteasome activity. Hence, the intracellular redox status, probably through the level of oxidized proteins, is an important element that can either activate or down-regulate the 20S proteasome chymotrypsin-like activity in living cells.     

Focal dysfunction of the proteasome: a pathogenic factor in a mouse model of amyotrophic lateral sclerosis

Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene are responsible for a familial form of amyotrophic lateral sclerosis (fALS). The present study demonstrated impaired proteasomal function in the lumbar spinal cord of transgenic mice expressing human SOD-1 with the ALS-causing mutation G93A (SOD-1(G93A)) compared to non-transgenic littermates (LM) and SOD-1(WT) transgenic mice. Chymotrypsin-like activity was decreased as early as 45 days of age. By 75 days, chymotrypsin-, trypsin-, and caspase-like activities of the proteasome were impaired, at about 50% of control activity in lumbar spinal cord, but unchanged in thoracic spinal cord and liver. Both total and specific activities of the proteasome were reduced to a similar extent, indicating that a change in proteasome function, rather than a decrease in proteasome levels, had occurred. Similar decreases of total and specific activities of the proteasome were observed in NIH 3T3 cell lines expressing fALS mutants SOD-1(G93A) and SOD-1(G41S), but not in SOD-1(WT) controls. Although overall levels of proteasome were maintained in spinal cord of SOD-1(G93A) transgenic mice, the level of 20S proteasome was substantially reduced in lumbar spinal motor neurons relative to the surrounding neuropil. It is concluded that impairment of the proteasome is an early event and contributes to ALS pathogenesis.     

盐胁迫下枸杞叶片细胞表面糖蛋白的变化

利用细胞化学的方法,对经过不同浓度盐胁迫的枸杞细胞表面糖蛋白的动态变化进行了标记定位电镜观察。枸杞盐胁迫处理后发现,盐胁迫过程中枸杞细胞表面糖蛋白层明显增厚、致密。随着胁迫时间的延长则变薄、脱落,游离到细胞间隙中。细胞璧糖蛋白的积累是枸杞对盐胁迫反应的重要生化机制,在枸杞的耐盐生理活动中起着重要作用。     

枸杞色素及多糖的不同提取次序对其得率及抗氧化活性的影响

为探究先后提取枸杞多糖及枸杞色素时对各自得率的影响。本研究通过分别考察这两种成分在提取过程中的提取溶剂、提取温度、料液比、提取次数及提取时间等因素对枸杞色素和枸杞多糖的得率影响,确定枸杞色素和枸杞多糖在提取次序不同时,两者的最佳提取工艺以及对DPPH·和·OH自由基清除率。结果表明,首先提取枸杞多糖后,枸杞色素的最佳提取工艺为采用正己烷,80℃时,料液比1∶10,提取2次,每次1 h,枸杞色素得率为2.48%,此时枸杞多糖得率为7.45%;而首先提取枸杞色素后,采用了超声辅助提取的方式提取枸杞多糖,发现超声效率为25%,料液比1∶10,提取20 min,枸杞多糖得率为5.23%,此时枸杞色素得率为3.93%。因此,首先提取枸杞多糖,使其平均得率为7.45%,而后提取枸杞色素,其平均得率为2.48%;总体上,枸杞色素1和枸杞多糖1对DPPH·自由基清除率都较高,枸杞多糖1对·OH自由基清除率较高,其抗氧化活性都接近Vc。     

Hypoglycemic and hypolipidemic effects and antioxidant activity of fruit extracts from Lycium barbarum

The hypoglycemic and hypolipidemic effects of Lycium barbarum fruit water decoction, crude polysaccharide extracts (crude LBP), and purified polysaccharide fractions (LBP-X) in alloxan-induced diabetic or hyperlipidemic rabbits were investigated through designed sequential trials and by measuring blood glucose and serum lipid parameters. Total antioxidant capacity was also assessed using trolox equivalent antioxidant capacity (TEAC) and oxygen radical absorbance capacity (ORAC) assay. It was found that the three Lycium barbarum fruit extracts/fractions could significantly reduce blood glucose levels and serum total cholesterol (TC) and triglyceride (TG) concentrations and at same time markedly increase high density lipoprotein cholesterol (HDL-c) levels after 10 days treatment in tested rabbits, indicating that there were substantial hypoglycemic and hypolipidemic effects. Hypoglycemic effect of LBP-X was more significant than those of water decoction and crude LBP, but its hypolipidemic effect seemed to be weaker. Total antioxidant capacity assay showed that all three Lycium barbarum extracts/fractions possessed antioxidant activity. However, water and methanolc fruit extracts and crude polysaccharide extracts exhibited stronger antioxidant activity than purified polysaccharide fractions because crude extracts were identified to be rich in antioxidants (e.g., carotenoids, riboflavin, ascorbic acid, thiamine, nicotinic acid). Lycium barbarum polysaccharides (glycocojugates), containing several monosaccharides and 17 amino acids, were major bioactive constituents of hypoglycemic effect. Both polysaccharides and vitamin antioxidants from Lycium barbarum fruits were possible active principles of hypolipidemic effect.     

外源甜菜碱对盐胁迫下枸杞光合功能的改善

研究了外源甜菜碱对盐胁迫下枸杞扦插苗叶片光合功能的影响。结果表明,外源甜菜碱能使盐胁迫下的枸杞叶片叶绿素荧光动力学参数Fo、Fm、Fv、Fv／Fm、Fm／Fo和Fv／Fo增高,使光合色素叶绿素a(Chla)、叶绿素b(Chlb)和类胡萝卜素(Car)含量明显增加,叶绿素a与b的比值(Chla／Chlb)升高,类胡萝卜素与叶绿素的比值(Car／Chl)降低,说明外源甜菜碱有利于植物对光能的捕获、吸收、传递和转换,提高叶片的光合活性,降低盐胁迫对植物的抑制作用。     

枸杞多糖的抗炎镇痛作用研究

为研究枸杞多糖的抗炎镇痛效应及其潜在分子机制,采用小鼠热板实验、小鼠福尔马林炎症疼痛模型和原代培养的背根神经节神经元细胞对疼痛指标和炎症相关分子进行了检测。实验结果显示枸杞多糖能显著缓解后足注射福尔马林引起的动物II相痛表现(P <0. 05),减少动物两后足重量差值(P <0. 001)和血液(P <0. 001)、脊髓(P <0. 05)组织中白细胞介素6的含量,而对小鼠热板潜伏期无明显影响;枸杞多糖降低原代培养的DRG细胞膜上TRPV1介导的钙信号(P <0. 05)。结果证明枸杞多糖具有良好的抗炎镇痛作用,其镇痛机制可能与降低炎症因子白细胞介素6表达及其引起的TRPV1活性增加有关。