灰黄霉素高产菌F208发酵过程蛋白质表达差异的研究

目的 为探寻灰黄霉素生物合成过程中的关键酶,以蛋白质组学技术手段分析灰黄霉素高产菌F208发酵过程蛋白质表达差异.方法 通过双向电泳联用质谱技术对F208发酵过程蛋白质组图谱进行比较分析.结果 研究发现灰黄霉素产生期( 192 h)F208表达的蛋白质与产生前期(72 h)有较大差异,并鉴定出在灰黄霉素产生高峰期蛋白表达量明显增加的两个特异点是丝氨酸羟甲基转移酶和S-腺苷甲硫氨酸合成酶.结论 成功建立灰黄青霉菌丝体总蛋白双向电泳技术体系.并鉴定出两个蛋白特异点,极可能与灰黄霉素生物合成有关.     

应用蛋白组方法筛选血浆中乙肝病毒PreS1结合蛋白

目的筛选血浆中乙型肝炎病毒PreS1结合蛋白。方法表达纯化了PreS1-谷胱甘肽-S-转移酶（glutathione—S-transferase,GST）融合蛋白,利用该蛋白与血浆进行Pull—down实验,并设立GST与血浆Pull—down,GST、PreS1-GST与PBS Pull—down对照,Pull-down产物进行双向电泳分离（2-DE）,差异蛋白点通过质谱鉴定。结果成功表达纯化出PreS1-GST融合蛋白,通过双向电泳分析发现一个PreS1特异结合蛋白,经质谱鉴定为含锚蛋白重复序列的蛋白57（ANKRD57）。结论锚蛋白重复序列的主要功能是介导蛋白质与蛋白质之间的相互作用,ANKRD57与PreS1特异结合后的生理功能值得深入研究。     

炭疽杆菌在家兔盲肠结扎模型内外培养的蛋白表达差异

【目的】建立家兔盲肠结扎模型,研究炭疽杆菌在家兔体内外不同培养条件下的蛋白表达差异。【方法】本实验通过进行家兔盲肠结扎模型对炭疽杆菌进行体内外培养,用不同方法提取胞外蛋白、细胞壁蛋白及全菌体蛋白,并经双向电泳分离和质谱鉴定。【结果】送检144个蛋白点,检出124个,其中包括上清蛋白19个,细胞壁蛋白29个,全菌体蛋白76个。【结论】经分析发现,与合成代谢相关的蛋白在体内主要呈下调趋势,包括多种氨基酰-tRNA合成酶、长链脂肪酸CoA连接酶等;在体内表达上调的蛋白则具有多种功能,其中包括分子伴侣DnaK、超氧化物歧化酶SodA、S-层蛋白等。     

失血性休克大鼠肝脏蛋白质的差异分析

 应用蛋白质双向凝胶电泳 (two-dimensional polyacrylamide gel electrophoresis, 2-DE) 技术,分析在急性重度失血性休克 (refractory hemorrhagic shock, RHS) 条件下,大鼠肝脏蛋白质组表达的差异.16 只雄性 Wistar 大鼠随机分成正常对照组 (sham hemorrhage shock, SHS) 和 RHS 模型组,每组 8 只.采用股动脉放血的方法制备模型,在规定时间内处死大鼠并分离肝脏,提取肝脏总蛋白质后进行 2-DE.运用 Image Master 2D Platinum v 5.0 凝胶图像分析软件对 2-DE 凝胶图像进行差异表达分析.有意义的差异蛋白质点用基质辅助激光解析电离飞行时间质谱进行肽质量指纹图谱分析,借助 Swiss-prot 数据库进行蛋白质搜索和鉴定.SHS 组和 RHS 组肝脏的 2-DE 图谱,分别平均识别到 698±11 和 700±13 个蛋白质点,SHS 组和 RHS 组肝脏间平均匹配率达88%～92 %.共发现 10 个差异有意义的蛋白质点,鉴定出了肿瘤抑制性抗原gp96、葡萄糖调节蛋白58、过氧还蛋白Ⅰ、细胞色素b5、谷胱甘肽转移酶、ATP合酶β亚单位、二磷酸果糖酶 B、三磷酸甘油醛脱氢酶等8种蛋白质.结果表明,以 2-DE 技术得到重复性和分辨率都较好的 2-DE图谱,并初步鉴定急性重度失血性休克后大鼠肝脏的差异表达蛋白质,为深入研究失血性休克的生理病理机制及寻找失血性休克预防和治疗的生物标志物提供了依据.     

大鼠脑红蛋白(NGB)可溶性原核表达和单克隆抗体制备及鉴定

脑红蛋白 (NGB)是新发现的脑特异的携氧蛋白 .为了对其功能进行深入的研究 ,应用已构建大鼠NGB基因编码区原核表达载体pGEX 4T 2 NGB转化的大肠杆菌BL2 1 ,获得可溶性表达产物 .用谷胱甘肽S 转移酶 (GST)亲和层析柱纯化后作为抗原 ,免疫Balb c小鼠 .通过传统的细胞融合方法制备了抗大鼠NGB的单克隆抗体 ,并对全部 4株单抗进行了亚类和亚型、效价和特异性鉴定 .为NGB结构与功能的深入研究提供了重要工具     

β-细辛醚对AD大鼠海马神经元蛋白质组图谱的影响

目的：探讨β-细辛醚对痴呆大鼠海马神经元蛋白质表达谱的影响。方法：SD大鼠随机分为正常时照组、模型组和β-细辛醚治疗组。模型组和β-细辛醚治疗组大鼠行脑立体定位注射术,模型组从大鼠右侧海马注入β淀粉样蛋白（Aβ1-40）5μg;治疗组：将Aβ1-40和β-细辛醚（0.12%）混合后注入大鼠右侧海马。抽提各组大鼠海马神经元组织蛋白质,行双向电泳,采用PDQuest-7.1.1分析软件解析各组之间有明显差异表达的蛋白质。结果：β-细辛醚治疗组α-烯醇化酶（α-enolase）、钙/钙调蛋白依赖蛋白激酶（Ca^2＋/Calmodulin-dependent protein kinase）的表达高于模型组,而尿激酶型纤溶酶原激活物（urokillasc plasminogen activator surface）、P53抑癌基因（P53 tumor suppressor）和未知蛋白（SSP6001）表达则低于模型组。结论：β-细辛醚可能通过上调或下调上述蛋白质的表达,参与促进大鼠海马受损神经元保护作用。     

双向电泳和质谱技术分析樟芝无性孢子萌发相关蛋白

【目的】采用双向电泳(2DE)、质谱技术和实时荧光定量PCR(RT-q PCR)技术分析樟芝无性孢子萌发相关蛋白。【方法】分别提取培养0 h和24 h的樟芝孢子总蛋白并进行双向电泳分离,再用PDQuest软件进行差异蛋白分析,并用MALDI-TOF-MS技术对差异蛋白进行鉴定;其次将鉴定成功的蛋白与孢子萌发相关蛋白的本地数据库进行比对,获得樟芝中的孢子萌发相关蛋白信息,最后用RT-qPCR技术对相关基因的转录水平进行分析。【结果】两组样品共有32个差异蛋白点,其中在24 h表达量上调的蛋白25个,下调的蛋白7个。将32个差异蛋白点挖取鉴定,成功鉴定24个。其中,与孢子萌发相关的蛋白有10个,分别为GerO、Ubc1、Cat-1、Snf1、Cas2、SfaD、Chaperonin、Fad5、Tyrosine-P和ChiA。【结论】该研究结果为进一步解析樟芝无性孢子萌发的分子机制提供了理论依据。     

双依他尼酸乙醇胺对谷胱甘肽S-转硫酶mu的选择性抑制作用

谷胱甘肽S-转移酶(GST)的同工酶mu(GSTM)高表达与卵巢癌顺铂耐药有关.以GST非选择性抑制剂依他尼酸设计二价潜抑制剂双依他尼酸乙醇胺(aminoethanol di-ethacrynic acid,ADEA),测定ADEA及其与还原型谷胱甘肽(glutathione,GSH)加合物对GST同工酶亚型A1、P1...     

温敏核不育水稻花药蛋白质组初步分析

采用固相pH梯度 SDS聚丙烯酰胺双向凝胶电泳对温敏核不育水稻 96 4 2S可育与不育条件下减数分裂期花药总蛋白进行了分离 ,通过银染显色 ,获得了分辨率和重复性较好的双向电泳图谱 .PDQuest 2DE图像分析软件可识别约 10 0 0个蛋白质点 .蛋白质点在 2D胶上的重复性为 :沿等电聚焦方向偏差为 1 4 5± 0 2 3mm(n =8) ,沿SDS PAGE方向偏差为 :1 15± 0 17mm(n =8) .对两种育性不同样品的 2D胶上部分共有的蛋白质点 ,采用基质辅助激光解析电离飞行时间质谱 (matrixassistedlaserdesorption ionizationtimeofflightmassspctrometry ,MALDI TOF MS)进行了肽质谱指纹图分析 .通过采用PeptIdent软件对SWISS PROT数据库的查询 ,有 5 0个蛋白质点在数据库得到归属鉴定 .对育性不同的2种样品 2D较上明显差异的蛋白质点进行了分析鉴定 .在不育变化为可育的过程中 ,明显表达上调的蛋白质点包括几丁质酶 ,酸性磷酸酶 ,胞浆激酶 ,谷蛋白前体 ,以及ESTSC72 61蛋白 ,明显下调的蛋白质包括β expansin前体 ,谷氨酸氨甲酰转移酶和 1种未知功能的蛋白质     

糠秕马拉色菌酵母态和菌丝态蛋白差异表达的研究

目的通过双向电泳及串联质谱技术鉴定糠秕马拉色菌酵母态及菌丝态差异蛋白,在蛋白水平探讨两态转化机制及致病机理。方法分别诱导糠秕马拉色菌标准株酵母态和菌丝态菌体,利用玻璃珠研磨和超声波破碎细胞壁,三氯乙酸/丙酮沉淀获取总蛋白。双向电泳分离蛋白,PDQuest软件比对找出差异蛋白点。电喷雾串联质谱对差异点进行肽段测序,用Mascot和NCBI的Blast软件经蛋白质数据库鉴定蛋白质。结果经双向电泳分离的糠秕马拉色菌酵母态、菌丝态蛋白各有800多个蛋白点、64个蛋白点表达量有3倍以上差异,其中11个为酵母态特有,9个菌丝态特有。在选取的40个差异点中,成功鉴定出22个点,共16个蛋白。经Mascot和Blast软件检索,有明确功能的蛋白中,肌动蛋白、丝切蛋白等9个蛋白在菌丝态上调,谷胱甘肽转移酶、细胞支架信号蛋白等5个蛋白下调。结论鉴定出16个蛋白分别与细胞代谢、运动、氧化应激等功能相关,为了解糠秕马拉色菌表型转换机制和致病机理提供重要信息。     

大鼠脑皮质表达蛋白质组学研究

文章用蛋白质组学方法初步分析大鼠脑皮质蛋白质的表达。提取大鼠脑皮质蛋白质,双向凝胶电泳分离,考马斯亮蓝染色,胰蛋白酶胶内酶解,用基质辅助激光解吸/电离飞行时间质谱对酶解后的肽段进行分析,根据肽质量指纹图谱,检索专业数据库(Swissprot),对蛋白质进行鉴定。鉴定出84个蛋白,分别属于代谢酶、细胞骨架蛋白、热休克蛋白、抗氧化蛋白、信号传导蛋白、蛋白酶体相关蛋白、神经元特异蛋白及神经胶质蛋白等。文章结果丰富了大鼠脑皮质蛋白质组数据库,为在大鼠模型上研究神经疾病奠定了基础。     

银杏叶提取物促进脑缺血半暗带神经元自噬提高神经保护作用

银杏叶提取物(ginkgo biloba extract-761,EGb-761)注射液在中国常作为辅助药物被用于治疗脑卒中,但是,其潜在的细胞和药理机制尚未完全了解.该研究旨在探讨EGb-761是否通过调节缺血性脑卒中半暗带神经元的自噬从而发挥保护作用.采用雄性SD大鼠大脑中动脉闭塞(middle cerebral ...     

Proteomics analysis of cerebral cortex in Wistar rats

To analyze the protein expression pattern of the cerebral cortex in Wistar rats using the proteomics approach, proteins were separated by two-dimensional gel electrophoresis, stained with Coomassie brilliant blue and digested with trypsin. Then, we analyzed the peptide section using a matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and identified the protein by indexing special database (SwissProt) according to the finger printing of the peptide quality. Eighty-four protein spots were identified, includ-ing metabolic enzymes, skeleton proteins, heat shock pro-teins, antioxidant proteins, signaling proteins, proteasome related proteins, neuron and glial specific proteins and serum associated proteins. The result of this study enriches the database of the proteome in the cerebral cortex of rats and lays a foundation for further research of neurological disorders in rat models.     

IGF-1对缺血性脑损伤大鼠脑内神经发生的影响

目的:建立大鼠单侧局灶脑缺血模型,观察胰岛素样生长因子-1(IGF-1)对局灶脑缺血后的鼠脑神经发生及增殖后细胞生存的影响.方法:用健康雄性SD大鼠建立大脑中动脉阻塞(MCAO)模型,随机分成假手术组,缺血对照组和IGF-1治疗组.各组再按不同的治疗时间分为7d、14d、28d、42d组.免疫组化法观察BrdU、PSA-NCAM的变化,免疫双标法观察BrdU/PSA-NCAM、BrdU/MAP2和BrdU/GFAP的共同表达变化.结果:BrdU标记细胞和PSA-NCAM标记细胞计数均在缺血后第7d最多,分别是缺血对照组的4.0倍和1.8倍,是假手术组的9.9倍和5.4倍.BrdU和PSA-NCAM双标细胞在缺血发生后双侧SVZ和DG区可以检测到,于第7d计数最多,之后逐渐降低;而BrdU和MAP2以及BrdU和GFAP双标细胞却从第14d开始逐渐增多,直到第42d.随着BrdU/PSA-NCAM双标阳性表达的逐渐降低,BrdU/MAP2双标阳性表达逐渐增高,呈现此消彼涨的变化.结论:IGF-1侧脑室注射后,在早期(7d内)诱导了缺血性脑损伤后神经细胞的增殖;在中期(7d-14d)诱导了新生细胞的迁移;在后期(14d后)伴随着迁移的进行新生细胞逐渐发生了分化.     

Upregulation of dihydropyrimidinase-related protein 2, spectrin alpha II chain, heat shock cognate protein 70 pseudogene 1 and tropomodulin 2 after focal cerebral ischemia in rats--a proteomics approach

In recent years, there are an increasing number of proteomics studies that investigated the alterations in the protein expression relevant to human diseases but none for stroke. We, therefore, attempted such a study in a paradigm of focal cerebral ischemia in rat. Rats were subjected to cerebral ischemia by unilateral occlusion of the middle cerebral artery. Global protein analysis was performed after 24h on the lesioned and sham-control cerebral cortex using two-dimensional gel electrophoresis. Protein spots with more than a 3-fold change in intensity were identified by mass spectrometry. Middle cerebral artery occlusion (MCAO) caused infarct volume of 18-22% predominantly in the cortex of the lesioned hemisphere. Two-dimensional gel electrophoresis resolved about 1500 protein spots of which only 12 were significantly upregulated by 3-46-fold. Three spots were identified to be dihydropyrimidinase-related protein 2 (DRP-2, also known as collapsin response mediator protein 2 (CRMP-2) or turned on after division, 64 kD protein (TOAD-64)). The spots varied in pI values only and this may reflect different phosphorylation status of the same protein. Two spots were identified as spectrin alpha II chain (rat fragment, also known as alpha-fodrin or non-erythroid alpha chain, SPNA-2); and one spot each for heat shock cognate protein 70 pseudogene 1 (HSC70-ps1, also known as heat shock protein 8 pseudogene 1), and tropomodulin 2 (Tmod2). The upregulation of protein expression was corroborated by observed upregulation of mRNA expression. The remaining five spots were not identified satisfactorily. As DRP-2, spectrin, and Tmod2 are involved in axonal and neurite growth as well as synaptic plasticity and maturation, the presently observed upregulation of the expression of these proteins may indicate active neuroregeneration and repair at 24h after the induction of cerebral ischemia.     

Upregulation of dihydropyrimidinase-related protein 2, spectrin α II chain, heat shock cognate protein 70 pseudogene 1 and tropomodulin 2 after focal cerebral ischemia in rats—A proteomics approach

In recent years, there are an increasing number of proteomics studies that investigated the alterations in the protein expression relevant to human diseases but none for stroke. We, therefore, attempted such a study in a paradigm of focal cerebral ischemia in rat. Rats were subjected to cerebral ischemia by unilateral occlusion of the middle cerebral artery. Global protein analysis was performed after 24 h on the lesioned and sham-control cerebral cortex using two-dimensional gel electrophoresis. Protein spots with more than a 3-fold change in intensity were identified by mass spectrometry. Middle cerebral artery occlusion (MCAO) caused infarct volume of 18–22% predominantly in the cortex of the lesioned hemisphere. Two-dimensional gel electrophoresis resolved about 1500 protein spots of which only 12 were significantly upregulated by 3–46-fold. Three spots were identified to be dihydropyrimidinase-related protein 2 (DRP-2, also known as collapsin response mediator protein 2 (CRMP-2) or turned on after division, 64 kD protein (TOAD-64)). The spots varied in pI values only and this may reflect different phosphorylation status of the same protein. Two spots were identified as spectrin α II chain (rat fragment, also known as α-fodrin or non-erythroid α chain, SPNA-2); and one spot each for heat shock cognate protein 70 pseudogene 1 (HSC70-ps1, also known as heat shock protein 8 pseudogene 1), and tropomodulin 2 (Tmod2). The upregulation of protein expression was corroborated by observed upregulation of mRNA expression. The remaining five spots were not identified satisfactorily. As DRP-2, spectrin, and Tmod2 are involved in axonal and neurite growth as well as synaptic plasticity and maturation, the presently observed upregulation of the expression of these proteins may indicate active neuroregeneration and repair at 24 h after the induction of cerebral ischemia.     

骨髓间充质干细胞移植对脑梗死大鼠神经功能恢复的影响

目的:观察骨髓间充质干细胞(BMSC)移植对脑梗死大鼠神经功能恢复的影响,并对其相关机制进行探讨。方法:90只大鼠随机分为3组:假手术组、对照组、BMSC移植组,每组30只。对照组和BMSC移植组建立大鼠大脑中动脉阻塞(MCAO)模型,假手术组只需要分离大鼠颈部组织,而不造MCAO模型。BMSC移植组在MCAO模型术后1天经尾静脉注射1 mL/3×10~6 BMSC,对照组注射同剂量的生理盐水,于MCAO术后1 d、3 d、7 d、14 d、21 d、28 d、35 d、42 d、49 d分别对各组大鼠进行神经功能评分(mNSS),术后2个月对BMSC移植组及对照组大鼠脑组织进行免疫组化染色,检测MAP2、TUJ1、Ⅷ因子、GFAP的表达情况。结果:在治疗后的第7天至第35天,BMSC移植组mNSS均显著低于对照组(P0.05)。术后2个月,BMSC移植组MAP2、TUJ1、Ⅷ因子表达量显著高于对照组,而GFAP表达量显著低于于BMSC对照组(P0.01)。结论:BMSC移植可以促进脑梗死神经功能的恢复。     

Protective Effects of 28-O-Caffeoyl Betulin (B-CA) on the Cerebral Cortex of Ischemic Rats Revealed by a NMR-Based Metabolomics Analysis

28-O-caffeoyl betulin (B-CA) has been demonstrated to reduce the cerebral infarct volume caused by transient middle cerebral artery occlusion (MCAO) injury. B-CA is a novel derivative of naturally occurring caffeoyl triterpene with little information associated with its pharmacological target(s). To date no data is available regarding the effect of B-CA on brain metabolism. In the present study, a ¹H-NMR-based metabolomics approach was applied to investigate the therapeutic effects of B-CA on brain metabolism following MCAO in rats. Global metabolic profiles of the cortex in acute period (9 h after focal ischemia onset) after MCAO were compared between the groups (sham; MCAO?+?vehicle; MCAO?+?B-CA). MCAO induced several changes in the ipsilateral cortex of ischemic rats, which consequently led to the neuronal damage featured with the downregulation of NAA, including energy metabolism dysfunctions, oxidative stress, and neurotransmitter metabolism. Treatment with B-CA showed statistically significant rescue effects on the ischemic cortex of MCAO rats. Specifically, treatment with B-CA ameliorated the energy metabolism dysfunctions (back-regulating the levels of succinate, lactate, BCAAs, and carnitine), oxidative stress (upregulating the level of glutathione), and neurotransmitter metabolism disturbances (back-regulating the levels of γ-aminobutyric acid and acetylcholine) associated with the progression of ischemic stroke. With the administration of B-CA, the levels of three phospholipid related metabolites (O-phosphocholine, O-phosphoethanolamine, sn-glycero-3-phosphocholine) and NAA improved significantly. Overall, our findings suggest that treatment with B-CA may provide neuroprotection by augmenting the metabolic changes observed in the cortex following MCAO in rats.

     

Synergistic Effect of Selenium and Melatonin on Neuroprotection in Cerebral Ischemia in Rats

The synergistic scavenger effects of selenium and melatonin collectively we called Se-Mel was studied on the prevention of neuronal injury induced by ischemia/reperfusion. Male Wistar rats were treated with sodium selenite (0.1 mg/kg, i.p.) and melatonin (10 mg/kg, i.p.) 30 min before the middle carotid artery occlusion (MCAO) and immediately after MCAO to male Wistar rats and was continued for 3 days once daily at the interval of 24 h. Behavioral activity (spontaneous motor activity and motor deficit) was improved in Se-Mel-treated rats as compared to MCAO group rats. The level of glutathione and the activity of antioxidant enzymes was depleted significantly while the content of thiobarbituric acid reactive substances, protein carbonyl, and nitric oxide radical (NO^·) was increased significantly in MCAO group. Systemic administration of Se-Mel ameliorated oxidative stress and improves ischemia/reperfusion-induced focal cerebral ischemia. Se-Mel also inhibited inducible nitric oxide synthase expression in Se-Mel+MCAO group as compared to MCAO group rats. Thus, Se-Mel has shown an excellent neuroprotective effect against ischemia/reperfusion injury through an anti-ischemic pathway. In conclusion, we demonstrated that the pretreatment with Se-Mel at the onset of reperfusion, reduced post-ischemic damage, and improved neurological outcome following transient focal cerebral ischemia in male Wistar rat.