一种快速分离纯化金葡萄球菌肠毒素C2的方法

目的 建立一种简单快捷的方法,用来从金黄色葡萄球菌的发酵液(以下简称金葡液)中纯化分离肠毒素C2(staphylococcal enterotoxin C2,SEC2).方法 金葡液首先经超滤浓缩,之后再分别经阳离子交换层析和阴离子交换层析进行选择性分离SEC2,用SDS-PAGE电泳、HPLC法检测纯度及相对分子质量,飞行质谱测定精确分子质量,对其进行N -端氨基酸序列分析.结果 分离得到的SEC2电泳纯及色谱分析纯度均达到质量分数98%以上,经各项理化指标检测均与文献一致,在等电点上表现出微不均匀性,等电点pI 为7.49和6.74,该蛋白的分子质量为27.58 ku,N -端氨基酸序列分析与文献报道的SEC2序列一致(ESQPDPTPDELHKSS),最大吸收波长为277.5 nm.结论 用该方法得到的SEC2纯度高,回收率质量分数高达50%,适宜于大批量制备SEC2.     

浙江眼镜蛇蛇毒中神经毒素的分离纯化及其镇痛作用研究

目的:研究浙江眼镜蛇(Najanajaatra)蛇毒的镇痛活性组分,为寻找镇痛效果好,而无成瘾性的新型镇痛药奠定基础.方法:通过两次CM-SephadexC-25和一次SephadexG-50柱层析对眼镜蛇毒进行了分离纯化.采用PAGE法对分离纯化后的眼镜蛇神经毒素(cobroneurotoxin,CNT)纯度进行鉴定;以SDS-PAGE法测定CNT的分子量;等电聚焦法(Isoelectrofocusing,IEF)测定CNT的等电点;以Lowry法测定其蛋白含量;用醋酸扭体法、热板法与电刺激法考察CNT的镇痛活性并通过测定LD50考察其毒性.结果:眼镜蛇毒经3次柱色谱后,CNT经鉴定为单一组分.经SDS-PAGE法测定其分子量为7000,等电点为10.2,蛋白含量为92%.CNT能显著降低小鼠对化学刺激与电刺激的敏感性;小鼠痛阈百分率的提高可达51.3%,并呈一定的量效关系;小鼠腹腔注射的LD50为81μg·kg-1,95%可信限为58～104μg·kg-1.结论:眼镜蛇毒经3次柱色谱后得到具有镇痛活性的单一组分(CNT).     

尖吻蝮蛇毒小分子多肽的分离及抗血小板聚集作用

目的从尖吻蝮蛇毒中分离纯化一种抗血小板聚集小分子多肽,研究其理化性质以及对ADP、胶原、凝血酶诱导的血小板聚集反应的影响。方法经SephadexG-75凝胶过滤,超滤,DEAE-SepharoseCL-6B离子交换层析法分离纯化蛇毒组分,采用高效液相鉴定纯度,用SDS-凝胶电泳(SDS-PAGE)测定其分子量,用比浊法测定其抗血小板聚集活性。结果从尖吻蝮蛇毒中分离相对分子质量约为7862u等电点为4.29的组分,该组分能抑制由ADP、胶原、凝血酶诱导的血小板聚集并成剂量依赖性。结论此法成功地从尖吻蝮蛇毒中纯化出抗血小板聚集组分。该组分与去整合素比较相似,可能属于去整合素家族。     

中药露蜂房水溶性蛋白NV-PP-4的分离纯化及部分理化性质鉴定

目的 :从中药露蜂房抗炎活性有效部分纯化得到一酸性蛋白NV PP 4 ,并鉴定了其部分理化性质。方法 :用SephadexG 50、DEAE SephadexA50、羟基磷灰石等柱层析及HPLC等方法纯化 ,并以HPLC、毛细管等电聚焦电泳 ,氨基酸自动分析等手段 ,鉴定其理化性质。结果 :NV PP 4经不同的HPLC柱和不同的流动相匀表明为一对称单一峰、毛细管等电聚焦电泳呈单一染色带。亲水型分子排阻HPLC测定其分子量为 8 71 1KD ,毛细管等电聚焦电泳测定其等电点为 1 79,氨基酸分析表明NV PP 4含有 87个氨基酸残基 ,其中Gly、Asp、Pro、Glu等含量较多。结论 :NV PP 4是从中药露蜂房中首次分离得到的酸性蛋白质成分。     

舟山眼镜蛇毒神经毒素的分离纯化及镇痛作用研究

目的从舟山眼镜蛇毒中分离纯化神经毒素,并测定其部分理化性质及镇痛活性。方法应用SP-Sephadex C-50离子交换色谱法分离眼镜蛇毒粗毒,用Sephadex G-50凝胶过滤色谱、CM-Sephadex C-25离子交换色谱纯化神经毒素;SDS-PAGE(Tris-Tricine系统)鉴定纯度;用Meier方法测定毒性;用热板法观察神经毒素的镇痛作用。结果应用SP-Sephadex C-50离子交换柱层析,从舟山眼镜蛇毒中分离得到14个蛋白峰,其中6个组分(NNAV-Ⅶ ~Ⅻ)经鉴定为含有神经毒素组分,将神经毒活性最强的组分XI经Sephadex G-50、CM-Sephadex C-25纯化得神经毒素纯品NTⅪ。该纯品分子量12.3kD,小白鼠静脉注射和腹腔注射的LD50分别为1.9875、2.2217 mg·kg-1。NTXI能显著提高小鼠对热板刺激的痛阈。腹腔注射0.2mg·kg-1NTXI可使小鼠的热板痛阈提高84.35%。NTXI的镇痛作用具有时效性,给药后2h起效,4h作用达峰值。结论舟山眼镜蛇毒中分离得到一个神经毒素纯品NTⅪ,具有镇痛作用。     

杜拉鲁肽类似药(27C7)的蛋白表达及理化性质分析

目的 真核表达杜拉鲁肽类似药胰高糖素样肽l（GLP-1）-Fc融合蛋白（27C7），并与杜拉鲁肽进行理化性质比较。方法 构建真核表达载体pCHO1.0-GLP-1-Fc，并用脂质体转染CHO细胞，经甲氨蝶呤和嘌呤霉素加压筛选，再单克隆筛选，得到1株高表达的细胞模型。采用Protein A亲和层析法纯化目的蛋白，SDS-PAGE检测蛋白大小，质谱仪进行高分辨分子量检测，高效液相色谱法检测蛋白纯度，毛细管等点聚焦电泳（CIEF）检测蛋白等电点，细胞活性功能评价杜拉鲁肽与27C7生物活性的一致性。结果 通过SDS-PAGE、样品高分辨分子量检测，27C7与杜拉鲁肽分子量一致；分子排阻色谱检测27C7与杜拉鲁肽单体纯度接近，CIEF显示27C7与杜拉鲁肽的等电点一致；体外活性检测结果表明，27C7与杜拉鲁肽的生物学活性一致。结论 类似药27C7与杜拉鲁肽具有一定的一致性，可进行下一步的类似药研发工作。     

蛇毒抗肿瘤蛋白心脏毒素的分离纯化与鉴定

目的:对蛇毒抗瘤蛋白的活性组分进行分离与鉴定。方法:采用 RP-HPLC 分离蛇毒抗瘤蛋白各组分,收集纯化组分Ⅱ。RP-HPLC 及 SDS-PAGE 测定组分Ⅱ的纯度;MALDI—TOF 质谱仪测定组分Ⅱ的相对分子质量;对组分Ⅱ进行 N-末端氨基酸序列测定。结果:蛇毒抗瘤蛋白组分Ⅱ经 RP-HPLC 和 SDS-PAGE 分析为单一成分,MALDI-TOF 质谱仪测得其相对分子质量为6719.49,其 N-末端氨基酸序列为 L-K-C-N-K-L-V-P-L-F-Y-K-T-C-P。结论:经分离鉴定,蛇毒抗瘤蛋白组分Ⅱ为心脏毒素。     

海胆黄多糖的分离、纯化及免疫活性测定

目的从光棘球海胆中分离纯化海胆黄多糖(polysaccharidefromtheeggsofStrongylocentrotusnu-dus,简称SEP),确定其纯度和分子量,并现察其免疫活性。方法海胆黄先经丙酮脱脂,根据正交实验和活性分析确定最佳热水提取条件,然后热水提取、去蛋白、醇沉得海胆黄粗多糖。粗多糖经超滤、DEAESepharoseFastFlow及SephacrylS-400柱层析纯化得多糖精品SEP。经高效液相色谱、聚丙烯酰胺凝胶电泳及纸层析鉴定其纯度。高效凝胶渗透色谱法(HPGPC)测定其分子量。体外脾淋巴细胞增殖实验测定其免疫活性。结果从海胆黄中分离纯化得到的均一多糖组分SEP,经检测其分子量为1950KD左右。脾淋巴细胞增殖实验表明SEP可显著促进脾淋巴细胞的增殖。结论从海胆黄中分离纯化得到的均一多糖组分SEP具有较强的体外免疫活性。     

鲨肝细胞再生刺激因子的分离纯化和特性

目的　从鲨鱼肝脏中分离纯化肝细胞再生刺激因子 ,并研究其分子特点和活性。方法和结果　健康鲨鱼肝脏经匀浆、冻融、热提、离心和超滤 ,在分子量小于 3万的组分中检出肝细胞再生刺激因子 (HRSF)的活性。此样品经DEAE Sepharosefastflow柱层析 ,FPLCResource 30Q、ResourceQ及MonoQ色谱 ,得到鲨肝细胞再生刺激因子 (sHRSF)纯品。SDS PAGE测定sHRSF的分子量为 14 6 0 0Da ,紫外扫描显示其特征吸收峰为 2 76nm ,等电点约为 5 1,含有 18种氨基酸 ,N端氨基酸序列为LVGPIGAVGPAGKDG。具有显著刺激肝再生生物活性。结论　获得了一种未知的新的活性蛋白 ,即鲨肝细胞再生刺激因子(sHRSF)     

蛇毒抗肿瘤蛋白化学成分的研究

对蛇毒抗肿瘤蛋白的化学成分进行研究。采用RP-HPLC分离蛇毒抗肿瘤蛋白各组分,收集纯化组分Ⅳ。非还原型SDS-PAGE和RP-HPLC鉴定组分Ⅳ的纯度;还原型SDS-PAGE与MALDI-TOF质谱仪测定组分Ⅳ的相对分子质量;测定组分ⅣN-末端氨基酸序列并进行Western blot鉴定;MTT法检测组分Ⅳ对体外培养的K562细胞的生长抑制作用。结果表明蛇毒抗肿瘤蛋白组分Ⅳ经SDS-PAGE和RP-HPLC分析为单一成分,MALDI-TOF质谱仪测得其相对分子质量为6714.22,其N-末端前5个氨基酸序列为L-K-C-N-K。经Western blot鉴定蛇毒抗肿瘤蛋白组分Ⅳ为细胞毒素,其对K562细胞的抑制作用呈明显的剂量依赖关系。     

Purification and characterization of a novel antinociceptive peptide from venom of Agkistrodon halys Pallas

Venom of Agkistrodon halys Pallas can control severe pain such as cancer pain and neuropathic pain, but it is made up of complicated components. Aim of this study is to separate major analgesic fraction from venom of A. halys Pallas, and to reveal its biochemical and pharmacological properties. Three steps with ion exchange column first and molecular sieve columns next were used to separate and purify the fractions of venom. Analgesic effects were evaluated by hot plate tests and writhing tests in mice. The molecular weight (MW), isoelectric point, amino acid sequence, purity were respectively determined by SDS-PAGE electrophoresis, isoelectric focusing, Edman degradation and HPLC. The dependence and tolerance were observed by withdrawal test in rats, and analgesic effects were observed in mice during 7 days administration. Fourteen fractions were obtained by separation; the best analgesic fraction named Pallanalgesin was selected by ED₅₀ and LD₅₀. It had single band in electrophoresis, relative purity 92.16 %, MW 16.6 kDa, isoelectric point 8.8, and former sequence of ten amino acids H-L-L-Q-F-R-K-M-I-K. It showed significant analgesic effect without tolerance and dependence. As a novel analgesic, Pallanalgesin has been found to explain the function of venom of A. halys Pallas on severe pain control in traditional uses.     

咪唑克生对吗啡镇痛、耐受和身体依赖的影响

目的:观察咪唑克生对吗啡镇痛及吗啡所致耐受和躯体依赖的影响.方法:采用小鼠醋酸扭体实验和55℃热板实验观察咪唑克生对基础痛阈及吗啡镇痛作用的影响;采用小鼠热辐射甩尾实验和小鼠55℃热板实验观察咪唑克生对吗啡耐受形成过程的影响;采用大鼠、小鼠身体依赖模型观察咪唑克生对吗啡所致身体依赖的影响.结果:咪唑克生(3-9mg/kg)能显著降低小鼠基础痛阈,抑制吗啡镇痛;加重吗啡所致耐受;诱发大、小鼠发生戒断综合征.结论:咪唑啉受体参与痛阈形成;咪唑克生能抑制吗啡镇痛,加重吗啡所致耐受;并诱发吗啡依赖性动物发生戒断综合征.     

Overexpression of type 7 adenylyl cyclase in the mouse brain enhances acute and chronic actions of morphine

The mechanisms by which morphine-induced analgesia and tolerance and physical dependence on morphine arise have been the subject of intense study, and much work has pointed to the involvement of cAMP-mediated events in the neuroadaptive phenomena leading to morphine tolerance and/or dependence. We overexpressed an opioid receptor-stimulatable form of adenylyl cyclase (type 7) in the central nervous system of mice and demonstrated significant effects of this manipulation on the animals' acute response to morphine, the development of morphine tolerance, and development of sensitization to morphine. Measurements of the acute analgesic response to morphine demonstrated that the ED(50) values for the transgenic mice were significantly lower than the ED(50) values determined for the "wild-type" animals. During chronic treatment with morphine, the transgenic mice developed tolerance more rapidly than the wild-type mice, and transgenic animals of the C57BL/6xSJL background showed a larger sensitization to morphine's effects on locomotor activity than did wild-type mice of the same background. These results indicated that cAMP-generating systems may simultaneously modulate the development of tolerance and sensitization. Interestingly, the signs of physical dependence on morphine in the transgenic mice did not differ from those in their wild-type litter mates, indicating that separate mechanisms may modulate opiate tolerance and opiate dependence.     

噻芬太尼的主要药效和致依赖性潜力的实验研究

本文研究噻芬太尼的镇痛药效和制动作用,评价其致身体依赖性潜力。小鼠热板法测得噻芬太尼的镇痛作用强度分别为吗啡、芬太尼和埃托啡的3260,22和1.5倍。以瘫痪作为制动指标测得噻芬太尼对大鼠、家兔、狗和猴制动作用强度为埃托啡的2～3倍。小鼠跳跃实验和大鼠饮药液自然戒断实验表明该药具有一定致身体依赖性潜力。大鼠ⅳ快速成瘾实验及长达20周的猴身体依赖性实验则未出现依赖性戒断症状。     

Chemical Composition and Antinociceptive Properties of Hyeronima alchorneoides. Leaves

Abstract

In the current study, we investigated the analgesic activity of a crude methanol extract and some fractions obtained from Hyeronima alchorneoides Allemao leaves and analyzed its phytochemical profile to determine active principles. All the fractions exhibited analgesic properties using the writhing test in mice, but the hexane fraction was the most active, causing 96.4 ± 1.0% inhibition at 10 mg/kg. Three compounds were isolated and identified based on their spectral data: simiarenol (1), β.-sitosterol (2), and amentoflavone (3). Compound 1, a rare terpene isolated for the first time from the family Euphorbiaceae, presented a calculated ID₅₀ value of 18.87 (14.6–24.4)µmol/kg with the writhing test, being about 8-fold more active than two known analgesic and anti-inflammatory drugs (aspirin and dipyrone). In the formalin test, it was particularly active against the second phase, with inhibition of 59.5 ± 9%, suggesting a peripheral activity. In the capsaicin and glutamate tests, it showed inhibition of 52.3 ± 4 and 52.1 ± 6%, respectively.     

Effects of glycine, beta-alanine and diazepam upon morphine-tolerant-dependent mice

The effects in mice of glycine, beta-alanine and diazepam on the analgesic response to morphine, on the intensity of tolerance and on the physical dependence on the analgesic have been examined. The two amino acids increased the analgesic response to morphine in a dose-related manner. However, both compounds were ineffective in the analgesic test (hot plate) when administered without morphine. Diazepam was ineffective in the analgesic test and it did not alter morphine analgesia, except when administered in a high dose which decreased and analgesic response. Glycine, either in single or repeated doses, did not modify tolerance to morphine, whereas beta-alanine induced a dose-related partial antagonism, which promptly reached a plateau. Diazepam induced a small decrease in the intensity of tolerance to the analgesic. The abstinence syndrome to morphine, induced by naloxone administration to primed mice, was reduced by single doses of glycine or beta-alanine. Diazepam behaved as a weak inhibitor of the abstinence syndrome when administered at a high dose. The potentiation of morphine analgesia and the antagonism of the abstinence syndrome induced by the amino acids may be related to their hyperpolarizing action in the c.n. system. The effects of beta-alanine on morphine tolerance cannot be explained by the same mechanism.     

Genetic variation in morphine analgesic tolerance: a survey of 11 inbred mouse strains

The present study assessed the analgesic potency of morphine in 11 inbred mouse strains before and after chronic morphine treatment. Using the 49 degrees C tail-withdrawal test, significant strain differences in morphine AD(50) estimates derived from cumulative dose-response curves were noted prior to tolerance induction on Day 1. AD(50) estimates were reassessed on Day 4, after three daily systemic morphine injections for 3 days using an escalating dose schedule (10, 20, and 40 mg/kg sc). In 9 of 11 strains, morphine potency was significantly reduced from 2-fold to as much as 11-fold. Two strains (129P3 and LP) displayed no evidence whatsoever of tolerance development. Neither initial baseline withdrawal latency nor morphine analgesic sensitivity was significantly correlated with tolerance magnitude. Also observed were strain-dependent alterations (mostly hyperalgesia) in baseline tail-withdrawal latencies as a result of chronic morphine treatment. The magnitude of hyperalgesia and analgesic tolerance was significantly correlated among strains, implicating common genetic substrates and supporting their proposed association. The present work demonstrates that the presence and magnitude of morphine analgesic tolerance is genotype-dependent and identifies strains with widely divergent liabilities that should facilitate identification of trait-relevant genes.     

The pharmacologic effect of flupirtine, a structurally new analgesic

The analgesic potency of ethyl-N-[2-amino-6-(4-fluorophenylmethylamino)pyridin-3-yl]carb ama te (flupirtine, D 9998) in mice and rats in Haffner's test, electro-pain test and Randall-Selitto test (inflammation induced pain) lies between the more potent dextromoramide and methadone and the more weakly active pethidine, dextropropoxyphene, codeine, phenacetin and paracetamol. In comparison to codeine flupirtine is up to 4 times more potent, up to 2 times more active than pethidine and 4 times more potent than dextropropoxyphene in the above-mentioned methods. With one exception of inflammation induced pain, where flupirtine shows an activity of about 1 1/2 times that of phenacetin and paracetamol, both analgesics are about 10 to nearly 30 times less active than flupirtine in other above-mentioned tests. In the hot plate test flupirtine is twice as active as codeine and approximately 10 times more active than phenacetin and paracetamol. The weakest analgesic activity of flupirtine is seen in acetic acid test where it is about half as active as codein and approximately as active as dextropropoxyphene. Nevertheless, flupirtine is up to 10 times more potent than phenacetin and paracetamol. The acetic acid test is claimed to be non-specific according to our own experience and to other authors. Flupirtine is enterally absorbed at a higher degree than the other tested centrally acting analgesics. In regard to the results of various analgesic investigations in mice and rats flupirtine can be classified as a medium to strong acting analgesic. The duration of action of flupirtine is comparable to that of codeine. Experiments with flupirtine suggest that there are some convincing criteria for a pronounced central acting component of its analgesic activity. These criteria are the strong efficacy in the hot-plate and Haffner's test, in which only centrally acting analgesics show distinct effects, and the finding that flupirtine increases the pain threshold for vocalisation in rats and mice excluding a pure reflex of the spinal cord. In current experiments concerning the mode of action flupirtine exhibits a distinct central analgesic component of action. In spite of its relatively high analgesic potency which corresponds to that of opiates flupirtine does not show any other signs of opiate properties and other potent analgesics. Thus, flupirtine does not develop tolerance in mice and rats after 19 or 17 days of daily administration.(ABSTRACT TRUNCATED AT 400 WORDS)     

利鲁唑对吗啡镇痛、耐受和依赖作用的影响(英文)

目的　研究利鲁唑对阿片镇痛、耐受及躯体功能的调节。方法　采用冰醋酸扭体 ,5 5℃热板法和热辐射甩尾法观察利鲁唑对小鼠痛阈及吗啡镇痛效应的影响 ;采用小鼠急性和慢性吗啡耐受模型及小鼠吗啡依赖模型 ,观察利鲁唑对吗啡耐受和依赖的作用。结果　单独皮下注射利鲁唑 2 .5～ 10mg·kg- 1在以上 3种模型无镇痛作用 ,然而能剂量依赖性地增强吗啡镇痛效应。利鲁唑 2 .5～ 10mg·kg- 1剂量依赖性地对抗吗啡引起的急性和慢性耐受。在小鼠吗啡依赖模型中 ,利鲁唑 2 .5～ 10mg·kg- 1剂量依赖性地抑制吗啡戒断症状的产生。结论　利鲁唑自身无镇痛作用 ,但能显著增强吗啡镇痛效应 ,并能预防吗啡所引起的耐受和依赖     

Pain and QOL--morphine-tolerance and morphine-resistant neuropathic pain

Morphine is now said to have no problematic side effects such as analgesic tolerance and physical dependence for cancer pain patients in clinic, as far as it is appropriately used. However, sub-sensitivity to morphine might be developed when higher doses of morphine are used for terminal cancer pain patients. Along with the severity of cancer, the nature of pain becomes changed to neuropathic pain, which is resistant to morphine or NSAIDS. In order to safely use morphine in the clinic, we need to know how morphine tolerance and neuropathic pain are developed and what adjuvants could be used to completely suppress the pain. Here I overview the proposed mechanisms for morphine tolerance and neuropathic pain in relation to the availability of analgesic adjuvants.