发形霞水母毒素溶血活性研究

目的 探讨发形霞水母毒素(Cyanea capillata full venom,CFV)的体外溶血活性及其影响因素.方法 超声破碎法分离得到CFV,测定其半数溶血分数(HU50),探讨温度、pH、放置时间对溶血活性的影响,并进一步研究不同动物红细胞对溶血活性的敏感性.结果 CFV溶血活性的HU50为5.82 μg·mL-1;溶血活性对温度和pH变化耐受性差,在40℃和pH 8.0时有最大溶血活性;25℃和37℃放置12 h CFV溶血活性迅速下降至18.6%和17.4%;对不同动物红细胞敏感性差异较大,其中以家犬红细胞最为敏感.结论 CFV有显著的溶血活性.     

荧光光谱法研究过氧化氢酶溶液稳定性

目的考察不同环境因素对过氧化氢酶(CAT)活性和构象的影响,为蛋白质制剂过程提供依据。方法改变温度、pH、变性剂浓度,用紫外分光光度法考察CAT溶液活性变化,并用荧光光谱法研究其构象变化。结果CAT溶液25℃p、H 7时,活性及荧光强度都达到最大;温度升高,荧光强度逐渐下降;pH降低或升高,荧光最大发射波长均红移;低浓度变性剂对CAT活性中心有稳定作用;浓度增加,荧光谱带红移,活性降低甚至丧失;脲和盐酸胍对CAT荧光有动态猝灭作用。结论用荧光光谱法研究CAT溶液稳定性,方法简便、准确、可靠。     

发形霞水母触手提取物活性的初步研究

目的 初步分离发形霞水母触手提取物并探讨其生物学活性.方法采用自溶、离心的方法去除发形霞水母触手刺丝囊并获取触手提取物.通过阶段梯度阳离子交换色谱,应用0%.20%,40%,100%B液4种比例洗脱液将触手提取物分成4个组分,分别观察分离各组分的溶血、心血管等活性,并与触手提取物平行对比分析.结果成功分离到具有溶血、心血管活性的发形霞水母触手提取物,4个洗脱组分中,0%B液组分无上述3种活性,20%B液组分具有溶血活性、40%B液组分具有心血管活性,而100%B液组分则含有色素.结论通过阳离子交换色谱.初步将发形霞水母触手提取物中具有溶血活性、心血管活性以及色素等组分分离开来.为后续进一步纯化其单一活性组分打下基础.     

三叶苷调控miR-539-5p表达保护缺氧缺糖心肌细胞损伤研究

摘要：目的:探讨三叶苷对缺氧缺糖心肌细胞损伤的保护作用及机制。方法:采用不同浓度的三叶苷作用于缺氧缺糖诱导的心肌细胞H9C2,试剂盒检测丙二醛（MDA）含量、乳酸脱氢酶（LDH）和超氧化物歧化酶（SOD）活性,流式细胞术、蛋白质印记（Western blot）检测细胞凋亡以及B细胞淋巴瘤-2（Bcl-2）和Bcl相关X蛋白（Bax）蛋白表达,实时荧光定量PCR（RTq PCR）检测miR-539-5p表达。转染miR-539-5p模拟物至H9C2细胞,检测过表达miR-539-5p对缺氧缺糖诱导的H9C2细胞损伤的影响。结果:三叶苷作用于缺氧缺糖诱导的H9C2细胞后,细胞中MDA含量、LDH释放量、细胞凋亡率和Bax蛋白表达水平显著降低,SOD活性、Bcl-2蛋白和miR-539-5p表达水平显著升高（P<0.05）。过表达miR-539-5p后,缺氧缺糖诱导的H9C2细胞中MDA含量、LDH释放量、细胞凋亡率和Bax蛋白表达水平显著降低,SOD活性、Bcl-2蛋白表达水平显著升高（P<0.05）。抑制miR-539-5p表达可减弱三叶苷对缺氧缺糖诱导的H9C2细胞MDA含量、LDH释放量、SOD活性、凋亡,以及Bax和Bcl-2蛋白表达的影响。结论:三叶苷可有效降低缺氧缺糖诱导的心肌细胞损伤,其机制与上调miR-539-5p表达有关。     

配制浓度对奥美拉唑滴注液稳定性的影响

目的探讨配置浓度对奥美拉唑滴注液稳定性的影响。方法使用氯化钠、葡萄糖注射液对奥美拉唑静脉滴注进行不同浓度的配制,使用高效液相色谱仪、p H仪对不同浓度的奥美拉唑滴注液稳定性进行分析。结果奥美拉唑经0.9%氯化钠溶液、5%葡萄糖溶液稀释后,经8h连续观察发现,不同组别滴注液的含量随时间的延长逐渐下降,但并未出现显著的变化,不同组别滴注液的p H随浓度增大逐渐降低,并随时间的延长逐渐降低,未发现有颜色上的变化发生。结论使用0.9%氯化钠溶液、5%葡萄糖溶液对奥美拉唑进行配制,浓度在0.08-0.8g/L范围内,时间在8h内,含量、p H值并未发生显著变化,稳定性较好。     

太白楤木活性成分竹节参皂苷Ⅳa抗氧化及抗衰老作用研究

目的探讨太白楤木皂苷中的重要成分竹节参皂苷Ⅳa(CHS)抗氧化及抗衰老作用。方法取5~8代的小鼠胚胎成纤维细胞(MEFs),使用过氧化氢(H2O2)诱导细胞氧化应激以及细胞衰老模型。MTT法分别检测不同浓度CHS及H2O2对细胞活力的影响;DCFH-DA染色流式细胞仪检测细胞总活性氧(ROS)水平;Mito SOX染色流式细胞仪检测细胞线粒体ROS水平;衰老相关β-半乳糖苷酶(SA-β-gal)染色检测细胞衰老状态;Western blot法检测衰老标志蛋白p53、p21蛋白表达水平以及关键抗氧化应激Nrf2通路(Nrf2、HO-1、GCLC、GCLM)蛋白表达。结果浓度≤160μg·m L-1的CHS对MEFs细胞无明显毒性作用;H2O2能够显著提高细胞总ROS和线粒体ROS水平,诱导细胞衰老指标SA-β-gal活性增加,提高p53、p21表达水平,提高抗氧化信号通路Nrf2通路表达水平。CHS能够剂量依赖地降低H2O2诱导的细胞总ROS和线粒体ROS水平,减轻H2O2诱导的SA-β-gal活性升高,抑制p53、p21以及Nrf2、HO-1、GCLC、GCLM蛋白表达水平的升高。结论 CHS具有显著的抗氧化应激和抗衰老作用。     

条斑紫菜藻胆蛋白对小鼠免疫功能及抗氧化活性的影响

目的本实验研究了条斑紫菜藻胆蛋白对小鼠免疫功能及抗氧化活性的影响。方法通过小鼠衰老模型探讨不同浓度藻胆蛋白提取液(CPP)灌胃30 d后,对小鼠免疫器官重量、血清超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性及肝组织丙二醛(MDA)生成的影响。同时,研究了纯化的R-藻红蛋白(R-PE)和C-藻蓝蛋白(C-PC)对二苯代苦味酸自由基(DPPH)的清除作用及对H2O2诱导的小鼠红细胞溶血和小鼠肝匀浆脂质过氧化生成的抑制作用。结果藻胆蛋白提取液可以提高免疫器官脏器指数,并且显著提高小鼠血清SOD和GSH-Px活性,显著降低了肝组织MDA生成。R-PE和C-PC浓度在1.4 mg.mL-1时,对DPPH的清除率达到最大,分别为89.01%和60.52%;在浓度为0.12 mg.mL-1时,对小鼠红细胞溶血的抑制率达到最大,分别为73.85%和63.42%;在浓度为0.2 mg.mL-1和0.3 mg.mL-1时,对肝匀浆脂质过氧化的抑制率分别达到66.63%和66.67%。以上活性均高于同浓度下的维生素C。结论条斑紫菜藻胆蛋白能提高小鼠的免疫功能,是一种具有良好应用前景的抗氧化剂。     

苦荞麦麸皮中类黄酮的抗氧化活性研究

选用山西黑苦荞的麸皮为原料,用70%乙醇加热提取其中的类黄酮物质,采用邻苯三酚自氧化法测定其类超氧化物歧化酶（L-SOD）的活性及不同环境条件对L-SOD活性的影响。苦荞麦麸皮提取物中的类黄酮（TBF）含量为8.1%,在350nm处有一个特征吸收峰,其L-SOD活性为660U/mg。TBF中L-SOD活性的最适温度在40℃左右,最适pH为8.0左右,在80℃左右,在80℃条件下仍可保持87%的活性;并且在碱性范围内的稳定性高于人血红细胞SOD。在一定浓度的Cu∧2 、Mg∧2 和Cd∧2 存在下,其L-SOD活性分别有不同程度的提高,表明苦荞麦麸皮中的类黄酮酮物质有较强的L-SOD活性。     

活性载体法从鱿鱼肝脏中提取核酸的最佳条件的研究

目的　探索用实验室构建的活性载体从鱿鱼肝脏中提取核酸的最佳条件。方法　首先在 3 7℃培养基 p H=6.0时 ,将 15 %、 10 %、 5 %、 3 %浓度的载体加入一定量肝匀浆分别培养 2 4、 3 6、 4 8、 72 h,经 4℃ 40 0 0 r/ m in离心处理 ,分别测其水相、沉淀中的核酸含量 ,并比较找出总核酸含量最高的载体浓度与培养时间条件 ;然后 ,利用得到的载体浓度和培养时间 ,将肝匀浆和载体的混合物置于不同培养温度、培养基 p H值、光照条件等情况下培养 ,再经 4℃ 4 0 0 0 r/ m in离心处理 ,测其水相与沉淀中核酸含量 ,比较找出总核酸含量最高的各种条件。结果　活性载体浓度 10 % ,培养 3 6h,测其总核酸含量最高。载体在无氧、无光照、p H=6.0、温度 3 7℃时其活性最高 ,测得其核酸含量最高。结论　实验室构建的活性载体法提取核酸的最佳条件为 :温度 3 7℃、p H =6.0、无氧、无光照、浓度 10 %、培养 3 6h     

小白菊内酯对宫颈癌细胞增殖、自噬及其作用机制研究

目的研究小白菊内酯(PTL)对宫颈癌Hela细胞增殖、自噬及其作用机制的影响。方法 CCK8法检测0、2.5、5、10、20、40μmol·L~(-1)浓度梯度的PTL对宫颈癌Hela细胞增殖的影响;荧光显微镜法检测PTL宫颈癌Hela细胞自噬情况;蛋白印迹检测凋亡相关的蛋白表达水平;ROS活性试剂盒检测细胞内活性氧(ROS)水平;蛋白质印迹检测凋亡相关的蛋白表达水平。结果不同浓度的PTL作用于Hela细胞后,细胞活性随着PTL浓度的增加显著降低;可导致自噬小体聚集及蛋白LC3、p62表达降低,细胞凋亡通路相关蛋白4E-BP1的表达明显降低,细胞内ROS水平显著增加。结论 PTL可通过ROS/4E-BP1信号通路促进Hela细胞的自噬,抑制其增殖。     

金针菇固态发酵产木聚糖酶的纯化及其性质研究

金针菇固态发酵产生木聚糖酶经硫酸铵盐析、透析、浓缩、Sephadex G-75分子筛层析等分离与纯化步骤,得到了一种相对分子质量约为59.7k的电泳纯木聚糖酶,其最终的活性收率为23.3%,纯化倍数为9.4。该木聚糖酶在pH值为6.0,50℃下的酶解效果最佳。酶的pH稳定性及热稳定性都比较好,在pH 4.0～8.0时基本稳定,80℃时相对酶活力仍保持在55.81%。Ca2+、Zn2+对该酶活性有一定的激活作用,而Hg2+、Cu2+则有较强的抑制作用。     

Purification and some properties of a hemolysin from the poisonous mushroom Rhodophyllus rhodopolius

A hemolytic protein which causes diarrhea and death to mice was purified from the fruit bodies of a poisonous mushroom species Rhodophyllus rhodopolius (Fries) by DEAE-cellulose column chromatography, ammonium sulfate precipitation, gel filtration on Sephadex G-200, and DEAE-Sephadex A-25 column chromatography. The mol. wt of the purified hemolysin was estimated to be 40,000 by SDS-polyacrylamide slab gel electrophoresis. The hemolytic activity of the purified hemolysin was destroyed by heating at 60 degrees C for 10 min, and partially reduced by pepsin, papain and 2-mercaptoethanol. Cholesterol and phosphatidylcholine did not inhibit the activity. The hemolysin was unstable below pH 7.0 but stable at pH 8.0. The optimal pH for hemolysis was 6.0. Hemolysis did not occur below 4 degrees C even though the hemolysin bound to the erythrocyte. Mouse, chicken, rat, horse and human erythrocytes were sensitive in this order, but sheep and cow erythrocytes were not lysed by the toxin.     

RP-HPLC法考察硫酸头孢噻利与奥美拉唑钠的配伍稳定性

目的观察硫酸头孢噻利(第4代头孢菌素类抗生素)与奥美拉唑钠(抗溃疡药)的配伍稳定性。方法用反相高效液相色谱法测定硫酸头孢噻利与奥美拉唑钠配伍后在不同温度下8 h内的相对百分含量,并考察其外观与pH值。结果硫酸头孢噻利与奥美拉唑钠配伍后,在4℃放置8 h、25℃放置3 h、37℃放置1 h,配伍溶液的外观、pH值及两者相对百分含量基本无变化。37℃放置2 h后,奥美拉唑钠相对百分含量变化约5%,配伍液不能使用。结论硫酸头孢噻利与奥美拉唑钠的配伍液在高温环境中1 h内稳定,2 h后稳定性差。     

节杆菌黄嘌呤氧化酶提取优化的初步研究

目的从节杆菌Arthrobactersp.XL2胞内中初步提取黄嘌呤氧化酶(XOD)。方法探讨了不同破壁方法和硫酸铵盐析提取粗酶的条件。采用响应面分析法优化了超声波法抽提XOD的条件。结果菌体用超声波破壁,在频率25 KHz,细胞浓度80 mg/mL下每次辐射2 s,间隔2 s,总辐射时间18 min,输出功率400 W,破碎产酶量达到(4.69±0.11)U/mL。用40%~60%饱和度硫酸铵阶段盐析提取粗酶效果最好。将盐析粗酶用Sephadex G-25脱盐后,比酶活达到6.95 U/mg,纯度提高了3.43倍,然后进行XOD粗酶部分酶学性质研究。XOD的稳定pH和温度范围分别为:pH 7.0~9.0和低于50℃。XOD的最适反应温度和pH分别为:40℃及pH 8.0。Zn2+,Cd2+,Cu2+,Pb2+,Ag+,Hg2+强烈抑制酶的活性。结论此粗酶提取工艺可以有效地将XOD从细胞内释放出来,提高了粗酶抽提得率。为后续的进一步纯化研究奠定了基础。     

Purification and properties of a toxin from the South African sea anemone, Pseudactinia varia

A comparison was made of the hemolytic potency of aqueous extracts prepared from five species of intertidal sea anemones from the coast of South Africa. The active agent in an extract of Pseudactinia varia was purified by ammonium sulfate precipitation, gel permeation chromatography and isoelectric focusing. The hemolytic toxin, termed variolysin, is a protein having a molecular weight of 19,500 and an isoelectric pH of 9.8. It retained appreciable activity after heating to 70 degrees for 40 min. Amino acid analysis revealed that it lacked methionine and cysteine. Its hemolytic activity was inhibited by sphingomyelin. The properties of variolysin show that it is broadly similar to cytolytic toxins isolated from a number of other anthozoans.     

Liquid high concentration IgG1 antibody formulations by precipitation

A manufacturing approach for liquid high concentration antibody formulations based on precipitation and subsequent re-dissolution was investigated. IgG1 antibody solutions were concentrated from 20 to 100 mg/mL by intermediate precipitation, with a recovery exceeding 95%, retention of the native secondary structure and binding activity as well as adequate stability. Quantitative, reproducible precipitation was performed using 1.45 M ammonium sulphate (pH 5.5 and 8.0), 0.67 M sodium citrate (pH 8.0) and 9% (w/v) PEG 4000 (pH 5.5 and 8.0). Scalability was confirmed from 1 to 100 mL. The concentrations achievable in the re-dissolution step were less affected by the re-dissolution medium, but limited by the residual precipitant. Both, improved removal of remaining precipitant liquid and larger precipitation scales were successful in increasing the final protein concentration. SEC and turbidity analysis directly after re-dissolution indicated that similar protein qualities were obtained, independent from the precipitant used. However, increased aggregate formation was observed after short term storage of the precipitated protein particles at either 2-8 degrees C or ambient temperature. An accelerated mechanical and thermal stability program verified comparable stability of the re-dissolved liquid 100 mg/mL formulations produced by intermediate precipitation to a control formulation obtained by standard ultrafiltration.     

一种具有纤溶活性的蛋白酶的分离纯化及性质

目的分离纯化BacillussubtilisJin2 1发酵产生的具有纤溶活性的蛋白酶 ,并对其性质进行研究。方法通过硫酸铵分级盐析 ,SerineSepharose 2B和SephacrylS— 2 0 0色谱柱进行分离纯化 ,用SDS PAGE测定纯度 ,SDS PAGE和凝胶层析测定相对分子质量 ,纤维蛋白平板法测定酶活力。结果分离得到了电泳纯的酶 ,该酶的相对分子质量为 2 8kD ,等电点约为 8 6 ,最适 pH为 7 5 ,最适温度为 4 0℃ ,在 4 0℃以下较稳定 ,超过 5 0℃时酶活力开始下降 ;金属离子Mn2 + 对酶有明显的激活作用 ,而Zn2 + 对酶有抑制作用 ,二异丙基磷酰氟 (DFP)和苯甲基磺酰氟 (PMSF)完全抑制酶活性。结论该酶是一种丝氨酸蛋白酶 ,与纳豆激酶相似 ,有望开发成为一种新型口服溶栓药物     

Monoamine oxidase (XXXVI). Characteristics of benzylamine oxidase in the dog serum]

Enzymic properties of monoamine oxidase (MAO) in dog serum were studied and the following results were obtained. Some of enzymic properties of MAO in dog serum differed from that of mitochondrial MAO. When dog serum was fractionated by ammonium sulfate, proteins were concentrated in two fractions, such as 25 approximately 33% and 67 approximately 80% of saturated ammonium sulfate fraction, while MAO activity was concentrated in 40 approximately 50% of saturated ammonium sulfate fraction. The reaction rate of MAO in dog serum was found to be proportional to enzyme concentration. The optimum pH of MAO in dog serum was 7.0 which differed from that of MAO in rabbit serum (pH 8.0). Tris-HCl buffer strongly inhibited MAO activity in dog serum. When benzylamine was used as substrate, the highest activity was obtained compared with the other substrate used. The activities with butylamine, amylamine, beta-phenylethylamine and tyramine showed about 30% while tryptamine and serotonin showed 3 approximately 10% compared to that with benzymlamine as substrate. The value of pI50 of catron was about 3 X 10(-6) M and that of harmaline was about 3 X 10(-5) M, but pargyline did not inhibit MAO activity in dog serum at the concentration of 1 X 10(-4) M.     

克班宁长循环脂质体的制备工艺

目的:研究克班宁长循环脂质体的处方筛选和制备工艺。方法:采用硫酸铵梯度法制备克班宁长循环脂质体,以包封率和载药量为评价指标,采用葡聚糖凝胶过滤法分离脂质体,紫外分光光度法测定克班宁含量,采用正交设计优化制备工艺。结果:最佳工艺为:药脂比为1:6,胆固醇与磷脂比为9:12,0.15 mol·L^-1的硫酸铵溶液,以PBS液(pH7.4)为透析介质,在40 ℃水浴,40 r·min^-1条件下孵化20 min。结论:硫酸铵梯度法制备的克班宁长循环脂质体处方合理,工艺可行,包封率较高。     

Rat brain nucleoside diphosphatase: Purification and properties of type L isozyme

Type L nucleoside diphosphatase of rat brain was purified with procedures of solubilization by Triton X-100, ammonium sulfate fractionation and 4 steps of column chromatography using DE-52, Con A-Sepharose, Sephadex G-150, and Phenyl-Sepharose CL-4B. The purified enzyme had a molecular weight of 45 KDa and an isoelectric point of 4.6. The optimum pH for enzyme activity was 7.5≈8.0 and the K _m value was 1.2 mM for GDP. The enzyme activity was markedly enhanced by ATP, the stimulatory effect being due to increase in the affinity of the enzyme for substrate. Inorganic pyrophosphate potently inhibited the activity. It was a competitive inhibitor at lower concentrations, while it also decreased the V_max value at higher concentrations.