聚乙二醇缀合熊果酸衍生物的合成及体外抗癌活性

目的:合成具有一定水溶性的聚乙二醇(PEG)缀合熊果酸衍生物并考察其体外抗癌活性。方法:以熊果酸(ursolic acid,UA)作为母核,采用PEG、琥珀酸对其C28位进行接合修饰,合成一系列PEG缀合熊果酸衍生物并考察其水溶性和抗癌活性;采用流式细胞术探讨其抗癌机制。结果:所合成系列PEG缀合熊果酸衍生物的水溶性较熊果酸有明显改善,同时具有更显著的体外抗癌活性;化合物8c对5株受试肿瘤细胞的IC50(48 h)均小于10μmol.L-1,将AGS细胞阻滞于G2/M期并具有诱导凋亡作用(凋亡率高于70%)。结论:将熊果酸C28位进行PEG修饰是保持和提高其抗癌活性的有效途径之一。     

口服聚乙二醇修饰固体脂质纳米粒的组织分布及抗肿瘤药效学研究

目的 研究口服固体脂质纳米粒(solid lipid nanoparticle,SLN)和经聚乙二醇(polyethylene glycol,PEG)修饰后的SLN(pSLN)在小鼠体内的组织分布及药效。方法 采用水性溶剂扩散法制备SLN,用聚乙二醇单硬脂酸酯(PEG2000-SA)修饰以提供亲水基团;测定其粒径、Zeta电位、表面元素、接触角和稳定性;以DiR为荧光标记物,测定SLN及pSLN制剂经口服给药后的体内组织分布;以阿霉素为模型药物,考察口服脂质纳米给药系统的体内抗肿瘤活性及安全性。结果 SLN经PEG修饰后,得到的pSLN制剂粒径降低,Zeta电位约为-20 mV,表面亲水性及体内稳定性增加;经口服给药后,pSLN在肿瘤组织有聚集,且经PEG修饰后的纳米粒在组织中的滞留时间可显著延长;在荷瘤裸鼠模型动物上的药效学结果显示,PEG修饰口服脂质纳米给药系统在改善药效的同时,降低药物的不良反应,提高给药系统的安全性。结论 PEG修饰改善了口服纳米给药系统的生物分布及药效,提高了给药系统的安全性。     

2种单硬脂酸甘油酯固体脂质纳米粒制剂的体内组织分布及药动学研究

目的:研究单硬脂酸甘油酯固体脂质纳米粒(MSLN)和经聚乙二醇2000(PEG2000)修饰后的MSLN(PEG-MSLN)在小鼠体内的组织分布及其在大鼠体内的药动学,考察PEG2000修饰对MSLN体内组织分布及药动学的影响。方法:采用溶剂扩散法制备MSLN,测定其粒径和Zeta电位;以罗丹明B为荧光标记物,测定和计算2种MSLN制剂经鼠尾静脉注射后的体内组织分布及药动学参数。结果:2种MSLN制剂粒径分布相似,Zeta电位约为—20mV;经鼠尾静脉注射后,MSLN靶向肝脏,且经PEG2000修饰后的纳米粒体循环时间可显著延长至2·2倍。结论:MSLN经PEG2000修饰后可改善体循环,其可作为肝脏靶向的药物载体。     

rmuGM-CSF增强巨噬细胞抗白念珠菌活性研究

目的： 研究重组小鼠粒细胞巨噬细胞集落刺激因子(rmuGM CSF)对巨噬细胞抗白念珠菌活性的调节作用。方法：分别将浓度为100，200，300，400 U·mL 1的rmuGM CSF与小鼠腹腔巨噬细胞共同培养2～5 d，然后测定巨噬细胞的抗白念珠菌活性。结果： rmuGM CSF可增强巨噬细胞的抗白念珠菌活性，作用呈剂量依赖性，且随着与巨噬细胞共培养时间的延长，增强效应不降低。结论： rmuGM CSF能增强巨噬细胞的抗白念珠菌活性，增强效应强而持久。     

重组人粒细胞集落刺激因子生物学活性工作标准品的标定

目的用世界卫生组织 (WHO)粒细胞集落刺激因子 (G CSF)生物学活性国际标准品对所制备的重组人粒细胞集落刺激因子 (rhG CSF)生物学活性工作标准品以及惠尔血 针剂 75进行标定。方法用NFS 6 0细胞采用MTT法进行rhG CSF生物学活性的测定 ,(4 ,4)法计算测定结果。结果制备rhG CSF生物学活性工作标准品 3批 ,两批为冻干剂 ,与G CSF生物学活性国际标准品配方一致 ,一批为水剂 ,与惠尔血 针剂 75配方一致 ;用G CSF生物学活性国际标准品对其标定 ,结果依次为 3.0 6 2× 10 6、4.2 76× 10 6IU/支及 1.6 35× 10 7IU/ml,样品为1.880× 10 7IU/ml。标定结果的FL %分别为 5 .5 2 9%、4.2 91%、4.2 44 %及 5 .175 %。结论所标定的rhG CSF生物学活性工作标准品可用于rhG CSF生物学活性测定。     

重组人粒细胞集落刺激因子生物学活性测定方法研究

目的用世界卫生组织国际标准品粒细胞集落刺激因子 (G CSF)生物学活性标准品研究NFS 6 0细胞测定重组人粒细胞集落刺激因子 (rhG CSF)生物学活性的方法。方法采用溴化四唑蓝 (MTT)染色法。结果NFS 6 0细胞染色体为 39条 ;细胞数在 1× 10 5～ 1× 10 7个 /ml间MTT染色显示梯度 ;将 (4 ,4)法引入rhG CSF生物学活性的测定 ,单次实验结果的平均可信限率为 13.5 6 0 % ,(4 ,4)法测定rhG CSF生物学活性的批内CV <10 % ,批间CV为 10 .10 9%。结论 (4 ,4)法可用于rhG CSF生物学活性的测定 ,对测定方法标准化后 ,测定结果可准确、有效地指导rhG CSF的生产和研究。     

rhGM-CSF抗系统性白念珠菌感染作用研究

目的研究重组人粒细胞巨噬细胞集落刺激因子（rhGM-CSF）抗系统性白念珠菌感染作用。方法首先制备系统性白念珠菌感染小鼠模型，用不同剂量rhGM—CSF作用于小鼠，观测其28d内的存活情况；另外，以一定剂量的rhGM-CSF作用于小鼠，检测其用药前后白细胞总数、中性粒细胞、淋巴细胞、单核细胞及血小板数量变化。结果应用25μg／kg的rhGM—CSF能显著延长小鼠28d内存活期，其白细胞总数、单核细胞显著增多（P〈0．05），而中性粒细胞的增多具有极显著性（P〈0．01）。结论rhGM-CSF有较好的抗系统性白念珠菌感染作用。     

蛋白质药物聚乙二醇修饰方法的生物优化

聚乙二醇 (polyethyleneglycol,PEG)修饰又称分子的PEG化。是用来解决或缓解蛋白质和多肽类在药用过程中存在的诸多问题的有效途径 ,PEG修饰对具有药用潜能的蛋白来说具有 :(1)增加稳定性 ,延长血浆半衰期 ;(2 )降低免疫原性和抗原性 ;(3)降低毒副作用。但是 ,很重要的一点是蛋白质经PEG修饰后会引起生物学活性的降低。PEG化引起生物学活性降低的原因是多方面的 ,其主要原因为终产品中引进的基团 ,包括PEG以及PEG和修饰蛋白质之间的连接键所致 ;也与修饰反应的条件、产生的副产物有关。不同的蛋白质影响差异很大 ,这也是PEG化个体…     

拟人参皂苷-F11对脂多糖所致急性肺损伤小鼠的保护作用

目的拟人参皂苷F11(PF11)是从西洋参茎、叶中提取分离的一种三萜皂苷。课题组前期研究表明,PF11具有抑制神经炎症、抗氧化应激的神经保护作用。但是,PF11是否对脂多糖(LPS)所致急性肺损伤(ALI)小鼠具有保护作用仍未报导。方法气管滴注脂多糖(3 mg·kg~(-1))构建小鼠ALI模型,在造模前连续3 d尾静脉注射PF11(3,10和30 mg·kg~(-1))。通过测定肺组织病理学变化、肺湿/干重比值、支气管肺泡灌洗液(BALF)中细胞数目及蛋白浓度来评价PF11对ALI小鼠的保护作用,随后采用酶联免疫吸附、免疫组织化学和流式细胞术等实验方法,对炎症反应、中性粒细胞募集、中性粒细胞凋亡以及凋亡后吞噬等方面进行研究。结果 PF11显著减轻LPS所致ALI小鼠肺组织病理损伤,逆转肺湿/干重比值及BALF中的细胞数目和蛋白浓度的增加。此外,PF11显著抑制LPS所致ALI小鼠BALF中炎症因子及趋化因子(IL-6,TNF-α,IL-1β,MIP-2)蛋白水平的升高、逆转肺组织中髓过氧化物酶活性和中性粒细胞浸润的增加,同时还能显著促进BALF内中性粒细胞的凋亡和巨噬细胞对凋亡中性粒细胞的吞噬。结论 PF11通过抑制中性粒细胞浸润和加速中性粒细胞清除发挥对LPS所致ALI小鼠的保护作用,有可能成为防治ALI的潜在药物。     

重组人粒细胞集落刺激因子临床应用进展

<正> 重组人粒细胞集落刺激因子(G—CSF)是应用生物工程技术生产的一种造血生长因子,它作用于骨髓中性粒细胞系造血前体细胞,促进其增殖、分化,并促进中性粒细胞的成熟和释放,增强成熟中性粒细胞的功能。自1991年在美国首先上市以来,已获越来越广泛的临床应用,G—CSF的临床应用研究总结如下: 1.肿瘤化疗引起的中性粒细胞减少症 G—CSF在化疗后给药,可升高外周血中性粒细胞计数,降低粒细胞减少症的发生率及持续     

The antiviral activity of naturally occurring proteins and their peptide fragments after chemical modification

Chemical modification of the proteins bovine serum albumin, alpha-lactalbumin, beta-lactoglobulin and chicken lysozyme by 3-hydroxyphthalic anhydride (3-HP) yielded compounds which exerted antiviral activity in vitro as compared with the native unmodified proteins. Of the three enveloped viruses tested, human herpes simplex virus type 1 (HSV-1), bovine parainfluenza virus type 3 and porcine respiratory corona virus, only HSV-1 proved sensitive to the 3-HP-proteins. All of the chemically modified proteins presented antiviral activity against HSV-1 when assayed before, during or after infection. However, to achieve HSV-1 inhibition, significantly higher concentrations of the modified proteins were required if present before infection as compared to during or after infection. Our results suggest that multiple mechanisms are involved in the inhibition of HSV-1 infection. Proteolytical digestion of albumin, alpha-lactalbumin, beta-lactoglobulin and lysozyme by trypsin, chymotrypsin and pepsin yielded several peptide fragments with antiherpetic activity. Chemical modification of these peptide fragments by 3-HP generated peptides with antiviral activity, however, this was almost always combined with a cytotoxic effect on the Vero cells. Overall, our results suggest that targeted chemical modification of some natural products might provide compounds effective against HSV-1 infection.     

Studies on the function of tryptophan-108 on lysozyme.

Chemical studies of selective modification of Trp-108 of lysozyme gave ambiguous results concerning its function on the catalytic activity, since the oxyndole derivative obtained with N-bromosuccinimide is inactive, whereas the kynurenine derivative obtained by oxidation with ozone is fully active. In order to explain this discrepancy, lysozyme has been modified with 2-nitro-4-carboxyphenylsulfenyl chloride (NCPS-Cl). This reagent reacts with the indole ring of tryptophan giving a 2-thioaryl-derivative. By chromatographic fractionation of the reaction mixture, a lysozyme derivative was isolated, that by sequence studies was proved to be modified only at Trp-108 retaining 10% of the lytic activity. Physico-chemical as well as kinetic studies indicate that the large decrease in activity following modification could be related to minor effects in the microenvironment of the active site, with a concomitant modification of the ionization constants of the groups involved in catalysis.     

几种因素在L-天冬酰胺酶化学修饰中对酶活力及抗原性的影响

为了寻求在较好地保持酶活力的同时解除L-天冬酰胺酶抗原性的方法,采用不同分子量的乙酸酐、右旋糖酐和单甲氧基聚乙二醇,作为修饰剂和不同的修饰方法对该酶进行了化学修饰。结果表明在保持酶活性和降低抗原性方面,大分子修饰剂右旋糖酐、单甲氧基聚乙二醇优于小分子乙酸酐,底物保护修饰优于直接修饰;活化PEG,优于活化PEG₁。在底物保护下的PEG,修饰酶其抗原性完全解除的同时,酶活力保持在30%以上。     

Batch purification of high-purity lysozyme from egg white and characterization of the enzyme modified by PEGylation

PEGylation is one of the most promising and extensively studied strategies for improving the pharmacological properties of proteins as well as their physical and thermal stability. Purified lysozyme obtained from hen egg white by batch mode was modified by PEGylation with methoxypolyethyleneglycol succinimidyl succinato (mPEG-SS, MW 5000). The conjugates produced retained full enzyme activity with the substrate glycol chitosan, independent of degree of enzyme modification, although lysozyme activity with the substrate Micrococcus lysodeikticus was altered according to the degree of modification. The conjugate with a low degree of modification by mPEG-SS retained 67% of its enzyme activity with the M. lysodeikticus substrate. The mPEG-SS was also shown to be a highly reactive polymer. The effects of pH and temperature on PEGylated lysozymes indicated that the conjugate was active over a wide pH range and was stable up to 50°C. This conjugate also showed resistance to proteolytic degradation, remained stable in human serum, and displayed greater antimicrobial activity than native lysozyme against Gram-negative bacteria.     

聚乙二醇修饰的牛血红蛋白血液代用品抗原性和免疫原性的研究

目的建立聚乙二醇修饰的牛血红蛋白血液代用品抗原性和免疫原性评价方法。方法用酶联方法测定供试品的抗原性残留量,用豚鼠过敏试验研究供试品的免疫原性,通过比较推算供试品抗原性残留量的安全性限值。结果供试品的抗原性残留量大于2 3%时,豚鼠会出现不同程度的过敏反应;供试品的抗原性残留量小于或等于2 3%时,豚鼠无过敏反应发生;供试品抗原性残留量的最高限值为2 3%。结论初步建立了牛血红蛋白血液代用品抗原性和免疫原性评价方法     

Biological and Clinical Correlates after Chemotherapy and Granulocyte Colony-Stimulating Factor Administration

Study Objective . To evaluate specific biological markers to improve understanding and use of granulocyte colony-stimulating factor (G-CSF) in patients receiving chemotherapy. Design . Prospective, randomized study. Setting . University-affiliated hospital and cancer center. Patients . Twenty-five patients randomized to begin G-CSF either 24 hours after chemotherapy (standard arm), or on the day the absolute neutrophil count (ANC) was below 1000/mm³ after chemotherapy (delayed arm). Interventions . To determine the effect of G-CSF on granulopoiesis, peripheral blood mononuclear cells were assayed by semisolid culture medium and flow cytometry for granulocyte progenitors and clonogenic CD34 antigen-positive cells. These biological markers were correlated with G-CSF administration schedules and the ANC. Measurements and Main Results . The effect of timing of G-CSF administration on rate of neutrophil recovery, duration of neutropenia, length of G-CSF therapy, delays of chemotherapy cycles, and neutropenic fever events was evaluated. Regardless of G-CSF schedule or chemotherapy regimen, the appearance of mobilized hematopoietic progenitors begins at the neutrophil nadir and parallels granulocyte recovery. Our data also demonstrate that proper timing of G-CSF administration produces similar rates of neutrophil recovery and comparable clinical outcomes. Conclusion . Based on the correlation between biological markers and ANC, we propose that the postchemotherapy ANC is a surrogate marker of renewed granulopoietic activity. The relevance of this finding in relationship to the clinical application of G-CSF remains to be further defined.     

Conformational aspects of human formyl-peptide receptor agonists

The conformation of several Met-Ile-Phe-Leu analogues was analyzed using circular dichroism and infra-red absorption. Their effect on human neutrophils was verified by receptor binding assays and measurements of lysozyme release. The results demonstrate that in amphipatic environments the compounds examined can be highly and weakly ordered in beta-turn structures, in dependence on their N-terminal substituents. The ability of the compounds to evoke neutrophil functions appears strongly and weakly influenced by N- and C-terminal modification, respectively.     

Impaired neutrophil chemotaxis in Crohn's disease relates to reduced production of chemokines and can be augmented by granulocyte-colony stimulating factor

BACKGROUND: Defective neutrophil recruitment has been described as a primary pathogenic abnormality in Crohn's disease. Cantharidin-induced blisters provide a novel investigative tool to assess cellular influx and inflammatory mediator production during acute inflammation and allows the effects of therapy on these parameters to be measured. AIMS: To determine whether reduced neutrophil tissue penetration in Crohn's disease relates to impaired production of inflammatory mediators, and whether it can be reversed by granulocyte-colony stimulating factor (G-CSF). METHODS: Neutrophil and monocyte/macrophage populations and inflammatory mediators were measured in cantharidin blisters at 24 h. Neutrophil chemotaxis was assessed in vitro using blister fluid as the chemoattractant. The effect of s.c. G-CSF on blister phenotype was determined. RESULTS: Significantly fewer neutrophils migrated into blisters in Crohn's patients. The production of neutrophil chemokines, but not other inflammatory mediators, was reduced. This significantly correlated with reduced chemotaxis in vitro. Differences were unrelated to caspase-recruitment domain 15 genotype. G-CSF significantly increased blister neutrophil concentrations in control subjects and Crohn's patients. CONCLUSIONS: Reduced neutrophil migration during acute inflammation in Crohn's disease is associated with impaired production of appropriate chemoattractants. G-CSF therapy increases neutrophil tissue migration, which may partially account for its observed therapeutic effect.     

Role of amino and carboxyl groups of cobrotoxin in the conformational stability and the interaction with acetylcholine receptor

To study the functional involvements of the common interaction of the Leu-1 α-amino group and Asp-58 in cobrotoxin, the lysine ε-amino groups of cobrotoxin were initially guanidinated with o-methylisourea. The α-amino group of Leu-I was then modified with TNBS after the guanidination of cobrotoxin. Both modified derivatives displayed no significant changes in the secondary structure and antigenicity of cobrotoxin, whereas the binding affinity for nicotinic acetylcholine receptor (nAChR) was pronouncedly decreased when Leu-1 was modified. Six out of seven free carboxyl groups and the remaining buried Glu-21 carboxyl group of cobrotoxin were modified with glycine methyl ester in the absence and presence of guanidine HCl, respectively. Alternation in the β-sheet secondary structure of cobrotoxin was observed with the carboxyl-group modified derivatives, which caused a decrease in the binding activity of the toxin molecule to the antibody and nAChR. Moreover, modification of the Glu-21 carboxyl group of cobrotoxin further reduced the nAChR binding activity, while the antigenicity remained unchange. Thus, our results conclude that the Glu-21 residue and the common interaction of the terminal Leu-1 α-amino group and the Asp-58 carboxyl group are related to the nAChR-binding activity of cobrotoxin, and the free carboxyl groups in cobrotoxin are conformation-essential. © Munksgaard 1995.     

T4溶菌酶在毕赤酵母中的诱导表达及抑菌活性测定

目的利用毕赤酵母诱导表达T4溶菌酶蛋白并测定其抗菌活性。方法T4溶菌酶（T4lysozyme）基因以N端融合的方式被准确插入到质粒pPIC9K的EcoR I-Not I位点内,得到分泌型重组表达载体 pPIC9K-T4L。该载体首先经限制性内切酶Sal I酶切,进而采用电击方法将线性化的重组质粒DNA导入到毕赤酵母中。经过梯度筛选得到多个单拷贝和多拷贝转化重组子。随机挑取部分重组子PCR扩增阳性克隆,经菌体培养和甲醇诱导后获得了分泌表达,表达产物存在于培养上清液中。结果表达蛋白经琼脂孔扩散抗菌实验显示抑菌圈明显;重组蛋白对金黄色葡萄球菌与肺炎链球菌均具有显著抑制作用;多拷贝与单拷贝重组表达子没有抗菌活性差异与蛋白表达量的差异;加热煮沸对于T4溶菌酶蛋白的抗菌活性无明显影响。结论T4溶菌酶在毕赤酵母中得以成功诱导与表达;表达产物不受拷贝数影响并具热稳定性。