修饰蚯蚓纤溶酶的药代动力学研究

环毛蚓纤溶酶经聚乙二醇（PEG）和β-环糊精（β-CD）修饰后，分别静脉注射到猪体内和兔体内进行药代动力学研究。结果表明PEG-EFE在猪体内的半衰期为0.96h，而原酶在猪体内的半衰期为0.59h，PEG-EFE较原酶提高了1.62倍。β-CD-EFE注射于兔体内的药代动力学证明，β-CD-EFE在兔体内的分布相半衰期为1.00h，而原酶仅为0.65h，β-CD-EFE较原酶的体内半衰期提高了1.52倍。说明2种修饰方法都是有效的。     

多重耐药大肠埃希菌氨基糖苷类修饰酶与Ⅰ类整合酶基因的研究

目的 探讨本地区临床分离多重耐药大肠埃希菌(Escherichia coli,E.coli)的氨基糖苷类修饰酶和Ⅰ类整合子基因存在情况.方法 纸片扩散法(K-B法)测定71株大肠埃希菌对常用抗菌药物的耐药性,应用PCR技术对E.coli进行氨基糖苷类修饰酶和Ⅰ类整合酶基因的检测.结果 71株大肠埃希菌对庆大霉素、左氧氟沙星、头孢噻肟、头孢他啶的耐药率分别分为66.20％、63.38％、59.15％、18.31％,其中23株为多重耐药株(32.39％).氨基糖苷类修饰酶基因aac(3)-Ⅱ阳性40株(56.34％),aac(6′)-Ⅰ阳性22株(30.99％),ant(3″)-Ⅰ阳性14株(19.72％),未检出aac(6′)-Ⅱ阳性株,氨基糖苷类修饰酶基因总检出率为74.65％;Ⅰ类整合酶基因(intI1)阳性53株(74.65％).多重耐药与非多重耐药大肠埃希菌中aac(3)-Ⅱ、aac(6')-Ⅰ、总氨基糖苷类修饰酶基因、intI1的阳性率有统计学差异(P＜0.05).结论 本地区多重耐药E.coli的氨基糖苷类修饰酶基因、Ⅰ类整合酶基因携带率高.     

鲍曼不动杆菌氨基糖苷类修饰酶和Ⅰ类整合酶基因研究

目的了解我院鲍曼不动杆菌（Acinetobacter baumannii，AB）氨基糖苷类修饰酶和Ⅰ类整合酶基因存在情况。方法根据美国CLSI／NCCLS2004年版要求进行抗菌药物敏感性试验，应用PCR技术对AB菌进行氨基糖苷类修饰酶和Ⅰ类整合酶基因检测。结果AB菌对13种抗菌药物耐药严重。27株AB菌中氨基糖苷类修饰酶基因aac（3）-I阳性23株（85．2％）、aac（6′）-I阳性18株（66．7％）、ant（3″）-I阳性22株（81．5％）。氨基糖苷类修饰酶基因总检出率为85．2％。Ⅰ类整合酶基因（intI1）阳性23株（85．2％）。结论我院AB菌不仅具有多重耐药性，而且氨基糖苷类修饰酶基因、Ⅰ类整合酶基因携带率高。     

聚二醇修饰牛胰核糖核酸酶

采用N－羟基珀酰亚胺活化酯法活化单甲氧基聚乙二醇,测定了乙二醇（PEG）的活化度为86．2％。以活化的PEG对牛胰核糖核酸酶进行化学修饰;分析了蛋白质被修饰程度。用毛细管电泳法给出了被修饰蛋白的修饰度与修饰蛋白分布的定量结果。比较了被修饰产物对大分子底物（酵母RNA）与小分子底物（2‘,3‘－环磷酸胞嘧啶）的降解活力,其表观酶活力分别保留了52．8％和66．3％。结合毛细管电泳定量分析得到的修正酶活力略低于表观酶活力。     

鲍曼不动杆菌老年患者分离株中发现16S rRNA甲基化酶基因新亚型

目的了解鲍曼不动杆菌老年患者分离株16SrRNA甲基化酶和氨基糖苷类修饰酶基因型。方法采用PCR检测20株鲍曼不动杆菌老年患者分离株12种16SrRNA甲基化酶和氨基糖苷类修饰酶基因。结果7株检出16SrRNA甲基化酶基因（armA），19株检出氨基糖苷类修饰酶基因[其中aac（3）-I阳性率为95％，ant（3″）-I为95％，aac（3）-II为40％，aac（δ′）-Ib为15％）]。6号株armA基因测得序列翻译成氨基酸序列与美国核酸库（GenBank）已登录的armA氨基酸不同，为新亚型。结论本组鲍曼不动杆菌老年患者分离株95％携带16SrRNA甲基化酶和氨基糖苷类修饰酶基因。     

聚乙二醇修饰牛胰核糖核酸酶

采用N-羟基珀酰亚胺活化酯法活化单甲氧基聚乙二醇,测定了聚乙二醇（PEG）的活化度为86.2%。以活化的PEG对牛胰核糖核酸酶进行化学修饰;分析了蛋白质被修饰程度。用毛细管电泳法给出了被修饰蛋白的修饰度与修饰蛋白分布的定量结果。比较了被修饰产物对大分子底物(酵母RNA)与小分子底物（2',3'-环磷酸胞嘧啶）的降解活力,其表观酶活力分别保留了52.8%和66.3%。结合毛细管电泳定量分析得到的修正酶活力略低于表观酶活力。     

肺炎克雷伯菌老年人分离株氨基糖苷类修饰酶和I类整合酶基因研究

目的 了解肺炎克雷伯菌(KPN)老年人分离株氨基糖苷类修饰酶和I类整合酶基因存在状况.方法 对80株PKN菌进行2种氨基糖苷类修饰酶基因和I类整合子的遗传标记-I类整合酶基因检测.结果 80株KPN菌中aac(3)-Ⅱ阳性60株(75%)、aac(6)-Ⅰb阳性5株(6.3%),共有60株检出氨基糖苷类修饰酶基因(75%),intll基因阳件62株(77.5%),与庆大霉素、妥布霉素、奈替米星、阿米卡星耐药率分别为75%、80%、65%和60%.结论 该80株老年人分离株PKN菌氨基糖苷类抗菌药物耐药与产氨基糖苷类修饰酶及I类整合酶基因密切相关.     

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。     

白眉蝮蛇蛇毒纤溶酶的聚乙二醇修饰

为提高纤溶酶的临床适用性，采用4种针对氨基修饰的mPEG试剂——mPEG—SPA5K、mPEG—SMB5K、mPEG—ButyrALD5K和mPEG2-NHS40K修饰蛇毒纤溶酶，以获得高比例的单修饰产物和较高的酶活性保留率。优化修饰反应条件，用Superdex75凝胶过滤色谱法分离纯化修饰产物。最终确定mPEG—SPA5K为最适宜的修饰剂。产物经sDs—PAGE和MALDI-TOF-MS法检测为单修饰，修饰产物酶活性收率为67．3％，修饰产物的热稳定性提高，免疫原性显著下降。     

多重耐药大肠埃希菌16S rRNA甲基化酶、氨基糖苷类修饰酶基因研究

目的了解多重耐药大肠埃希菌(ECO)氨基糖苷类药物耐药机制。方法采用PCR检测20株多重耐药ECO菌11种16S rRNA甲基化酶和氨基糖苷类修饰酶基因。结果1株检出16S rRNA甲基化酶基因(5.0%),并为新亚型;16株检出氨基糖苷类修饰酶基因(80.0%)。15号株rmtB基因测得序列翻译成氨基酸序列与美国核酸库(GenBank)已登录的rmtB氨基酸不同,为新亚型。结论本组多重耐药ECO菌氨基糖苷类耐药机制与产16S rRNA甲基化酶和产氨基糖苷类修饰酶相关。多重耐药ECO菌存在新的氨基糖苷类药物耐药机制。     

化学修饰对组织型纤溶酶原激活剂的纤溶活性和体内半衰期的影响

用肝素、右旋糖酐和聚乙二醇对重组组织型纤溶酶原激活剂(rt-PA)进行化学修饰。不同的化学修饰物对rt-PA体外纤维蛋白溶解活性的影响不同。肝素修饰小PA的纤维蛋白溶解活性提高63％;右旋糖酐和聚乙二醇修饰rt-PA的纤维蛋白溶解活性则分别降低了36％和63％。rt-PA经化学修饰后半衰期均有所延长,其中聚乙二醇修饰的小PA的半衰期由修饰前的3．4min延长至6．3mim。     

Characterization and cytotoxicity of mixed PEG-DSG modified liposomes

It is known that polyethyleneglycol (PEG) modification of the liposome surface leads to the formation of a fixed aqueous layer around the liposomes due to interaction between the PEG polymer and water molecules, which prevents the attraction of opsonins. When a combination of PEG-distearolyglycerol (PEG-DSG) whose characteristics are remarkably different is used, interaction between molecules occurs, leading to increased fixed aqueous layer thickness (FALT). From this speculation, we studied the effect of both modification of PEG900-DSG and PEG2000-DSG modified liposome on FALT, cell uptake and biodistribution. The FALT of mixed PEG modified liposome increased, compared to that of each single PEG modified liposome. In this mixed modification, maximum FALT was shown at liposome modified by added PEG-2000:PEG-900=2:1. This most suitable additional ratio was equal to actual incorporated ratio. On the other hand, cell uptake of mixed modified liposome containing doxorubicin (DOX) was similar with that of PEG2000 modified liposome. Furthermore, mixed PEG modification of liposome was tendency to increase cytotoxicity, compared to that of other modifications. After DOX contained liposome treatment, DOX distribution in the tumor and antitumor activity of DOX increase by mixed PEG modification. In conclusion, it was suggested that mixed PEG liposome (PEG-2000:PEG-900=2:1) was useful for cancer chemotherapy.     

Effect of the covalent modification of horseradish peroxidase with poly(ethylene glycol) on the activity and stability upon encapsulation in polyester microspheres

Encapsulation of proteins in polyester microspheres by coacervation methods frequently causes protein inactivation and aggregation. Furthermore, an often-substantial amount of the encapsulated proteins is released within the first 24 h from the microspheres. To overcome these problems poly(ethylene glycol) (PEG) was employed as excipient and protein-modifying agent. The model protein horseradish peroxidase (HRP) was chemically modified or co-lyophilized with PEG of differing molecular weights, namely PEG(5000), PEG(20000), and PEG(40000). The lyophilized preparations were encapsulated in poly(D,L-lactide-co-glycolic) acid (PLGA) microspheres by a coacervation method. Covalent modification of HRP with PEG increased the encapsulation efficiency (EE) from 83% to about 100% while PEG when used as an excipient reduced the EE. Encapsulation caused aggregation of ca. 5% of non-modified HRP and the residual specific activity was only 57%. Covalent modification with PEG reduced HRP aggregation to less than 1% and improved its residual activity to more than 95%. When PEG was used as excipient similar results were found with respect to a reduction in encapsulation-induced aggregation, but no more than 80% of residual activity was obtained even for the best formulation after encapsulation. It was also found that covalent modification of HRP with PEG substantially reduced the unwanted initial "burst" release observed during the initial 24 h of in vitro release from about 70% to 23%. Furthermore, HRP activity and stability were also improved during in vitro release for HRP-PEG conjugates. The data show that covalent modification of proteins with PEG might be useful to improve protein stability during coacervation encapsulation and subsequent release as well as to increase EE and reduce the burst release.     

Effect of the covalent modification with poly(ethylene glycol) on alpha-chymotrypsin stability upon encapsulation in poly(lactic-co-glycolic) microspheres

The effectiveness of the covalent modification of alpha-chymotrypsin with methoxy poly(ethylene glycol) (PEG) to afford its stabilization during encapsulation in poly(lactic-co-glycolic) acid (PLGA) microspheres by a solid-in-oil-in-water method was investigated. alpha-Chymotrypsin was chemically modified with PEG (M(w) = 5000) using molar ratios of PEG-to-chymotrypsin ranging from 0.4 to 96. Various conjugates were obtained and the amount of PEG modification was determined by capillary electrophoresis. In this investigation, only those conjugates with PEG/chymotrypsin molar ratios between approximately 1 and 8 were considered because higher levels of modification caused protein instability even before encapsulation. The stability and functionality of the chymotrypsin formulations were investigated before encapsulation by measuring enzyme kinetics, thermal stability, and tertiary structure intactness, and after the initial lyophilization process by determining the secondary structure content. These stability parameters were related to select ones after encapsulation in PLGA microspheres (specifically, the amount of insoluble aggregates, residual enzyme activity, and magnitude of protein structural perturbations). The results show that the more stable the protein conformation before encapsulation was, the higher was the retention of the specific activity after encapsulation. In contrast, no relationship was found between the protein stability before encapsulation and the magnitude of encapsulation-induced protein aggregation. Even the lowest level of modification (PEG-to-chymotrypsin molar ratio of 0.7) drastically reduced the amount of insoluble aggregates from 18% for the nonmodified protein to 4%. The results demonstrate that PEG modification was able to largely prevent chymotrypsin aggregation and activity loss upon solid-in-oil-in-water encapsulation in PLGA microspheres. It is demonstrated that it is essential to optimize the degree of protein modification to ascertain protein stability upon encapsulation.     

基因壳聚糖纳米粒表面修饰和转染研究

目的：研究递送基因纳米粒表面修饰对体外基因转染的影响。方法：利用末端活化的聚乙二醇(PEG)制备PEG化基因壳聚糖纳米粒;通过两端活化的PEG将糖蛋白配基连接到纳米粒表面,完成肝靶向纳米粒的制备;用透射电镜观察表面修饰对纳米粒粒径大小、粒子形态的影响;使用蛋白质测定试剂盒测算纳米粒表面蛋白连接量;利用体外转染实验考察表面修饰对纳米粒转染活性的影响;用倒置荧光显微镜观察并用流式细胞仪测定转染结果。结果：纳米粒PEG化使转染效率大幅度升高,半乳糖基牛血清白蛋白(Galn—BSA)使体系的转染效率比PEG化纳米粒略有下降,但比不经修饰的纳米粒转染活性高。壳聚糖纳米粒的表面PEG化能提高纳米粒的体外稳定性,从而提高体外转染效率,并适合于进行冷冻干燥。结论：长循环壳聚糖基因递送纳米粒在基因治疗研究中可能会发挥重要作用。     

聚乙二醇对溶菌酶和粒细胞集落刺激因子的初步化学修饰

目的考察聚乙二醇 (PEG)修饰对溶菌酶和粒细胞集落刺激因子 (G CSF)活性的影响。方法以不同方法活化的PEG修饰溶菌酶 ,通过正交试验和单因素考察确定合适的修饰条件 ;溶菌酶活力测定采用溶壁小球菌法 ,抗原性测定采用试管沉淀法 ;G CSF活性测定以小鼠血浆中中性粒细胞数目增殖情况反映。结果当偶联上一个PEG分子时 ,溶菌酶的活性保留 13% ,抗原性显著减弱 ;G CSF经PEG修饰后对小鼠中性粒细胞数目的增加有显著促进作用 ,且作用时间明显延长。结论PEG修饰对G CSF血循环半衰期的延长有显著作用     

PEGylated recombinant human tumor necrosis factor alpha: pharmacokinetics and anti-tumor effects.

The aim of the present work was to investigate and assess the merit of PEGylated recombinant human tumor necrosis factor-alpha (rHuTNF-alpha) following our previous work. The rHuTNF-alpha was modified using activated polyethylene glycol (PEG), N-succinimidyl succinnate monomethoxy polyethylene glycol (SS-PEG). The pharmacokinetics and anti-tumor effect were investigated. The experimental results showed that PEGylated rHuTNF-alpha could obviously alter in vivo behavioral characteristics of rHuTNF-alpha. Among the synthesized PEG-rHuTNF-alphas with different PEG molecules, PEG20000-rHuTNF-alpha demonstrated the longest circulating half-life (24.8 h) which was about 50 times longer than that of rHuTNF-alpha (28.8 min). In addition, there was much more PEG20000-rHuTNF-alpha distributed into tumor tissues than other PEG-rHuTNF-alphas or rHuTNF-alpha with time, and PEG20000-rHuTNF-alpha also showed the highest anti-tumor potency. These results indicated that PEG20000-rHuTNF-alpha was a useful long circulating molecule with selective localization in tumor tissues and enhanced anti-tumor activity of rHuTNF-alpha.     

番麻蛋白酶活性中心基团的初步研究

在不引起酶变性的条件下,用半胱氨酸(Cys)专一性化学修饰试剂碘乙酸、对氯汞苯甲酸(?)(CMB),5,5′-二硫双硝基苯甲酸(DTNB)对酶分子进行化学修饰后,酶活性显著下降。发现酶的失活程度与修饰剂浓度间存在着化学计量关系,同时底物(酪蛋白)对酶活性有明显的保护作用。说明被修饰的部位(Cya残基)是酶的活性中心基团。Cys对酶有明显的激活作用;同时,(?)CMB、DTNB、碘乙酸对酶活性有明显的抑制作用(?)CMB抑制或O_2氧化而失活的酶溶液,又可被Cys重新激活,而恢复其活力,进一步证明其活性中心存在着Cys残基。以上事实均与典型的植物巯基蛋白酶——木瓜蛋白酶一致,说明番麻蛋白酶是一种巯基蛋白醇,其活性中心存在着Cys。     

聚乙二醇化重组人肿瘤坏死因子-α:制备和抗肿瘤活性

AIM: To assess the merits of polyethylene glycol-modified recombinant human tumor necrosis factor alpha (PEG-rHuTNF-alpha). METHODS: The rHuTNF-alpha was modified with N-succinimidyl succinate monomethoxy polyethylene glycol (SS-PEG) of three different molecular weights. The PEG-rHuTNF-alpha was separated into fractions of various molecular weights by gel filtration chromatography. In vitro activities of various fractions were determined with L929 cell assay and in vivo anti-tumor potencies of main fractions were studied with respect to necrosis of S-180 solid tumor. RESULTS: The rHuTNF-alpha could be modified using SS-PEG under mild conditions. The main fraction of PEG5000-rHuTNF-alpha contained four PEG molecules, and PEG12000-rHuTNF-alpha and PEG20000-rHuTNF-alpha contained two PEG molecules, respectively. There was a higher activity when rHuTNF-alpha was coupled to less numbers of the same molecular weight PEG molecules. When PEG-rHuTNF-alpha was of the same molecular weight, rHuTNF-alpha modified with bigger molecular weight PEG molecules had a higher activity. PEG-rHuTNF-alpha was resistant to proteolysis, and over 70 % activity remained after 8 h, but the activity of rHuTNF-alpha was time-dependently diminished by incubation with bovine trypsin. PEG5000-rHuTNF-alpha (1500 IU per mouse) had a similar anti-tumor potency compared with rHuTNF-alpha (3000 IU per mouse). PEG12000-rHuT NF-alpha (1500 IU per mouse) had an increased anti-tumor potency compared with rHuTNF-alpha (3000 IU per mouse). In particular, PEG20000-rHuTNF-alpha at a dose of 1500 IU per mouse had a higher anti-tumor potency than rHuTNF-alpha at a dose of 6000 IU per mouse. CONCLUSION: PEG-modified rHuTNF-alpha could be more suitable for therapeutic use