α-干扰素治疗慢性病毒性肝炎的现状和展望

长期以来α－干扰素治疗慢性乙型和丙型病毒型肝炎是有效的,临床治疗中已得到承认。但大剂量发现副作用明显,口含天然人α－干扰素无副作用,如果能替代α－干扰素并联合其它药物治疗,对病毒性疾病和恶性肿瘤将提供新的治疗方法．     

奶牛α干扰素在毕赤酵母中的分泌表达及其生物活性测定

提取经新城疫病毒诱导培养的奶牛外周血淋巴细胞总RNA,应用RT-PCR方法扩增出奶牛α干扰素成熟蛋白基因,然后将特异性片段连接到pMD18-T载体,测序结果表明,扩增片段为牛α干扰素成熟蛋白序列,与Genebank上发表的α1干扰素序列同源性为98%。然后将特异性片段连接到含分泌信号肽序列的Pichiapastoris表达载体pPICZαA上,将重组质粒经SacI酶切线性化后电转化导入毕赤酵母菌株X-33。转化子经PCR分析鉴定后利用甘油增菌和甲醇诱导,实现了BoIFN-α在毕赤酵母系统中的分泌表达。SDS-PAGE结果显示表达产物分子量约为23kDa,比其推导结果(20kDa)略大,推测可能是因为表达产物发生了一定程度的糖基化。细胞病变抑制法结果表明,重组牛IFN-α具有较高的干扰素活性,约为4.1×10⁵U/ml,经微量蛋白测量仪测得,其表达量约为80μg/ml,比活为5.1×10⁶U/mg。     

HPLC法测定重组人干扰素α1b粉雾剂的含量

采用高效液相色谱法测定重组人干扰素α1b粉雾剂的含量及均匀度。结果表明溶液中重组人干扰素α1b浓度在92.50~462.50μg/ml时峰面积（Y）与相应浓度（X）呈线性关系,（r=0.999 9）。并且在成品的含量测定时,重组人干扰素α1b的色谱峰峰形良好,杂质与流动相无干扰。本方法精密度、准确度高,稳定性良好,能够准确、迅速的测定重组人干扰素α1b粉雾剂成品中的主药含量及均匀度。     

花生黄曲霉抗性与SR和α-微管蛋白相关性

信号识别颗粒受体在分泌性蛋白合成和分泌过程中起重要作用。微管蛋白对细胞内各种生命活动都是必需的。将数据库中花生抗黄曲霉和敏感品种的两类α-微管蛋白α和α7和SR蛋白进行序列比较分析发现,在种子发育早期5,α-微管蛋白在敏感品种各有三条EST,抗性品种各有一条EST,晚期7只有抗性品种各有一条EST。SR只在抗性品种的6、7时期各有一条EST,敏感品种中没有。用荧光定量PCR方法对抗性品种KB153与敏感品种JH1012发育中不同时期的果实和部位进行差异表达分析,结果表明在果实发育早期的小果期,SR、α-微管蛋白α和α7在抗性品种中都显著上调表达,说明SR介导的内质网蛋白质运输途径与黄曲霉抗性有关。SR基因在抗性品种的子叶中的表达也上调,这与抗性品种中一些贮藏蛋白含量尤其是蛋白酶抑制剂高于敏感品种的现象一致。     

人α干扰素基因在家蚕细胞中的高表达

用家蚕核多角体病毒(NPV)为载体,使人α干扰素基因正确装配到NPV多角体蛋白基因启动子控制下,构成重组质粒,又与NPVDNA共转染家蚕细胞。反复病毒空斑纯化后,得到重组病毒。     

α-突触核蛋白N-端结构域参与线粒体功能的调控

目的：确定α-Synuclein蛋白与线粒体相互作用的功能结构域,并检测该结构域对线粒体功能的影响。方法：构建重组融合蛋白质粒pCMV-myc/α-Syn-WT、pCMV-myc/α-Syn-N和pCMV-myc/α-Syn-△N,转染人胚肾HEK293T细胞,通过免疫共沉淀明确α-Synuclein蛋白与线粒体相互作用的功能结构域。使用表达α-Synuclein蛋白的病毒上清感染小鼠多巴胺能神经细胞MN9D,通过免疫荧光、流式细胞术检测线粒体膜电位及Cytochrome c释放。结果：成功构建了pCMV-myc/α-Syn-WT、pCMV-myc/α-Syn-N和pCMV-myc/α-Syn-△N融合蛋白质粒,免疫共沉淀明确α-Synuclein N-端为其功能结构域,JC-1染色发现N-端使线粒体膜电位降低,流式细胞术证实N-端使Cytochrome c释放明显增加。结论：α-Synuclein N-端是其与线粒体相互作用的功能结构域,N-端降低线粒体功能。     

重组野猪α-干扰素基因的高效表达和下游工艺的研发

利用PCR方法,从东北野猪和长白猪的肌肉基因组DNA中分别扩增出一条501bp的基因片断 ,与GenBank上登载的猪α-干扰素基因序列相比,核苷酸同源性达到98.4%以上。以其中野猪α-干扰素基因片断为参考模板,经过密码子优化、5?和3?区域的mRNA二级结构优化,全基因合成得到编码野猪α-干扰素基因成熟肽的基因。克隆该基因到原核表达载体pWL之中.随后转化至宿主菌DH-5α。经过SDS-PAGE电泳检测,发酵后的重组野猪IFN-α-干扰素基因的蛋白表达量大于25%。复性纯化后的重组蛋白,经过VSV-WISH系统检测,其生物活性为4.45×106IU/mg。     

人趋化因子MIP-3α的原核可溶性表达及趋化活性分析

目的:克隆人趋化因子MIP3α,进行原核表达并初步鉴定其趋化活性。方法:从人扁桃体中提取总RNA,进行RTPCR,扩增MIP3α成熟蛋白基因,重组于pET32a(+)载体,转化大肠杆菌BL21TrxB(DE3),进行融合表达,Westernblot验证融合蛋白,金属离子亲和层析,肠激酶酶切,弱阳离子交换层析,得到纯化的MIP3α蛋白,趋化试验鉴定其趋化活性。结果:成功构建了MIP3α天然蛋白的硫氧还蛋白融合表达载体,表达并纯化出MIP3α蛋白,Westernblot证明融合蛋白能与羊抗人MIP3α抗体结合,纯化的MIP3α蛋白能趋化HEK293CCR6稳定转染细胞。结论:构建的天然MIP3α融合表达载体以可溶性蛋白的方式稳定表达MIP3α,初步纯化得到的MIP3α具有趋化HEK293CCR6稳定转染细胞的活性。     

原子力显微镜检测过表达α-突触核蛋白引起的线粒体结构变化

目的：利用原子力显微镜等方法,明确α-突触核蛋白（α-synuclein, α-syn）过表达对线粒体的影响。方法：将腺相关病毒（AAV）载体介导表达的α-syn (AAV /α-syn)病毒包装颗粒,给大鼠脑内黑质区定位注射,建立α-syn过表达的大鼠模型,并以AAV介导表达的报告基因LacZ (AAV/LacZ)为动物对照组。提取大鼠黑质脑组织的线粒体,并通过JC1染色检测线粒体膜电势（ΔΨ）变化和Western Blot检测线粒体中α-syn蛋白量的变化;采用原子力显微镜观测线粒体表面结构的变化。结果：实验组大鼠黑质脑组织过表达α-syn基因,第16周时,Western blot显示线粒体中α-syn蛋白量增加约2倍;JC1染色发现ΔΨ下降;原子力显微镜观测可见线粒体体积增大,线粒体膜表面出现较多孔道样改变。结论：大鼠黑质脑组织过表达α-syn基因,第16周时,可导致α-syn蛋白在线粒体积聚;并可引起线粒体增大、膜形成孔道样改变、ΔΨ下降。     

帕金森病相关蛋白PINK1和α-突触核蛋白相互作用研究

目的 利用蛋白质组学方法和激光扫描共聚焦显微镜来鉴定帕金森病相关蛋白PINK1和α-突触核蛋白（α-synuclein）之间的相互作用关系及两种蛋白在MN9D细胞中的分布。方法 将α-synuclein基因插入pGEX-4T-1载体,经DNA测序证明形成GST融合蛋白,经大肠杆菌BL-2l表达纯化后,与MN9D细胞裂解液共孵育,检测PINK1与α-synuclein蛋白间的相互作用。并应用MN9D细胞的内源性PINK1与α-synuclein进行免疫共沉淀实验,进一步验证两蛋白之间的相互作用。MN9D细胞经免疫细胞化学染色,利用激光扫描共聚焦显微镜观察两种蛋白的细胞内分布及共定位状态。结果 利用GST-α-synuclein融合蛋白捕获及免疫共沉淀技术,检测到特异的PINK1条带。激光扫描共聚焦显微镜检测到两者存在部分共定位。结论 PINK1和α-synuclein在体外和细胞内均可发生相互作用并在MN9D细胞中存在共定位关系。     

长效重组蛋白药物的研究进展

重组蛋白药物经静脉和皮下注射后通常半衰期较短,目前延长蛋白药物半衰期的方法主要基于三种原理：1、增大蛋白药物分子量;2、利用血浆药物平衡;3、减少免疫原性。本文针对构建突变体、PEG化修饰和与血清白蛋白融合三种延长重组蛋白药物半衰期的方法,及其已上市的和正在研发中的长效重组蛋白药物的特征、半衰期和免疫原性问题进行了综述。     

Nebulization of Liposomal rh-Cu/Zn–SOD with a Novel Vibrating Membrane Nebulizer

Liposomes are potential drug carriers for pulmonary drug delivery: They can be prepared from phospholipids, which are endogenous to the respiratory tract as a component of pulmonary surfactant, and at an appropriate dose liposomes do not pose a toxicological risk to this organ. Among the various categories of drug that benefit from liposomal entrapment is the anti-inflammatory enzyme superoxide dismutase, thus prolonging its biological half-life. The delivery of liposomes by nebulization is hampered by stability problems, like physical and chemical changes that may lead to chemical degradation and leakage of the encapsulated drug. Here we present data of liposomes aerosolized with a novel electronic nebulizer based on a vibrating membrane technology (PARI eFlow?), which amends drawbacks like liposomes degradation and product release. The data acquisition included aerosol properties such as aerodynamic particle size, nebulization efficiency, and liposome leakage upon nebulization. In conclusion, this study shows the ability of the PARI eFlow? to nebulize high amounts of liposomal recombinant human superoxide dismutase with reduced vesicle disruption tested in an enclosing experimental protocol.     

Nebulization of liposomal rh-Cu/Zn-SOD with a novel vibrating membrane nebulizer

Liposomes are potential drug carriers for pulmonary drug delivery: They can be prepared from phospholipids, which are endogenous to the respiratory tract as a component of pulmonary surfactant, and at an appropriate dose liposomes do not pose a toxicological risk to this organ. Among the various categories of drug that benefit from liposomal entrapment is the anti-inflammatory enzyme superoxide dismutase, thus prolonging its biological half-life. The delivery of liposomes by nebulization is hampered by stability problems, like physical and chemical changes that may lead to chemical degradation and leakage of the encapsulated drug. Here we present data of liposomes aerosolized with a novel electronic nebulizer based on a vibrating membrane technology (PARI eFlow), which amends drawbacks like liposomes degradation and product release. The data acquisition included aerosol properties such as aerodynamic particle size, nebulization efficiency, and liposome leakage upon nebulization. In conclusion, this study shows the ability of the PARI eFlow to nebulize high amounts of liposomal recombinant human superoxide dismutase with reduced vesicle disruption tested in an enclosing experimental protocol.     

泛素特异性蛋白酶18抑制IFNα抗HBV活性但并不抑制IFNλ抗HBV的活性

干扰素α（IFN-α）是临床最常用的抗乙型肝炎病毒（HBV）药物之一。泛素特异性蛋白酶18（USP18）被证实是抑制IFN-α抗HBV活性的因子,但USP18是否对干扰素λ(IFN-λ)抗HBV有影响还尚未可知。为了明确USP18对IFNλ抗HBV活性的影响,本研究以HepG2.2.15细胞作为乙肝体外模型,采用脂质体转染法分别向细胞转染pEGFP-USP18、PEGFP-N1经 48 h,再经IFN-α和IFN-λ处理24 h,分为阴性对照组﹑USP18过表达+IFN-α组﹑空载组+IFN-α组﹑USP18过表达+IFN-λ组﹑空载组+IFN-λ组。采用Western印迹、RT-qPCR和ELISA检测各组的乙肝病毒标志物、STAT1/pSTAT1和下游的干扰素刺激基因(ISGs)的表达。结果显示,与阴性对照组和空载组相比,USP18蛋白在过表达组明显升高（P<0.05）,过表达细胞模型构建成功;在IFN-α处理的两组中,空载组中HBsAg、HBeAg、HBcAg及HBV-DNA的表达均低于USP18过表达组,差异有统计学意义（P<0.05）。而IFN-λ处理组中,乙肝病毒标志物的差异不明显。在IFN-α处理组中,空载组的ISG15、MxA、IFIT1和pSTAT1表达均高于USP18过表达组,差异有统计学意义（P<0.05）,而在IFN-λ处理组中ISGs和 pSTAT1的表达无明显差异。上述结果证实,USP18可通过抑制JAK/STAT信号通路的激活来减弱IFN-α抗HBV的活性。研究还证实,IFN-λ可发挥抗HBV的作用,USP18不通过JAK/STAT信号通路抑制其抗HBV活性。     

Heparin potentiates the action of acidic fibroblast growth factor by prolonging its biological half-life

The mechanism(s) by which heparin influences the biological activities of acidic and basic fibroblast growth factors (aFGF and bFGF) is not completely understood. One mechanism by which heparin could alter the biological activities of aFGF and bFGF is by altering their biological half-lives. We investigated the possibility that heparin potentiates aFGF-induced neurite outgrowth from PC12 cells by prolonging its biological half-life. Under conditions where heparin potentiated aFGF-induced neurite outgrowth, we observed that heparin increased the biological half-life of aFGF from 7 to 39 hr. We determined that greater than 25 hr of exposure to active aFGF was required for induction of neurite outgrowth. If aFGF activity was maintained for greater than 25 hr by periodic readdition of factor, heparin no longer potentiated aFGF-induced neurite outgrowth. These observations strongly suggest that heparin potentiates the activity of aFGF by prolonging its biological half-life. The protease inhibitors hirudin, leupeptin, and pepstatin A did not potentiate aFGF-induced neurite outgrowth, indicating that proteases inhibited by these inhibitors are not responsible for the loss of aFGF activity that we observed. However, aprotinin potentiated aFGF neurite-promoting activity approximately sevenfold, indicating that proteases that are inhibited by aprotinin are at least partially responsible for aFGF inactivation. These observations suggest that heparin regulates the activity of aFGF by regulating its proteolytic degradation, thereby regulating its biological half-life.     

Non-phagocytic uptake of intravenously injected microspheres in rat spleen: influence of particle size and hydrophilic coating

A recent development in prolonging the circulation time of drug carriers, such as liposomes and microspheres, has been to minimize their removal by macrophages of the reticuloendothelial system by covering their surface with hydrophilic polymers such as poloxamers, poloxamines and poly(ethyleneglycols). Here we demonstrate that this strategy may not necessarily prolong the circulatory half-life of drug carriers in all animal models. In rats, as opposed to rabbits, a non-phagocytic mechanism in the spleen may be triggered to remove efficiently from the blood drug carriers coated with hydrophilic coatings. Both the size of particle and its hydrophilic coating may act synergistically to trigger this non-phagocytic mechanism. In rats, a remarkable log to log relationship between particle size and spleen uptake was observed for both uncoated and polymeric coated microspheres. The potential implication of these observations in site-specific delivery of drug carriers is discussed.     

多肽类药物制剂研究现状

多肽类药物制剂研究所面临的主要问题是多肽的不稳定性、体内半衰期短和生物膜透过性差。本文综述了(1)引起多肽不稳定性的原因;(2)提高多肽稳定性的方法;(3)多肽类药物制剂货架时间的确定;(4)多肽类药物的分析手段;(5)多肽类药物的控释研究;(6)多肽的非注射途径给药研究。最后提出了多肽类药物制剂研究的展望。     

The evolution of drug-resistant malaria: the role of drug elimination half-life

This paper seeks to define and quantify the influence of drug elimination half-life on the evolution of antimalarial drug resistance. There are assumed to be three general classes of susceptibility of the malaria parasite Plasmodium falciparum to a drug: Res0, the original, susceptible wildtype; Res1, a group of intermediate levels of susceptibility that are more tolerant of the drug but still cleared by treatment; and Res2, which is completely resistant to the drug. Res1 and Res2 resistance both evolve much faster if the antimalarial drug has a long half-life. We show that previous models have significantly underestimated the rate of evolution of Res2 resistance by omitting the effects of drug half-life. The methodology has been extended to investigate (i) the effects of using drugs in combination, particularly when the components have differing half-lives, and (ii) the specific example of the development of resistance to the antimalarial pyrimethamine-sulphadoxine. An important detail of the model is the development of drug resistance in two separate phases. In phase A, Res1 is spreading and replacing the original sensitive forms while Res2 remains at a low level. Phase B starts once parasites are selected that can escape drug action (Res1 genotypes with borderline chemosensitivity, and Res2): these parasites are rapidly selected, a process that leads to widespread clinical failure. Drug treatment is clinically successful during phase A, and health workers may be unaware of the substantial changes in parasite population genetic structure that predicate the onset of phase B. Surveillance programs are essential, following the introduction of a new drug, to monitor effectively changes in treatment efficacy and thus provide advance warning of drug failure. The model is also applicable to the evolution of antibiotic resistance in bacteria: in particular, the need for these models to incorporate drug pharmacokinetics to avoid potentially large errors in their predictions.