乳糖化-去甲斑蝥素磷脂复合物及其pH敏感型脂质体的制备

目的:合成乳糖化-去甲斑蝥素磷脂复合物,并制备其pH敏感型脂质体。方法:将乳糖化-去甲斑蝥素与磷脂聚合成药物磷脂复合物,并采用FT-IR、DSC和1H-NMR对其进行表征。逆向蒸发法制备药物磷脂复合物脂质体;利用羧甲基壳聚糖与脂质体表面的静电吸附作用,使羧甲基壳聚糖吸附在脂质体表面,制备乳糖化-去甲斑蝥素磷脂复合物pH敏感型脂质体;考察了药物与磷脂的复合率,磷脂复合物脂质体的包封率,粒径大小和分布,以及体外释药特性。结果:药物磷脂复合率为(97.2±2.01)%,磷脂复合物脂质体的平均包封率为(70.00±1.30)%,平均粒径为(47.18±4.16)nm,粒径跨距为(0.70±0.07),电镜显示其形态圆整,体外释药符合Weibull方程。结论:乳糖化-去甲斑蝥素磷脂复合率高,制成的pH敏感型脂质体性质稳定,且具有缓释特性。     

肝细胞刺激物质及其自身抗体与特异性免疫复合物的测定与研究

从人胎肝中提取出肝细胞刺激物（ＨＳＳ）用ＥＬＩＳＡ方法分别对肝病患者及自身免疫病，心血管病及慢性肾病血清中细胞刺激物质的含量，抗人肝细胞激物（ＨＳＳ－Ａｂ）及其人肝细胞的刺激物－抗人肝细胞刺激物特异免疫复合物（ＨＳＳ－ＩＣ）进行测定。结果表明丙型肝炎患者血清中ＨＳＳ含量，ＨＳＳ－Ａｂ和ＨＳＳ－ＩＣ阳性率均高于其它对照组。     

猕猴桃籽油对D-半乳糖衰老大鼠肝细胞凋亡的影响

目的研究猕猴桃籽油对D-半乳糖拟衰老大鼠肝细胞凋亡的影响。方法30只SD大鼠随机分为3组。正常对照组：每鼠胃灌服生理盐水和背部皮下注射150ms／（kg·d）生理盐水；模型组：每鼠胃灌服生理盐水和背部皮下注射150ms／（kg·d）25％D-半乳糖溶液；猕猴桃籽油干预组：每鼠胃灌服猕猴桃0．670/（kg·d）和背部皮下注射150ms／（kg·d）25％D-半乳糖溶液；每组时间均为8周。8周后制备大鼠肝组织石蜡切片，且行末端脱氧核苷酸转移酶介导的dUTP缺13末端标记（TUNEL）法，检测肝细胞凋亡指数（AI），并对染色结果行计算机图像分析。结果模型组大鼠肝细胞凋亡指数比正常组显著增加（P〈0．01），猕猴桃籽油干预组肝细胞凋亡指数与模型组相比明显减少（P〈0．01）。结论猕猴桃籽油可减少D-半乳糖衰拟老大鼠肝细胞凋亡的发生。     

ε-聚赖氨酸对白念珠菌抑菌活性及机制研究

目的 研究ε-多聚赖氨酸(ε-poly-L-lysine, ε-PL)对白念珠菌(Candida albicans)的抑菌活性及抑菌机制。方法 以白念珠菌的标准菌株ATCC64548(氟康唑敏感株)、ATCC64550(氟康唑耐药株)以及临床收集的50株菌为实验菌株,按照CLISI- M27文件中的微量稀释法测定ε-多聚赖氨酸的MIC、MFC和SMIC50值;绘制48h内的浮游菌株时间-生长曲线和生物膜抑制-时间曲线;连续观测并记录4h内的芽管形成率和芽管长度;测定药物处理前后白念珠菌的丙二醛和活性氧(ROS)含量。结果 ε-PL对白念珠的最低抑菌浓度(MIC)为512μg/mL,最低杀菌浓度(MFC)为1024μg/mL,SMIC50为512μg/mL,ε-PL对白念珠菌浮游菌及生物膜的抑菌作用随着浓度的升高作用愈明显。抑菌-时间曲线结果表明ε-PL对白念珠菌的浮游菌和生物膜在12h左右即产生明显抑制作用。芽管实验的结果表明高浓度的ε-PL对白念珠菌的芽管形成率及芽管长度具有明显抑制作用。ε-PL作用于白念珠菌后MDA和ROS含量呈现上升趋势,并且与药物浓度大小成正相关。结论 ε-PL对白念珠菌浮游菌株及生物膜均有良好的抑制作用,高浓度ε-PL对白念珠菌主要毒力菌丝有明显抑制作用,ε-PL作用导致白念珠菌内产生大量的活性氧(ROS)以及产生一定程度的脂质氧化,提示ε-PL可能通过氧化作用发挥抑菌效果。     

ε-聚赖氨酸对耐甲氧西林金黄色葡萄球菌USA300的作用机制

摘要：目的 研究ε-聚赖氨酸(ε-polylysine,ε-PL)对耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)标准菌株USA300的抑菌作用及其机制。方法 依据CLSI微量肉汤稀释法测定最低抑菌浓度(minimum inhibitory concentration, MIC)和最低杀菌浓度(minimum bactericidal concentration, MBC);绘制24 h内不同浓度ε-PL作用后USA300菌株时 间-抑菌曲线;SYBR Green I/PI检测ε-PL处理后USA300的生存情况;测定ε-PL处理后菌液电导率、胞外ATP含量、可溶性蛋白 含量的变化;利用扫描电镜(scanning electron microscopy,SEM)观察ε-PL对USA300形态的影响。结果 ε-PL对USA300的MIC、 MBC分别为5.12和10.24 mg/mL。ε-PL处理后细菌死/活比例,菌液电导率,胞外ATP含量,胞外可溶性蛋白含量均增加,表明菌 膜破损;经SEM进一步确证。结论 ε-PL对USA300生长有良好的抑制作用,且与ε-PL浓度呈正相关。ε-PL处理后细胞膜结构破 坏、通透性改变,导致细胞内容物大量渗出,其抑菌机制可能与破坏细菌菌体结构有关。     

ε-聚赖氨酸微生物生产及其应用研究进展

ε-聚赖氨酸是一种同聚酰胺生物聚合物,由25～35个L-赖氨酸残基通过α-羧基和ε-氨基缩合形成的酰胺键连接而成。因其独特的结构,ε-聚赖氨酸具有许多优异的性能,如可食性、水溶性、热稳定性、可降解性、广谱抗菌活性和无毒性等。因此,ε-聚赖氨酸已在日本、韩国、美国和中国等国家被广泛用作食品防腐剂。目前,ε-聚赖氨酸主要由白色链霉菌发酵生产。自ε-聚赖氨酸于1977年由日本学者发现以来,ε-聚赖氨酸发酵生产得到了很大提高,对其应用研究也由最初的食品领域拓展到了许多其他的领域。本文首先概述了ε-聚赖氨酸生物合成机理、菌种选育和发酵生产策略,在简要介绍其抑菌机制基础上,着重介绍了ε-聚赖氨酸在食品、医学和材料等领域应用研究进展,最后,对相关研究进行了展望,以期为ε-聚赖氨酸微生物生产及其应用研究提供有益的借鉴。     

ε-聚赖氨酸对金黄色葡萄球菌生长及生物膜形成的影响#br#

目的 研究ε-聚赖氨酸(ε-poly-L-lysine, ε-PL)对金黄色葡萄球菌(Staphylococcus aureus, S. aureus)生长及生物膜形成的影响。方法 以金黄色葡萄球菌标准菌株ATCC25923及社区获得性耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus, MRSA)USA300为试验菌株,常量肉汤稀释法(试管)测定最低抑菌浓度(minimum inhibitory concentration, MIC),酶标仪检测吸光度法观察各株金黄色葡萄球菌的生长曲线,96孔板结晶紫染色法检测各株菌生物膜的形成并用扫描电子显微镜(SEM)观察生物膜的形成情况。结果 ε-PL对ATCC25923和USA300的MIC均为31.25mg/mL。ε-PL浓度升高时对两株菌生长的抑制作用随之增强。1/2MIC ε-PL能显著降低ATCC25923和USA300生物膜的形成能力。 结论 ε-PL作为一种安全、高效的天然防腐剂,能有效抑制金黄色葡萄球菌细菌的生长及生物膜的形成,为新型药物的研发提供了理论依据。     

天然活性成分磷脂复合物的研究新思路-α-甘草酸二铵脂质复合物

以甘草酸二铵磷脂复合物为重点,论述近年来国内外天然活性成分磷脂复合物药学方面的研究进展情况。总结了天然活性 成分磷脂复合物的制备方法、理化性质、结构特点,α-甘草酸二铵磷脂酰胆碱脂质复合物药理学方面的作用特点、生物利用度, 以其对新的磷脂复合物研究提供参考。     

不同β-受体阻滞剂对心力衰竭患者β-受体与β1-受体基因表达干预性研究

目的 研究心力衰竭患者外周血淋巴细胞β-受体（β-AR）密度和β1-受体基因（β1-AR mRNA）表达水平的变化规律，探讨不同β-受体阻滞剂对β-AR和β1-AR mRNA表达水平的影响。方法 将104例心衰患者随机分为非β-受体阻滞剂治疗组（35例）、美托洛尔治疗组（34例）和卡维地洛治疗组（35例），2个月后复测β-AR和β1-AR mRNA表达水平。结果 心衰组β-AR密度和β1-AR mRNA表达水平较正常人明显下降（P〈0．01），心功能Ⅳ级患者下降更明显（P〈0．01）。冠心病与扩张型心肌病两组之间差异无显著意义（P〉0．05）。治疗后，美托洛尔治疗组β-AR密度和β1-AR mRNA表达水平明显高于卡维地洛治疗组和非β-受体阻滞剂治疗组（P〈0．01），卡维地洛治疗组与非β-受体阻滞剂治疗组差异无显著意义。结论 心衰时外周血淋巴细胞β-AR密度和β1-AR mRNA表达水平下调、下调幅度与心衰严重程度有关，与病因无关。应用美托洛尔能明显上调β-AR密度和β1-AR mRNA表达水平，卡维地洛则无此作用。     

Internalization of Fractionated 3H-Heparin by the Scavenger-Like Receptor in Rat Liver Parenchymal Cells in Primary Culture

The present study was aimed at clarifying the uptake mechanisms of fractionated ³H-heparin (FH) in rat liver parenchymal cells in an effort to explore further the clinical applications of mucopolysaccharides, including their utilization in drug delivery. The internalization and surface binding of FH were determined by removing surface-bound FH by the NaCl wash method in uptake experiments in rat liver parenchymal cells in primary culture. Initial and transient peaks were observed in the time course of the surface binding of FH, suggesting the involvement of receptor-mediated endocy tosis (RME) and downregulation of the receptors. Consistent with this suggestion, internalization of FH was reduced by lowering the temperature from 37 to 4°C, while total association was unchanged. Although the internalization of FH was slow and concentration independent, both total association and internalization were inhibited by ligands of the scavenger receptor and some anions, but not by inhibitors of the RME of polypeptides. All these results collectively suggest the involvement of the scavenger receptor or similar substance in terms of substrate specificity in the uptake of FH in rat liver parenchymal cells. This is the first suggestion of the existence of the scavenger-like receptor in liver parenchymal cells. Macromolecular compounds such as heparin have recently been increasingly investigated for their clinical applications, including utilization as carriers for drug delivery (Nishikawa et al. 1995). Although there have been a number of studies concerning the disposition of polypeptides in the body and the utilization of macromolecules as drug carriers, more extensive studies are required to exploit a variety of macromolecules for clinical applications. Heparin, a macromolecular drug with molecular weights ranging from 3000 to 30,000 d., has been used as an anticoagulant drug. The disposition of heparin after intravenous administration depends on the molecular weight     

Matrix Metalloproteinase Gene Delivery for Liver Fibrosis

The resolution of advanced liver fibrosis has been recently recognized to be possible, if the causative stimuli are successfully removed. However, whether complete resolution from cirrhosis, the end stage of liver fibrosis, can be achieved is still questionable. Delivery of interstitial collagenases, such as matrix metalloproteinase (MMP)-1, in the liver could be an attractive strategy to treat advanced hepatic fibrosis from the view point that the imbalance between too few interstitial collagenases and too many of their inhibitors is the main obstacle to the resolution from fibrosis. Remodeling of hepatic extracellular matrix by delivered interstitial collagenases also facilitates the disappearance of activated hepatic stellate cells, the main matrix-producing cells in the liver, and promotes the proliferation of hepatocytes. This review will focus on the impact of the gene delivery of MMPs for the treatment of advanced liver fibrosis while discussing other current therapeutic strategies for liver fibrosis, and on the need for the development of a safe and effective delivery system of MMPs.     

Regional Delivery of Model Compounds and 5-Fluorouracil to the Liver by Their Application to the Liver Surface in Rats: Its Implication for Clinical Use

Purpose The purpose of this study was to examine drug distribution in the liver after drug application to the rat liver surface.Methods Phenolsulfonphthalein (PSP) and fluorescein isothiocyanate dextran (MW 4400, FD-4) as model compounds or 5-fluorouracil (5-FU) was applied to the rat liver surface by employing a cylindrical diffusion cell (i.d. 9 mm, 0.64 cm²). Then, blood and the remaining solution in the diffusion cell were collected at selected times, followed by excision of the liver. The excised liver was divided into three sites: the region under the diffusion cell attachment site (site 1), the applied lobe except for site 1 (site 2), and non-applied lobes (site 3).Results In the case of i.v. administration, there were no differences in PSP concentrations among the three sites of the rat liver, and the concentrations rapidly decreased. On the other hand, the PSP concentration in site 1 after application to the rat liver surface was considerably higher than in site 2 and site 3. In addition, the area under the curve (AUC) value (AUC_site1), calculated from the PSP concentration profile in site 1, was about 10 times larger than that in site 3. A similar trend of regional delivery advantage by liver surface application was observed in the case of the macromolecule model FD-4, with a marked AUC_site1 of about 5 times larger than the other two sites. Moreover, we clarified that the anticancer drug 5-FU preferentially distributed in site 1 after application to the rat liver surface.Conclusion These results demonstrate the possibility of regional delivery of drugs to the liver by application to the liver surface.     

Contribution of Parenchymal and Non-Parenchymal Liver Cells to the Clearance of Hepatocyte Growth Factor From the Circulation in Rats

Purpose. The distribution of ¹²⁵I-hepatocyte growth factor (HGF) to either liver parenchymal cells (PC) or non-parenchymal cells (NPC) was investigated in rats. Methods. After injection of a trace amount of ¹²⁵I-HGF, the distribution of radioactivity determined by microautoradiography closely resembled that of ¹²⁵I-epidermal growth factor which distributes mainly to PC. Results. The uptake clearance of ¹²⁵I-HGF estimated by determining the radioactivity of isolated liver cells was three times higher for PC than for NPC. This suggests that HGF distributes mainly to PC at relatively low doses. On the other hand, the uptake clearance by PC fell on coadministering an excess (80 µg/kg) of unlabeled HGF, while no change was observed for NPC, indicating that a saturable process for the hepatic handling of HGF exists only in PC where the HGF receptor is expressed. Conclusions. At such a dose the uptake clearance was comparable for both PC and NPC showing that HGF distributes to both cell types although NPC have few HGF receptors. Since the distribution to NPC was relatively non-specific and heparin-sensitive, it may be that heparin-like substances, which are believed to exist on PC and/ or the extracellular matrix, also exist on NPC.     

Reduced Fluoride Sensitivity of Liver Cells from Rats Chronically Exposed to Fluoride

Abstract: The fluoride sensitivity, determined as effect on protein synthesis (incorporation of ¹⁴C-leucine), of liver and kidney cells in suspension culture was explored. The cells were freshly prepared by collagenase perfusion from rats given drinking water with or without addition of 100 p.p.m. (5.26 mM) fluoride for 9–28 weeks. The fluoride sensitivity of the liver cells from rats given fluoridated water for more than 9 weeks was decreased compared to cells from control rats, whereas the fluoride sensitivity of the kidney cells from fluoride exposed and control rats appeared similar. Fluoride resistance (i.e. decreased sensitivity) may thus develop also in cells in vivo. When exposed to 3 mM NaF for 1 hour the intracellular concentration of fluoride in liver cells from fluoride exposed and control animals was similar.     

肝靶向微粒给药系统的研究进展

目的对近年来微粒给药系统在肝靶向治疗的研究进展做一综述。方法根据国内外文献资料进行整理归纳。结果纳米粒、微球、脂质体及微乳等微粒系统具有被动靶向于肝的趋势,利用肝细胞表面某些受体则可特异性靶向于肝达到主动靶向作用。结论微粒给药系统在肝靶向治疗领域具有重要意义。     

The Use of Sonication for the Efficient Delivery of Plasmid DNA into Cells

Purpose. Ultrasonic methods have considerable potential for the introduction of macromolecules into cells. In this paper we demonstrate that, under controlled conditions, application of 20kHz ultrasound to a suspension of yeast cells facilitates the delivery of plasmid DNA into these cells. Methods. Aliquots of growing yeast cells (Saccharomyces cerevisae, strain AH22) were suspended in buffer and exposed to 20kHz ultrasound from a laboratory (probe-type) sonicator in the presence of microgram quantities of plasmid DNA. Efficiency of DNA delivery was scored as the number of cells transformed. Results. Cell transformation was optimal at 30 seconds sonication using an output of 2.0 watts and resulted in a 20 fold enhancement over control values. At extended sonication times, fewer cells showed evidence of transformation because of reduced cell viability. The increased DNA uptake and the decreased cell viability were both attributable to acoustic cavitation events during sonication. The extent of acoustic cavitation was measured and it was found that there was an increase in cavitation events with increased sonication time. Cell viability was shown to be directly related to the number of cavitation events. The effects of sonication on plasmid DNA were investigated and indicated that the structural integrity of plasmid DNA was unaffected by the sonication conditions employed. Conclusions. Under controlled conditions, ultrasound is an effective means of delivering plasmid DNA into cells. The subsequent expression of DNA molecules in cells depends upon a balance between transient cell damage and cell death.     

高浓度大鼠肝实质细胞与非实质细胞共培养的细胞功能研究

目的:高浓度肝实质细胞与非实质细胞原代共培养,研究其功能活性。方法:应用原位胶原酶灌流法分离大鼠肝细胞,获得有活性的肝细胞,并应用高浓度实质和非实质肝细胞共培养的方法原代培养（共培养组）,并以微囊肝细胞培养为对照组（微囊组）进行了比较。结果:两组均维持白蛋白分泌、尿素合成功能7天;共培养组的白蛋白分泌与微囊组一样为下降趋势,共培养组从（0.870±0.102）降至（0.492±0.040）g·L-1·10-6cells·24h-1,微囊组从（1.147±0.099）降至（0.375±0.012）g·L-1·10-6cells·24h-1;共培养组的肝细胞第4天后维持在一个较稳定的水平,而微囊组肝细胞前3天明显高于共培养组,而后两天显著低于共培养组（P<0.05）。共培养组的尿素合成功能,由（4.50±0.56）降至（4.37±0.19）μmol·L-1·10-6cells·90min-1,微囊组由（5.42±0.81）降至（3.60±0.33）μmol·L-1·10-6cells·90min-1,前3天微囊组高于共培养组,后2天共培养组明显高于微囊组（P<0.05）。共培养2～7天肝细胞的对氨基苯甲酸（PABA）浓度稳定在 7.2～9.9mg/L·10-6cells·24h-1。结论:高浓度肝实质细胞与非实质细胞共培养,可使肝细胞的特异性功能维持7d,而且较微囊肝细胞更适合应用于中空纤维舱型生物人工肝。     

In Vitro Gene Transfection in Human Glioma Cells Using a Novel and Less Cytotoxic Artificial Lipoprotein Delivery System

Purpose. To develop and evaluate a novel artificial lipoprotein delivery system for in vitro gene transfection in human glioma cells. Method. Nanoemulsion was formulated with similar lipid compositions present in natural lipoproteins. The oil phase of nanoemulsion was composed of triolein (70%), egg phosphatidylcholine (22.7%), lysophosphatidylcholine (2.3%), cholesterol oleate (3.0%), and cholesterol (2.0%). To replace the surface protein as in natural lipoprotein, poly-L-lysine was modified to add palmitoyl chains at a basic condition and was incorporated onto the nanoemulsion particles through hydrophobic interaction. A model plasmid DNA, pSV--Gal containing a reporter gene for -galactosidase was carried by the nanoemulsion/poly-L-lysine particles. The charge variation of so-formed complex was examined by agarose gel electrophoresis and zeta potential measurement. In vitro transfection was conducted on human SF-767 glioma cell line using this new system. After standard X-Gal staining, transfected cells were observed under light microscope. The effect of chloroquine on the transfection was examined and, finally, the cytotoxicity of this new system was evaluated in comparison with commercial Lipofectamine gene transfection system. Results. The plasmid DNA was effectively carried by this artificial lipoprotein delivery system and the reporter gene was expressed in the glioma cells. Transfection efficiency was significantly increased by the treatment of chloroquine, indicating that endocytosis possibly was the major cellular uptake pathway. Compared to Lipofectamine system, this new delivery system demonstrated similar transfection efficiency but a much lower cytotoxicity. In the experiment, the cell viability showed up to 75% using this system compared to only 24% using Lipofectamine system. Conclusion. A new artificial lipoprotein delivery system was developed for in vitro gene transfection in tumor cells. The new system showed similar transfection efficiency but a much lower cytotoxicity compared with commercial Lipofectamine system.