新型阳离子基因传递载体PEI-PBLG的细胞转染研究

对新型阳离子聚合物PEI(10kD)-PBLG进行研究,重点考察其基因转染效率与细胞毒性,探讨其作为基因载体的可能性。通过粒径分析及扫描电镜(SEM)观察PEI(10kD)-PBLG与质粒pEGFP自组装形成的颗粒形态及粒径,预测其进入细胞的可能性。使用MTT比色法分析PEI(10kD)-PBLG、PEI(25kD)-PBLG、PEI(10kD)和PEI(25kD)的细胞毒性差异。选用表达增强型绿色荧光蛋白的质粒pEGFP作为报告基因模型,将其与PEI(10kD)-PBLG自组装后,分别转染真核细胞株Hela、COS-7、Vero-E6和ECV304,应用流式细胞术检测细胞转染效率,并比较了血清、缓冲液、细胞谱等多种因素对基因转染效率的影响。PEI(10kD)-PBLG可包裹质粒形成粒径100~120nm的纳米复合物,适合介导质粒进入细胞。该纳米粒复合物对转染缓冲液的敏感度较低,并能够在10%血清存在的条件下,转染全部实验用细胞株,尤其对Hela的转染效率最高,其次是COS-7、Vero-E6和ECV304;其中PEI-PBLG(10kD)/pEGFP复合物转染Hela细胞的比率为45.02%,高于PEI(10kD)/pEGFP的29.16%;PEI(10kD)-PBLG的细胞毒性作用显著低于PEI(25kD)、PEI(10kD)和PEI(25kD)-PBLG。新型阳离子多聚物PEI(10kD)-PBLG在提高PEI介导的基因转染效率的同时降低了其细胞毒性,提高了生物相容性,有望成为基因转移的有效载体。     

Tubular Membrane Formation of Binary Giant Unilamellar Vesicles Composed of Cylinder and Inverse-Cone-Shaped Lipids

We have succeeded in controlling tubular membrane formations in binary giant unilamellar vesicles (GUVs) using a simple temperature changing between the homogeneous one-phase region and the two-phase coexistence region. The binary GUV is composed of inverse-cone (bulky hydrocarbon chains and a small headgroup) and cylinder-shaped lipids. When the temperature was set in the two-phase coexistence region, the binary GUV had a spherical shape with solidlike domains. By increasing the temperature to the homogeneous one-phase region, the excess area created by the chain melting of the lipid produced tubes inside the GUV. The tubes had a radius on the micrometer scale and were stable in the one-phase region. When we again decreased the temperature to the two-phase coexisting region, the tubes regressed and the GUVs recovered their phase-separated spherical shape. We infer that the tubular formation was based on the mechanical balance of the vesicle membrane (spontaneous tension) coupled with the asymmetric distribution of the inverse-cone-shaped lipids between the inner and outer leaflets of the vesicle (lipid sorting).     

Tubular Membrane Formation of Binary Giant Unilamellar Vesicles Composed of Cylinder and Inverse-Cone-Shaped Lipids

We have succeeded in controlling tubular membrane formations in binary giant unilamellar vesicles (GUVs) using a simple temperature changing between the homogeneous one-phase region and the two-phase coexistence region. The binary GUV is composed of inverse-cone (bulky hydrocarbon chains and a small headgroup) and cylinder-shaped lipids. When the temperature was set in the two-phase coexistence region, the binary GUV had a spherical shape with solidlike domains. By increasing the temperature to the homogeneous one-phase region, the excess area created by the chain melting of the lipid produced tubes inside the GUV. The tubes had a radius on the micrometer scale and were stable in the one-phase region. When we again decreased the temperature to the two-phase coexisting region, the tubes regressed and the GUVs recovered their phase-separated spherical shape. We infer that the tubular formation was based on the mechanical balance of the vesicle membrane (spontaneous tension) coupled with the asymmetric distribution of the inverse-cone-shaped lipids between the inner and outer leaflets of the vesicle (lipid sorting).     

Structure Relationship of Cationic Lipids on Gene Transfection Mediated by Cationic Liposomes

The aim of this study was to investigate the transfection efficiency of cationic liposomes formulated with phosphatidylcholine (PC) and novel synthesized diethanolamine-based cationic lipids at a molar ratio of 5:1 in comparison with Lipofectamine™ 2000. Factors affecting transfection efficiency and cell viability, including the chemical structure of the cationic lipids, such as different amine head group (diamine and polyamine; and non-spermine and spermine) and acyl chain lengths (C14, C16, and C18) and the weight ratio of liposomes to DNA were evaluated on a human cervical carcinoma cell line (HeLa cells) using the pDNA encoding green fluorescent protein (pEGFP-C2). Characterizations of these lipoplexes in terms of size and charge measurement and agarose gel electrophoresis were performed. The results from this study revealed that almost no transfection was observed in the liposome formulations composed of cationic lipids with a non-spermine head group. In addition, the transfection efficiency of these cationic liposomes was in the following order: spermine-C14 > spermine-C16 > spermine-C18. The highest transfection efficiency was observed in the formulation of spermine-C14 liposomes at a weight ratio of 25; furthermore, this formulation was safe for use in vitro. In conclusion, cationic liposomes containing spermine head groups demonstrated promising potential as gene carriers.Key words: cationic lipids, cationic liposomes, gene transfection     

Novel Cationic Phosphonolipids Agents for in Vitro Gene Transfer to Epithelial Cell Lines

Abstract

The use of cationic liposomes is one of the main approaches currently investigated to introduce into a cell a gene with therapeutic properties. This study presents in vitro results obtained with a new family of gene transfer agents, the phosphonolipids. We have synthesized 37 members of this family and optimized the conditions of in vitro gene transfer targeted at a lung epithelial cell line (CFT1 cells) by using a reporter gene (β-galactosidase). Two quantitative tests, a CPRG¹ (Chlorophenol red galactopyranoside) test and a Flow cytometric assay (FACS-Gal¹ assay), have been used to determine the percentage of transfected cells. The cationic phosphonolipids were tested alone or formulated with 50% DOPE¹ (Dioleoylphosphatidylethanolamine) (w:w). The results obtained with the CPRG test led us to select 7 compounds that were more efficient than the commercialized lipids Lipofectin, Lipofectamine and Transfectam. The transfer kinetics of the transgene were studied and showed that more than 20 % of cells were still positive at day 7 after transfection.     

In Vitro Evaluation of Proniosomes as a Drug Carrier for Flurbiprofen

The purpose of the present investigation is to formulate and evaluate proniosomal transdermal carrier systems for flurbiprofen. Proniosomes were prepared using various non-ionic surfactants, namely span 20 (Sp 20), span 40 (Sp 40), span 60 (Sp 60) and span 80 (Sp 80) without and with cholesterol at percentages ranging from 0% to 50%. The effect of surfactant type and cholesterol content on drug release was investigated. Drug release was tested by diffusion through cellophane membrane and rabbit skin. Drug release from the prepared systems was compared to that from flurbiprofen suspensions in distilled water and HPMC (hydroxypropylmethylcellulose) gels. In case of Sp 20 and Sp 80, the added amount of cholesterol affected the preparation type to be either proniosomal alcoholic solutions or liquid crystalline gel systems. On the other hand, both Sp 40 and Sp 60 produced gel systems in presence or absence of cholesterol. Microscopic observations showed that either proniosomal solutions or gel formulations immediately converted to niosomal dispersions upon hydration. Due to the skin permeation barrier, rabbit skin showed lower drug diffusion rates compared to cellophane membrane. The proniosomal composition controlled drug diffusion rates to be either faster or slower than the prepared flurbiprofen suspensions in HPMC gels or distilled water, respectively. In conclusion, this study demonstrated the possibility of using proniosomal formulations for transdermal drug delivery.     

Following the Fate In Vivo of COPI Vesicles Generated In Vitro

COPI vesicles are a class of transport carriers that function in the early secretory pathway. Their fate and function are still controversial. This includes their contribution to bidirectional transport within the Golgi apparatus and their role during cell division. Here we describe a method that should address several open questions about the fate and function of COPI vesicles in vivo . To this end, fluorescently labeled COPI vesicles were generated in vitro from isolated rat liver Golgi membranes, labeled with the fluorescent dyes Alexa-488 or Alexa-568. These vesicles appeared to be active and colocalized with endogenous Golgi membranes within 30 min after microinjection into mammalian cells. The COPI vesicle-derived labeled membrane proteins could be classified into two types that behaved like endogenous proteins after Brefeldin A treatment.     

The Novel Analogue of Hirsutine as an Anti-Hypertension and Vasodilatary Agent Both In Vitro and In Vivo

In this paper, an analogue of hirsutine (compound 1) has been synthesized and evaluated as an anti-hypertension agent, which exhibits extraordinary effects on the contractile response of thoracic aorta rings from male SD rats in vitro (IC₅₀ = 1.129×10^-9±0.5025) and the abilities of reducing the systolic blood pressure (SBP) and heart rate (HR) of SHR in vivo. The mechanism investigation reveals that the vasodilatation induced by compound 1 is mediated by both endothelium-dependent and -independent manners. The relaxation in endothelium-intact aortic rings induced by compound 1 can be inhibited by L-NAME (1×10^-6 mol•L^-1) and ODQ (1×10^-6 mol•L^-1). Moreover, compound 1 can also block Ca²⁺ influx through L-type Ca²⁺ channels and inhibit intracellular Ca²⁺ release while no effect on K⁺ channel has been observed. All these data demonstrated that the NO/cyclic GMP pathway can be involved in endothelium-dependent manner induced by compound 1. Meanwhile the mechanism on the vasodilatation of compound 1 probably also related to blockade of Ca²⁺ influx through L-type Ca²⁺ channels and inhibition of intracellular Ca²⁺ release may have no relationship with K⁺ channels.     

Characterization of Fast-Scan Cyclic Voltammetric Electrodes Using Paraffin as an Effective Sealant with In Vitro and In Vivo Applications

Fast-scan cyclic voltammetry (FSCV) is a powerful technique for measuring sub-second changes in neurotransmitter levels. A great time-limiting factor in the use of FSCV is the production of high-quality recording electrodes; common recording electrodes consist of cylindrical carbon fiber encased in borosilicate glass. When the borosilicate is heated and pulled, the molten glass ideally forms a tight seal around the carbon fiber cylinder. It is often difficult, however, to guarantee a perfect seal between the glass and carbon. Indeed, much of the time spent creating electrodes is in an effort to find a good seal. Even though epoxy resins can be useful in this regard, they are irreversible (seals are permanent), wasteful (epoxy cannot be reused once hardener is added), hazardous (hardeners are often caustic), and require curing. Herein we characterize paraffin as an electrode sealant for FSCV microelectrodes. Paraffin boasts the advantages of near-immediate curing times, simplicity in use, long shelf-life and stable waterproof seals capable of withstanding extended cycling. Borosilicate electrode tips were left intact or broken and dipped in paraffin embedding wax. Excess wax was removed from the carbon surface with xyelenes or by repeated cycling at an extended waveform (-0.4 to 1.4V, 400 V/s, 60 Hz). Then, the waveform was switched to a standard waveform (-0.4 to 1.3V, 400 V/s, 10 Hz) and cycled until stable. Wax-sealing does not inhibit electrode sensitivity, as electrodes detected linear changes in dopamine before and after wax (then xylenes) exposure. Paraffin seals are intact after 11 days of implantation in the mouse, and still capable of measuring transient changes in in vivo dopamine. From this it is clear that paraffin wax is an effective sealant for FSCV electrodes that provides a convenient substitute to epoxy sealants.     

Development and Evaluation of Curcumin-loaded Elastic Vesicles as an Effective Topical Anti-inflammatory Formulation

Curcumin has diverse biological activities including antioxidant and anti-inflammatory activity. However, its clinical use for topical application is limited due to its poor aqueous solubility and thus, minimal cutaneous bioavailability. Elastic vesicles (EVs) of curcumin were prepared to improve its cutaneous bioavailability and to use it for topical anti-inflammatory effect. Ex vivo skin permeation and retention studies were performed to check if incorporation of curcumin into EVs could improve its permeation into and retention in the skin. Evaluation of acute and chronic anti-inflammatory effect was done using xylene-induced acute ear edema in mice and cotton pellet-induced chronic inflammation in rats, respectively. A significant improvement in flux (nine times) across murine skin was observed when aqueous dispersion of curcumin (flux − 0.46 ± 0.02 μg/h/cm²) was compared with curcumin-loaded EVs (flux − 4.14 ± 0.04 μg/h/cm² ). Incorporation of these curcumin-loaded EVs into a hydrophilic ointment base resulted in higher skin retention (51.66%) in contrast to free curcumin ointment (1.64%) and a marketed formulation (VICCO® turmeric skin cream). The developed ointment showed an effect similar (p < 0.05) to the marketed diclofenac sodium ointment (Omni-gel®) in suppression of acute inflammation in mouse; a significant inhibition (28.8% versus 3.91% for free curcumin) of cotton pellet-induced chronic inflammation was also observed. Thus, curcumin-loaded EVs incorporated in hydrophilic ointment is a promising topical anti-inflammatory formulation.KEY WORDS: anti-inflammatory, curcumin, elastic vesicles, topical formulation     

Dynamic Storage of Dopamine in Rat Brain Synaptic Vesicles In Vitro

Abstract: The dynamics of catecholamine storage were studied in highly purified, small synaptic vesicles from rat brain both during active uptake or after inhibiting uptake with reserpine, tetrabenazine, or removal of external dopamine. To assess turnover during active uptake, synaptic vesicles were allowed to accumulate [³H]dopamine ([³H]DA) for ～10 min and then diluted 20-fold into a solution containing unlabeled DA under conditions such that active uptake could continue. After dilution, [³H]DA was lost with single exponential kinetics at a half-time of ～4 min at 30°C in 8 mM Cl^? medium, in which both voltage and H⁺ gradients are present in the vesicles. In 90 mM Cl^? medium, in which high H⁺ and Cl^? gradients but no voltage gradient are present, [³H]DA escaped at a half-time of ～7 min. In both high and low Cl^? media, ～40% of [³H]DA efflux was blocked by reserpine or tetrabenazine. The residual efflux also followed first-order kinetics. These results indicate that two efflux pathways were present, one dependent on DA uptake (and thus on the presence of external DA) and the other independent of uptake, and that both pathways function regardless of the type of electrochemical H⁺ gradient in the vesicles. The presence of both uptake-dependent and -independent efflux was observed in experiments using DA-free medium, instead of uptake inhibitors, to prevent uptake. Uptake-independent efflux showed molecular selectivity for catecholamines; [¹⁴C]DA was lost about three times faster than [³H]norepinephrine after adding tetrabenazine directly (without dilution) to vesicles that had taken up comparable amounts of each amine. In addition, the first-order rate constant for uptake-independent efflux showed little change over a 60-fold range of internal DA concentrations, which suggests that this pathway had a high transport capacity. All efflux was blocked at 0°C, suggesting that efflux did not occur through a large pore. There was little or no change in the proton gradient in synaptic vesicles, monitored by [¹⁴C]methylamine equilibration, during the experimental manipulations used here. Thus, the driving force for catecholamine uptake remained approximately constant. The physiological role of uptake-independent efflux could be to allow the monoamine content of synaptic vesicles to be regulated over a time range of minutes and, thereby, control the amount released by exocytosis. These results imply that catecholamines turn over with a half-time of minutes during active uptake by brain synaptic vesicles in vitro.     

Use of Bottom Ash of Waste Coal as an Effective Microbial Carrier

An experiment was done to determine the efficacy of waste bottom ash as an effective microbial carrier. Bottom ash found to be a suitable microbial carrier. The average of viable cells of Paenibacillus polymyxa GS01 (as a test biocontrol agent) in bottom ash samples was about 10⁸ cfu/10±2 mg. The surface of bottom ash coated with 5% PVA w/v was most effective for improvement of cell viability. TSB medium containing 50 mg/L of MnSO₄·H₂O was the best for spore production of P. polymyxa GS01. Thus waste bottom ash coating with 5% PVA is likely to be suitable for use as a microbial carrier.     

Incorporation In Vivo and In Vitro of Radiolabeled Sphingolipid Precursors into Paramecium tetraurelia Lipids

ABSTRACT Paramecium tetraurelia contains high concentrations of ethanolamine sphingolipids, especially in its ciliary membrane. Three ethanolamine sphingophospholipids with different long chain bases (dihydrosphingosine, sphingosine and phytosphingosine), and their phosphonyl analogs, were previously identified and characterized. In the present study, radiolabeling experiments on lag- and log-phase cells were performed to investigate the extent of sphingolipid biosynthetic capacities of the ciliate. Long chain bases of sphingolipids are formed by an initial condensation reaction of serine with a fatty-coenzyme A. Thus, radiolabeled palmitic acid, stearic acid and serine were used as precursor compounds in these experiments. The results indicated that (1) sphingolipid precursors were incorporated into every major lipid fraction. (2) ethanolamine sphingophosphonolipids accumulated faster than the ethanolamine sphingophospholipids, (3) in contrast to these sphingolipids, the glycerolipid, phosphatidyethanolamine. accumulated faster than its phosphono analog, and (4) palmitic acid, but not stearic acid, was incorporated into the long chain bases of ethanolamine sphingophospho- and sphingophosphonolipids. consistent with an earlier report demonstrating that these lipids contain only C,g long chain bases. Since P. tetraurelia takes up serine and other water-soluble substrates very slowly, and catabolizes fatty acids rapidly, label is randomized in intact cells. Thus, cell-free protocols provide useful experimental systems for studies of sphingolipid biosynthesis than do intact organisms, when the uptake of precursor substrates are slow.     

In Vitro Heat Stability of Plasma Membrane Vesicles and Tonoplast Membrane Vesicles Isolated From the Hypocotyle of Phaseolus vulgaris

Microsome, plasma membrane vesicles and tonoplast membrane vesicles were isolated from the hypocotyles of Phaseolus vulgaris L. 85CT-49762, with very high heat tolerance potential. Comparing the H+-pump heat stability in vitro of the vesicles from the heat acclimated cells and the cells in which protein synthesis was inhibited by actidion during heat acclimation with that of normal cells, the authors found that heat acclimation could increase the heat stability of membrane vesicles, and that the heat shock proteins synthesized during heat acclimation were related to the effect. The authors further analysed the role of membrane peripheral proteins on H+-pump thermotolerance of membrane vesicles, and proved that heat shock protein HSP 70 and low molecular weight heat shock protein (LMW HSP) were able to protect H+-pump from heat destruction.     

新型非病毒三元复合DNA载体的研究

基因治疗是未来临床医学最具潜力的治疗方式,目前阻碍临床基因治疗发展的主要因素是缺乏安全和高效的基因载体,因此研究理想的非病毒转基因载体具有重要的意义.构建了由质粒DNA(D)-抗DNA抗体(A)-阳离子脂质体(C)组成的三元复合纳米基因载体(DAC),研究表明,三组分在磷酸缓冲液中可通过分子组装形成复合纳米胶束,DAC在细胞培养中表现出显著高效的基因表达,DAC在血管平滑肌细胞中的基因转染效率比不含抗DNA抗体的二元组合(DC)高4倍,比不含阳离子脂质体的二元组合(DA)约高11倍.激光共聚焦荧光显微观察证明,DAC细胞摄取量和DNA进入细胞核的量均明显高于对照组,而DC二元组合(不含抗DNA抗体)的DNA很少进入细胞核,细胞在DAC存在下生长正常.未发现细胞毒性.研究结果提示,DAC的作用机理主要是三元复合胶束中DNA的装载量比二元载体大得多,抗DNA抗体与阳离子脂质体的协同作用明显有利于DNA被细胞摄取和胞吞,从而提高了基因的转染和表达.     

干湿纺聚乳酸／壳聚糖纤维交织织物作为细胞体外培养载体的实验观察

在体外培养的条件下观察干湿纺聚乳酸/壳聚糖纤维交织织物与成骨细胞的相容性,探讨其作为人工胸壁支架材料和人工骨支架材料的可行性。将hFOB1.19人SV40转染的成骨细胞与干湿纺聚乳酸/壳聚糖纤维交织织物体外联合培养。用扫描电镜对体外联合培养早期细胞的形态学进行观察。结果表明,成骨细胞与干湿纺聚乳酸/壳聚糖交织织物间黏附良好,具有良好的相容性,干湿纺聚乳酸/壳聚糖纤维交织织物有可能成为一种理想的可用于修复胸壁缺损和骨缺损的成骨细胞载体。     

A Novel Micro-Linear Vector for In Vitro and In Vivo Gene Delivery and Its Application for EBV Positive Tumors

Background

The gene delivery vector for DNA-based therapy should ensure its transfection efficiency and safety for clinical application. The Micro-Linear vector (MiLV) was developed to improve the limitations of traditional vectors such as viral vectors and plasmids.

Methods

The MiLV which contained only the gene expression cassette was amplified by polymerase chain reaction (PCR). Its cytotoxicity, transfection efficiency in vitro and in vivo, duration of expression, pro-inflammatory responses and potential application for Epstein-Barr virus (EBV) positive tumors were evaluated.

Results

Transfection efficiency for exogenous genes transferred by MiLV was at least comparable with or even greater than their corresponding plasmids in eukaryotic cell lines. MiLV elevated the expression and prolonged the duration of genes in vitro and in vivo when compared with that of the plasmid. The in vivo pro-inflammatory response of MiLV group was lower than that of the plasmid group. The MEKK1 gene transferred by MiLV significantly elevated the sensitivity of B95-8 cells and transplanted tumor to the treatment of Ganciclovir (GCV) and sodium butyrate (NaB).

Conclusions

The present study provides a safer, more efficient and stable MiLV gene delivery vector than plasmid. These advantages encourage further development and the preferential use of this novel vector type for clinical gene therapy studies.     

Cytomegalovirus Replicon-Based Regulation of Gene Expression In Vitro and In Vivo

There is increasing evidence for a connection between DNA replication and the expression of adjacent genes. Therefore, this study addressed the question of whether a herpesvirus origin of replication can be used to activate or increase the expression of adjacent genes. Cell lines carrying an episomal vector, in which reporter genes are linked to the murine cytomegalovirus (MCMV) origin of lytic replication (oriLyt), were constructed. Reporter gene expression was silenced by a histone-deacetylase-dependent mechanism, but was resolved upon lytic infection with MCMV. Replication of the episome was observed subsequent to infection, leading to the induction of gene expression by more than 1000-fold. oriLyt-based regulation thus provided a unique opportunity for virus-induced conditional gene expression without the need for an additional induction mechanism. This principle was exploited to show effective late trans-complementation of the toxic viral protein M50 and the glycoprotein gO of MCMV. Moreover, the application of this principle for intracellular immunization against herpesvirus infection was demonstrated. The results of the present study show that viral infection specifically activated the expression of a dominant-negative transgene, which inhibited viral growth. This conditional system was operative in explant cultures of transgenic mice, but not in vivo. Several applications are discussed.     

Effects of Iron Deficiency on Serotonin Uptake In Vitro by Rat Brain Synaptic Vesicles

Abstract: The effects of moderate and severe degrees of iron deficiency on brain and liver nonhaem iron levels and 5-hydroxytryptamine (serotonin; 5-HT) uptake by synaptic vesicles in vitro were investigated in experimental rats. Data obtained suggested that in both moderate and severe forms of iron deficiency, 5-HT uptake by brain synaptic vesicles is decreased and is accompanied by a reduction in brain and liver nonhaem iron levels. On repletion with iron for 4 weeks, the deficient group of rats showed a normalisation of 5-HT uptake by synaptic vesicles and liver nonhaem iron content, whereas the brain nonhaem iron concentration still showed a significant deficit. The data thus suggest that changes in the uptake of 5-HT by brain synaptic vesicles that accompany iron depletion and repletion are more rapid than changes in the total nonhaem iron concentration in the brain. The observation that 5-HT uptake by brain synaptic vesicles is decreased in iron deficiency suggests a probable role for iron in 5-HT storage in rat brain.