Addition of ascorbic acid to the extracellular environment activates lipoplexes of a ferrocenyl lipid and promotes cell transfection

The level of cell transfection mediated by lipoplexes formed using the ferrocenyl lipid bis(11-ferrocenylundecyl)dimethylammonium bromide (BFDMA) depends strongly on the oxidation state of the two ferrocenyl groups of the lipid (reduced BFDMA generally mediates high levels of transfection, but oxidized BFDMA mediates very low levels of transfection). Here, we report that it is possible to chemically transform inactive lipoplexes (formed using oxidized BFMDA) to “active” lipoplexes that mediate high levels of transfection by treatment with the small-molecule reducing agent ascorbic acid (vitamin C). Our results demonstrate that this transformation can be conducted in cell culture media and in the presence of cells by addition of ascorbic acid to lipoplex-containing media in which cells are growing. Treatment of lipoplexes of oxidized BFDMA with ascorbic acid resulted in lipoplexes composed of reduced BFDMA, as characterized by UV/vis spectrophotometry, and lead to activated lipoplexes that mediated high levels of transgene expression in the COS-7, HEK 293T/17, HeLa, and NIH 3T3 cell lines. Characterization of internalization of DNA by confocal microscopy and measurements of the zeta potentials of lipoplexes suggested that these large differences in cell transfection result from (i) differences in the extents to which these lipoplexes are internalized by cells and (ii) changes in the oxidation state of BFDMA that occur in the extracellular environment (i.e., prior to internalization of lipoplexes by cells). Characterization of lipoplexes by small-angle neutron scattering (SANS) and by cryogenic transmission electron microscopy (cryo-TEM) revealed changes in the nanostructures of lipoplexes upon the addition of ascorbic acid, from aggregates that were generally amorphous, to aggregates with a more extensive multilamellar nanostructure. The results of this study provide guidance for the design of redox-active lipids that could lead to methods that enable spatial and/or temporal control of cell transfection.     

Folate-targeted,cationic liposome-mediated gene transfer into disseminated peritoneal tumors

A folate-targeted, cationic lipid based transfection complex was developed and found to specifically transfect folate receptor-expressing cells and tumors. These liposomal vectors were comprised of protamine-condensed plasmid DNA, a mixture of cationic and neutral lipids, and a folic acid-cysteine-polyethyleneglycol-phosphatidylethanolamine (FA-Cys-PEG-PE) conjugate. Pre-optimization studies revealed that inclusion of low amounts (0.01 to 0.03%) of FA-Cys-PEG-PE yielded the highest binding activity of dioleoylphosphatidylcholine/cholesterol liposomes to folate receptor-bearing cells. In contrast, higher amounts (>0.5%) of FA-Cys-PEG-PE progressively decreased cellular binding of the liposomes. In vitro studies with cationic lipid/dioleoylphosphatidylethanolamine formulations indicated that as little as 0.01 to 0.3% of FA-Cys-PEG-PE was needed to produce optimal targeted expression of plasmid DNA. Similarly, using a disseminated intraperitoneal L1210A tumor model, maximum in vivo transfection activity occurred with intraperitoneally administered formulations that contained low amounts (0.01 mol%) of the FA-Cys-PEG-PE targeting lipid. Overall, folate-labeled formulations produced an eight- to 10-fold increase in tumor-associated luciferase expression, as compared with the corresponding non-targeted cationic lipid/DNA formulations. These results collectively indicate that transfection of widespread intraperitoneal cancers can be significantly enhanced using folate-targeted techniques.     

Lipoxygenase contributes to the oxidation of lipids in human atherosclerotic plaques.

Oxidized LDL is present in human atherosclerotic lesions, but the mechanisms responsible for oxidation in vivo have not been definitively demonstrated. Circumstantial evidence has implicated the enzyme 15-lipoxygenase as a contributor to the formation of oxidized lipids in this disease. To assess whether oxidized lipids are indeed formed by the action of 15-lipoxygenase on polyunsaturated fatty acids (PUFAs) in vivo, we have used a sensitive and specific method (chiral phase HPLC) to analyze the lipid oxidation products present in human atherosclerotic lesions. Human 15-lipoxygenase is an omega-6 lipoxygenase that has previously been shown to oxidize esterified PUFA in a stereospecific manner, forming predominantly cholesteryl hydroperoxy-octadecadienoate (13(S)-HPODE) from cholesteryl linoleate substrate in LDL. This property allows its activity to be distinguished from nonenzymatic oxidation, which results in the formation of equal quantities of the S and R stereoisomers of the same oxidation product. A total of 80 specimens of human atherosclerotic plaque were analyzed. Esterified, oxidized linoleate was purified from human atherosclerotic lesions and from LDL oxidized by copper, and the chirality of these oxidation products was compared. There was significantly greater stereospecificity of oxidation in the oxidized linoleate from human atherosclerotic lesions. Even greater stereospecificity was detected in the HPODE derived from cholesteryl ester, purified from human lesions. Cholesteryl HPODE is the primary oxidation product from cholesteryl linoleate, the major esterified PUFA that accumulates in atherosclerotic vessels. Cholesteryl HPODE and its reduced form, cholesteryl hydroxy-octadecadienoate, were detected in all lesions analyzed. Neither the stereospecificity of oxidation nor the percentage of available substrate oxidized to primary oxidation products was correlated with the stage of disease of the lesions examined. We conclude that 15-lipoxygenase contributes to the formation of oxidized lipids in human atherosclerotic lesions.     

Single histidine residue in head-group region is sufficient to impart remarkable gene transfection properties to cationic lipids: evidence for histidine-mediated membrane fusion at acidic pH

Presence of endosome-disrupting multiple histidine functionalities in the molecular architecture of cationic polymers, such as polylysine, has previously been demonstrated to significantly enhance their in vitro gene delivery efficiencies. Towards harnessing improved transfection property through covalent grafting of endosome-disrupting single histidine functionality in the molecular structure of cationic lipids, herein, we report on the design, the synthesis and the transfection efficiency of two novel nonglycerol-based histidylated cationic amphiphiles. We found that L-histidine-(N,N-di-n-hexadecylamine)ethylamide (lipid 1) and L-histidine-(N,N-di-n-hexadecylamine,-N-methyl)ethylamide (lipid 2) in combination with cholesterol gave efficient transfections into various cell lines. The transfection efficiency of Chol/lipid 1 lipoplexes into HepG2 cells was two order of magnitude higher than that of FuGENE(TM)6 and DC-Chol lipoplexes, whereas it was similar into A549, 293T7 and HeLa cells. A better efficiency was obtained with Chol/lipid 2 lipoplexes when using the cytosolic luciferase expression vector (pT7Luc) under the control of the bacterial T7 promoter. Membrane fusion activity measurements using fluorescence resonance energy transfer (FRET) technique showed that the histidine head-groups of Chol/lipid 1 liposomes mediated membrane fusion in the pH range 5-7. In addition, the transgene expression results using the T7Luc expression vector convincingly support the endosome-disrupting role of the presently described mono-histidylated cationic transfection lipids and the release of DNA into the cytosol. We conclude that covalent grafting of a single histidine amino acid residue to suitable twin-chain hydrophobic compounds is able to impart remarkable transfection properties on the resulting mono-histidylated cationic amphiphile, presumably via the endosome-disrupting characteristics of the histidine functionalities.     

Discovery of siRNA Lipid Nanoparticles to Transfect Suspension Leukemia Cells and Provide In Vivo Delivery Capability

As a powerful research tool, siRNA''s therapeutic and target validation utility with leukemia cells and long-term gene knockdown is severely restricted by the lack of omnipotent, safe, stable, and convenient delivery. Here, we detail our discovery of siRNA-containing lipid nanoparticles (LNPs) able to effectively transfect several leukemia and difficult-to-transfect adherent cell lines also providing in vivo delivery to mouse spleen and bone marrow tissues through tail-vein administration. We disclose a series of novel structurally related lipids accounting for the superior transfection ability, and reveal a correlation between expression of Caveolins and successful transfection. These LNPs, bearing low toxicity and long stability of >6 months, are ideal for continuous long-term dosing. Our discovery represents the first effective siRNA-containing LNPs for leukemia cells, which not only enables high-throughput siRNA screening with leukemia cells and difficult-to-transfect adherent cells but also paves the way for the development of therapeutic siRNA for leukemia treatment.     

Enhanced cationic liposome-mediated transfection using the DNA-binding peptide mu (mu) from the adenovirus core

Promising advances in nonviral gene transfer have been made as a result of the production of cationic liposomes formulated with synthetic cationic lipids (cytofectins) that are able to transfect cells. However few cationic liposome systems have been examined for their ability to transfect CNS cells. Building upon our earlier use of cationic liposomes formulated from 3beta-[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol (DC-Chol) and dioleoyl-L-alpha-phosphatidyl-ethanolamine (DOPE), we describe studies using two cationic viral peptides, mu (mu) and Vp1, as potential enhancers for cationic liposome-mediated transfection. Mu is derived from the condensed core of the adenovirus and was selected to be a powerful nucleic acid charge neutralising and condensing agent. Vp1 derives from the polyomavirus and harbours a classical nuclear localisation signal (NLS). Vp1 proved disappointing but lipopolyplex mixtures formulated from pCMVbeta plasmid, mu peptide and DC-Chol/DOPE cationic liposomes were able to transfect an undifferentiated neuronal ND7 cell line with beta-galactosidase reporter gene five-fold more effectively than lipoplex mixtures prepared from pCMVbeta plasmid and DC-Chol/DOPE cationic liposomes. Mu was found to give an identical enhancement to cationic liposome-mediated transfection of ND7 cells as poly-L-lysine (pLL) or protamine sulfate (PA). The enhancing effects of mu were found to be even greater (six- to 10-fold) when differentiated ND7 cells were transfected with mu-containing lipopolyplex mixtures. Differentiated ND7 cells represent a simple ex vivo-like post-mitotic CNS cell system. Successful transfection of these cells bodes well for transfection of primary neurons and CNS cells in vivo. These findings have implications for experimental and therapeutic uses of cationic liposome-mediated delivery of nucleic acids to CNS cells.     

Trimethylated chitosans as non-viral gene delivery vectors: cytotoxicity and transfection efficiency.

Chitosans are linear polysaccharides of natural origin that show potential as carriers in drug and gene delivery. Introducing quaternisation on the chitosan backbone renders the polymer soluble over a wider pH range and confers controlled cationic character. This study aims to investigate the effect of increasing quaternisation and therefore, positive charge on cell viability and transfection. Oligomeric and polymeric chitosans were trimethylated, the toxicity and transfection efficiency of these derivatives were tested with respect to increasing degree of trimethylation. The cytoxicity of polymer and oligomer derivatives alone and of their complexes with plasmid DNA were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay on COS-7 (monkey kidney fibroblasts) and MCF-7 (epithelial breast cancer) cells. Transfection efficiency was investigated using the pGL3 luciferase reporter gene on the same cell lines. Complexes were characterised for their stability by gel electrophoresis. Cytotoxicity results showed that all derivatives were significantly less toxic than linear polyethylenimine (PEI). A general trend of increasing toxicity with increasing degree of trimethylation was seen. However, higher toxicity was seen in polymeric chitosan derivatives over oligomeric chitosan derivatives at similar degrees of trimethylation. All derivatives complexed pGL3 luc plasmid DNA efficiently at 10:1 ratio and three (TMO44, TMC57 and TMC93) were able to transfect MCF-7 cells with greater efficiency than PEI; 16, 23 and 50-fold, respectively. TMC57, TMC93 and all TMOs gave appreciable transfection of COS-7 cells.     

Dicationic amphiphiles bearing an amino acid head group with a long-chain hydrophobic tail for in vitro gene delivery applications

Amino acid-based cationic lipids, which have proven their efficacy as plasmid DNA nanocarriers, were employed as dicationic forms to transfect genes into cancer and non-cancer cells in this study. Proline, methionine, and serine amino acids are involved as hydrophilic moieties and the hydrocarbon long-chain serves as a hydrophobic tail. In a multicultural investigation, cationic lipids were employed as nano-vectors in conjunction with the helper lipid DOPE. To quantify the lipid efficient size, charge, and pDNA binding, biophysical analyses such as hydrodynamic diameter, zeta potential, agarose gel electrophoresis, and serum stability were done primarily. The liposomal particle composition was examined by scanning electron microscopy (SEM). Synthesized dicationic vector lipoplex formulations with reporter genes were found to be non-toxic to the cells investigated by MTT assay, and in addition, therapeutic gene p53 transfected into oral and brain cancer cells causing cell death was examined. In vitro investigations further validated that the proline-based lipid (C14-P) has high gene knockdown efficacy than methionine-based lipid (C14-M) and serine-based lipid (C14-S) at optimal N/P ratios as measured by β-galactosidase protein and eGFP expression. C14-P lipid shows superior cellular internalization compared to C14-M and C14-S in HEK-293 and CAL-27 cells attested by confocal study. These findings could include the proline-based lipid vector''s exceptional gene delivery activity.

C14-P, C14-M, and C14-S lipids formed lipoplexes using pDNA. The lipoplex cellular uptake into the cells resulted in the release of nucleic acids. C14-P lipid showed superior eGFP transfection in non-cancer cell line and more apoptosis cell death in cancer cell line.     

Apoptotic cells with oxidation-specific epitopes are immunogenic and proinflammatory

Oxidation of low density lipoprotein (LDL) generates a variety of oxidatively modified lipids and lipid-protein adducts that are immunogenic and proinflammatory, which in turn contribute to atherogenesis. Cells undergoing apoptosis also display oxidized moieties on their surface membranes, as determined by binding of oxidation-specific monoclonal antibodies. In the present paper, we demonstrated by mass spectrometry that in comparison with viable cells, membranes of cells undergoing apoptosis contain increased levels of biologically active oxidized phospholipids (OxPLs). Indeed, immunization of mice with syngeneic apoptotic cells induced high autoantibody titers to various oxidation-specific epitopes of oxidized LDL, including OxPLs containing phosphorylcholine, whereas immunization with viable thymocytes, primary necrotic thymocytes, or phosphate-buffered saline did not. Reciprocally, these antisera specifically bound to apoptotic cells through the recognition of oxidation-specific epitopes. Moreover, splenocyte cultures from mice immunized with apoptotic cells spontaneously released significant levels of T helper cell (Th) 1 and Th2 cytokines, whereas splenocytes from controls yielded only low levels. Finally, we demonstrated that the OxPLs of apoptotic cells activated endothelial cells to induce monocyte adhesion, a proinflammatory response that was abrogated by an antibody specific to oxidized phosphatidylcholine. These results suggest that apoptotic cell death generates oxidatively modified moieties, which can induce autoimmune responses and a local inflammatory response by recruiting monocytes via monocyte-endothelial cell interaction.     

Induction of apoptosis in macrophages by air oxidation of dioleoylphosphatidylglycerol.

Dioleoylphosphatidylglycerol (DOPG) containing unsaturated sites is the target of oxidation during preparation, storage, or in vivo use of anionic liposomes. We investigated the biological effect of air oxidation of DOPG on RAW 264.7 murine macrophage-like cells. Oxidation was induced by exposing DOPG to air for 24-72 h. The extent of air oxidation was confirmed using Matrix-Assisted Laser Desorption and Ionization with Time-of-Flight (MALDI-TOF) mass spectrometry. The product of the air oxidation of DOPG was identified as the addition of one oxygen atom to one of the symmetrical fatty moieties of DOPG at m/z 814.77. The treatment of DOPG with air oxidation produced dose-dependent cytotoxicity in macrophages. RAW 264.7 cells exposed to oxidized DOPG exhibited morphological features of apoptosis, such as chromatin condensation and cell shrinkage. Typical apoptotic ladders were observed in DNA extracted from RAW 264.7 cells treated with oxidized DOPG. Flow cytometric analysis demonstrated an increase in the hypodiploid DNA population (sub-G1), indicating that DNA cleavage occurred after treatment with oxidized DOPG. In addition, we showed that pretreating RAW 264.7 cells with zVAD-fmk, a general caspase inhibitor, did not prevent apoptosis induced by oxidized DOPG, suggesting that apoptosis in macrophage cells follows a caspase-independent pathway. These results point to a need for precaution in formulating DOPG liposomes for drug delivery and therapeutic purposes.     

Structural variation of cationic lipids: minimum requirement for improved oligonucleotide delivery into cells.

In vivo transfection efficiency (TE) using cationic liposome/oligonucleotide (ODN) complexes is often hampered by interactions with serum components. Novel cationic lipids with different hydroxyethyl or dihydroxypropyl ammonium backbones, esterified hydrocarbon chains and hydroxy substituents have been synthesized and applied in cationic liposome formulations with and without the helper lipid DOPE (1:1, m/m). Their properties for cellular ODN delivery were determined using fluorescently labeled ODNs (F-ODNs). Cationic lipids with hydrocarbon chains esterified to non-glycerol backbones in non-vicinal configuration were completely ineffective in nuclear ODN-delivery. Instead, an increased cytoplasmic localization of F-ODNs was observed. Cationic lipids equipped with only one hydrocarbon were completely incompetent for cellular ODN delivery. In the absence of serum, all cationic lipids tested with hydrocarbon chains in vicinal configuration esterified to a glycerol backbone (the respective N-(1,2-diacyl-dihydroxypropyl)-N,N,N-trimethyl-ammoniumchlorides or N-(1,2-diacyl-dihydroxypropyl)-N(hydroxyethyl)-N,N-dimethyl-ammoniumchlorides as well as N-(1,2-diacyl-dihydroxypropyl)-N(1,2-dihydroxypropyl)-N,N-dimethyl-ammoniumchlorides with lauroyl, myristoyl, palmitoyl, stearoyl and erucoyl chains) were able to transfect cells when combined with DOPE (20-80% nuclear fluorescence). Remarkably, only the analog esterified with two myristoyl chains was equally effective even in the absence of DOPE. By adding hydroxy groups to the N-alkyl residue, TE under serum conditions was improved yielding transfection rates of 55%, 75% and 90% for 0, 1 or 2 substituted hydroxy groups, respectively. For plasmid DNA, different requirements were identified. Again, the analog with two myristoyl chains was most effective but only in the presence of DOPE. However, the addition of hydroxy groups had no influence on the TE in the presence of serum.     

Development of a non-viral multifunctional envelope-type nano device by a novel lipid film hydration method.

The development of a multifunctional envelope-type nano device (MEND) for use in a non-viral gene delivery system using a novel lipid film hydration method is described. This packaging method involves three steps: (i) DNA condensation with a polycation, (ii) lipid film hydration for the electrostatic binding of the condensed DNA, and (iii) sonication to package the condensed DNA with lipids. For DNA condensation, the optimum ratio of DNA to poly-L-lysine (PLL) was determined by titrating DNA and PLL. The condensed DNA/PLL complex (DPC) was coated with a lipid bilayer by lipid film hydration followed by sonication, which was confirmed by sucrose density gradient centrifugation. Packaging of DPC with lipids increased the transfection activity 10-fold over that of DPC. MEND, having octaarginine on the envelope as a device for membrane penetration to enhance cellular uptake, showed a 1000-fold higher transfection activity than DPC. The MEND developed in this study represents a promising non-viral gene delivery system.     

Synthesis of a single-tailed cationic lipid and investigation of its transfection.

Single-tailed cationic lipids were originally reported to have low transfection efficiency and high toxicity in plasmid delivery. We hypothesized that particular single-tailed cationic lipids may also function in plasmid transfection. To test this hypothesis, we synthesized a new cationic lipid-oleoyl ornithinate (OLON). To decrease cytotoxicity, we then introduced a potential biodegradable ester bond in the tail of lipid yielding 6-lauroxyhexyl ornithinate (LHON). The data demonstrated that the cytotoxicity of LHON was lower than that of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or OLON. To investigate the transfection activity of the new lipids and determine the cellular uptake of DNA/liposome complexes, we compared the transfection of liposomes produced from double-tailed 1',2'-dioleyl-sn-glycero-3'-succinyl-1, 6-hexanediol ornithine conjugate (DOGSHDO) with an ornithine headgroup, single-tailed OLON with an ornithine head group, double-tailed DOTAP with quaternary amine group, and single-tailed cetyltrimethylammonium bromide (CTAB) with a quaternary amine group. At the optimal ratios as defined in transfection experiments, OLON/DOPE had more than 10 times the transgene expression than other liposomes even though the DNA uptake was not necessarily greater. In the experiments comparing the release of DNA from DNA/liposome complexes by anionic substances, a greater fraction of DNA was released from DNA/OLON/DOPE complexes than that from DNA/DOTAP/DOPE complexes.     

Modified Tat peptide with cationic lipids enhances gene transfection efficiency via temperature-dependent and caveolae-mediated endocytosis

The HIV-1 Tat peptide has been successfully used for intracellular gene delivery. Likewise, various lipid-based methods have shown increased endocytosis and can influence endosomal escape. This study combines the favorable properties of Tat peptide with that of lipid systems for DNA delivery. We combined the lipid FuGENE HD (FH) with the Tat peptide sequence modified with histidine and cysteine residues (mTat). mTat/FH transfection was evaluated by luciferase expression plasmid in five cell types. mTat/FH produced significant improvement in transfection efficiency of all cell lines when compared to FH or mTat. Treatment with chloroquine, associated with energy-dependent endocytosis, significantly increased transfection efficiency with mTat/FH while incubation at low temperature decreased it. The zeta potential of mTat/FH/DNA was significantly higher compared to FH, mTat, or their DNA combination in the presence of serum, and it was correlated with transfection efficiency. The particle size of the FH/DNA complex was significantly reduced by addition of mTat. Filipin III, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/FH transfection, but transfection was increased by chlorpromazine, an inhibitor of clathrin-mediated endocytosis. These findings demonstrated the feasibility of using a combination of mTat with lipids, utilizing temperature-dependent and caveolae-mediated endocytosis, as a potentially attractive non-viral gene vector.     

THP-1单核细胞不同转染方法的比较

背景：人单核细胞系THP-1细胞是典型的悬浮细胞系,其转染并瞬时表达外源蛋白比一般贴壁细胞相对困难。其中DEAE-葡聚糖介导的细胞转染方法虽然建立较早,但在国内应用很少。目的：采用不同质粒转染方法介导质粒绿色荧光蛋白真核表达质粒（pEGFP-N1）转染人THP-1单核细胞,以获得较高转染效率的方法。方法：分别采用DEAE-葡聚糖、Lipofectamine 2000、FuGENE 6、梭华-Sofast转染试剂不同方法介导质粒绿色荧光蛋白真核表达质粒进行转染,测定不同方法的转染效率、及其对细胞活力的影响。结果与结论：荧光显微镜下观察及流式细胞仪检测结果均显示DEAE-Dextran介导的转染效率最高,Lipfectamine 2000脂质体次之,FuGENE 6和梭华-Sofast则明显低于前二者。将质粒绿色荧光蛋白真核表达质粒体外成功转染人THP-1单核细胞中,通过优化转染方法提高转染效率、降低对细胞活力的影响。     

短发夹RNA诱导survivin基因缄默对Jurkat细胞凋亡和增殖的影响

目的研究应用载体表达短发夹 RNA(short hairpin RNA,shRNA)干扰技术抑制人 T 淋巴细胞白血病细胞株 Jurkat 细胞 survivin 基因的表达,探讨 survivin 基因表达缄默对 Jurkat 细胞凋亡和增殖的影响。方法构建针对 survivin 基因的 shRNA 重组质粒并转染至 Jurkat 细胞,分别用多重 PCR和 Western blot 法检测瞬时转染和稳定转染细胞 survivin 基因 mRNA 和蛋白表达水平的变化;流式细胞术检测瞬时转染和稳定转染细胞凋亡指数的变化,绘制细胞生长曲线,探讨 survivin shRNA 转染对细胞生长和增殖的影响。结果多重 PCR 结果示与无功能(对照组)shRNA 处理组和磷酸盐缓冲液处理组比较,survivin shRNA 瞬时转染和稳定转染细胞 surivivin 基因 mRNA 表达均显著下降,抑制率分别为66.675% 和60.69%(P<0.05);Western blot 结果显示 survivin shRNA 瞬时转染和稳定转染细胞survivin 蛋白表达水平亦显著降低,抑制率分别为63.41% 和60.18%(P<0.05)。流式细胞术检测瞬时转染和稳定转染细胞的凋亡率显著增加,分别为(22.41±2.83)% 和(20.73±2.56)%(与对照组比较,P均<0.05),细胞倍增时间显著延长。生长曲线显示稳定转染细胞的生长显著减慢。结论shRNA 重组质粒介导的 RNA 干扰能明显抑制 Jurkat 细胞 survivin 基因 mRNA 和蛋白产物的表达,诱导细胞凋亡和生长抑制。     

Extracellular and intracellular barriers in non-viral gene delivery.

Complexes of DNA with cationic lipids and cationic polymers are frequently used for gene transfer. Extracellular interactions of the complexes with anionic glycosaminoglycans (GAGs) may interfere with gene transfer. Interactions of GAGs with carrier DNA complexes have been studied using tests for DNA relaxation (ethidium bromide intercalation), DNA release (electrophoresis), and transfection (pCMVbGal transfer into RAA smooth muscle cells). Several cationic lipid formulations (DOTAP, DOTAP/Chol, DOTAP/DOPE, DOTMA/DOPE, DOGS) and cationic polymers (fractured dendrimer, polyethylene imines 25 and 800 kDa, polylysines 20 and 200 kDa) were tested. Polycations condensed DNA more effectively than monovalent lipids. Hyaluronic acid did not release or relax DNA in any complex, but it inhibited transfection by some polyvalent systems (PEI, dendrimers, DOGS). Gene transfer by other carriers was not affected by hyaluronic acid. Sulfated GAGs (heparan sulfate, chondroitin sulfates B and C) completely blocked transfection, except in the case of liposomes with DOPE. Sulfated GAGs relaxed and released DNA from some complexes, but these events were not prerequisites for the inhibition of transfection. Furthermore, preliminary results suggest that cell surface GAGs, particularly heparan sulfate, inhibit gene transfer by cationic lipids and polymers.     

Lipid synthesis in macrophages derived from the human cell line THP-1: modulation of the effects of native and oxidized chylomicron-remnant-like particles by oestrogen

The effects of native and oxidized chylomicron remnants on the synthesis of cholesteryl ester and triacylglycerol in macrophages, and the way that this is influenced by exposure of the cells to oestrogen, was investigated using the human monocyte cell line THP-1 and chylomicron-remnant-like particles containing human apolipoprotein E (CRLPs). Synthesis of the lipids was measured by the incorporation of [(3)H]oleate into cholesteryl ester and triacylglycerol. CRLPs (5-40 microgram of cholesterol/ml) containing either trilinolein or triolein as the triacylglycerol component caused a dose-dependent decrease in cholesteryl ester formation, while triacylglycerol production was unchanged. After oxidation of the CRLPs, the level of thiobarbituric acid-reactive substances was increased by 6.3-fold and 2.2-fold in particles containing trilinolein and triolein respectively. Furthermore, CRLPs containing oxidized trilinolein lost their ability to down-regulate cholesterol esterification, while CRLPs containing oxidized triolein did not. Both types of oxidized CRLPs decreased triacylglycerol synthesis. Treatment of the macrophages with 17beta-oestradiol caused increases of approx. 94% and 34% in the synthesis of cholesteryl ester and triacylglycerol respectively in the absence of CRLPs. The differences between control and oestrogen-treated cells were abolished, however, when CRLPs (40 microgram of cholesterol/ml) were added to the incubations. In addition, in contrast with their lack of effect in control cells, CRLPs containing oxidized trilinolein decreased cholesterol esterification in oestrogen-treated cells by approx. 48%. These findings with CRLPs suggest that chylomicron remnants have significant effects on cholesteryl ester and triacylglycerol synthesis in macrophages, which may be modulated both by the oxidation state of the particles and by oestrogen.     

Degradation and oxidation of methionine enkephalin by human neutrophils

Met5-enkephalin, tyr-gly-phe-met, is an endogenous pentapeptide, with morphine agonist activity. In this study, we demonstrated that met5-enkephalin was degraded with the release of tyrosine by resting human PMN, whereas it was degraded as well as oxidized to its sulfoxide derivative, met5-(O)-enkephalin, by phagocytosing PMN. PMN also degraded met5-(O)-enkephalin but to a lesser extent. Bacitracin at 1 gm/L inhibited the degradation and oxidation of met5-enkephalin without affecting the production of superoxide and viability of PMN. The oxidation of met5-enkephalin by phagocytosing PMN was inhibited by catalase or NaN3 but not by SOD. This suggests that the oxidation of met5-enkephalin by phagocytosing PMN was, at least in part, dependent on the MPO system (MPO-H2O2-halide). Using purified canine MPO, we further demonstrated that MPO-H2O2-CI- oxidized met5-enkephalin to met5-(O)-enkephalin. The MPO-mediated oxidation of met5-enkephalin was inhibited by methionine but not by methionine sulfoxide, tyrosine, glycine, or phenylalanine, confirming that it was the methionine moiety of met5-enkephalin which was oxidized. Since both the sulfoxide derivative and the degradation products met5-enkephalin have reduced opiate agonist activity, oxidation and degradation of met5-enkephalin by PMN may contribute to the pain at the site of inflammation. (J Lab Clin Med 99:418, 1982.)     

Protective effect of high density lipoprotein associated paraoxonase. Inhibition of the biological activity of minimally oxidized low density lipoprotein.

Our group has previously demonstrated that oxidized phospholipids in mildly oxidized LDL (MM-LDL) produced by oxidation with lipoxygenase, iron, or cocultures of artery wall cells increase monocyte-endothelial interactions and this sequence of events is blocked by HDL. To obtain further insight into the mechanism by which HDL abolishes the activity of MM-LDL we investigated the effect of the HDL-associated ester hydrolase paraoxonase (PON). Treatment of MM-LDL with purified PON significantly reduced the ability of MM-LDL to induce monocyte-endothelial interactions. Inactivation of PON by pretreating HDL with heat or EDTA reduced the ability of HDL to inhibit LDL modification. HPLC analysis of phospholipids isolated from MM-LDL before and after treatment with purified PON showed that the 270 nm absorbance of phospholipids was decreased, while no effect was observed on 235 nm absorbance. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC) and specific fractions of Ox-PAPC isolated by HPLC induced the same monocyte-endothelial interactions as did MM-LDL. Biologically active and inactive HPLC fractions of Ox-PAPC were compared by fast atom bombardment-mass spectrometry which revealed that active fractions possessed ions with a mass to charge [correction of change] ratio greater than native PAPC by multiples of 16 D suggesting the addition of 3 and 4 oxygen atoms to PAPC. Comparison of Ox-PAPC by fast atom bombardment-mass spectrometry before and after PON treatment showed that PON destroyed these multi-oxygenated molecules found in biologically active fractions of Ox-PAPC. These results suggest that PON in HDL may protect against the induction of inflammatory responses in artery wall cells by destroying biologically active lipids in mildly oxidized LDL.