共查询到20条相似文献,搜索用时 15 毫秒
1.
Gene transfer into primary rat hepatocytes was performed by employing cationic liposome as DNA carrier and the specific ligand of hepatic asialoglycopmtein receptor (ASGPR), asialofetuin, as liver-targeting ligand. The results showed that asialofetuin, when added to the gene transfer complexes, could significantly increase the hepatocyte transfection efficiency, and alleviate the cellular toxicity of Lipofectin. Several synthetic ligands of ASGPR (galactosyl albumin) could also increase the transfection efficiency of hepatocyte like asialofetuin. It was proved that ASGPR and cationic liposome could synergistically mediate the gene transfer into primary rat hepatocytes. This novel gene delivery system provided a safer, more simple and efficient gene transfer method for primary hepatocytes, and showed prospecting application in hepatic gene therapy. 相似文献
2.
《Bioorganic & medicinal chemistry》2014,22(19):5212-5219
Highly efficient drug carriers targeting hepatocyte is needed for treatment for liver diseases such as liver cirrhosis and virus infections. Galactose or N-acetylgalactosamine is known to be recognized and incorporated into the cells through asialoglycoprotein receptor (ASGPR) that is exclusively expressed on hepatocyte and hepatoma. In this study, we synthesized a galactose-modified lipid with aromatic ring with click chemistry. To make a complex with DNA, termed ‘lipoplex’, we prepared a binary micelle composed of cationic lipid; dioleoyltrimethylammoniumpropane (DOTAP) and galactose-modified lipid (D/Gal). We prepared lipoplex from plasmid DNA (pDNA) and D/Gal and examined the cell specificity and transfection efficiency. The lipoplex was able to interact with ASGPR immobilized on gold substrate in the quartz-crystal microbalance (QCM) sensor cell. The lipoplex induced high gene expression to HepG2 cells, a human hepatocellular carcinoma cell line, but not to A549 cells, a human alveolar adenocarcinoma cell line. The treatment with asialofetuin, which is a ligand for ASGPR and would work as a competitive inhibitor, before addition of the lipoplexes decreased the expression to HepG2 cells. These results indicate that D/Gal lipoplex was incorporated into HepG2 cells preferentially through ASGPR and the uptake was caused by galactose specific receptor. This delivery system to hepatocytes may overcome the problems for gene therapy and be used for treatment of hepatitis and hepatic cirrhosis. 相似文献
3.
Gene delivery into human hepatocytes remains a critical issue for the development of liver-directed gene therapy. Gene delivery based on non-viral vectors is an attractive approach relative to viral vectors. In this report, novel delivery system of preS/liposome/DNA virus-like particle (VLP) was developed for gene transfection into hepatocytes in vivo and in vitro. Plasmid pCMVbeta, expressing beta-galactosidase, was encapsulated with cationic liposome, and then the histidine-tagged preS domain of hepatitis B virus was coated on the surface of liposome/DNA to form preS/liposome/ DNA VLP. Transfection efficiencies of preS/liposome/DNA, liposome/DNA, naked DNA and preS were analyzed using several different human cell lines. The highest transfection efficiency was found using preS/liposome/DNA VLP as the transfection reagent in human hepatocyte (HH) cell line. Results show that preS domain of hepatitis B virus coated on liposome/DNA can be used for highly efficient gene transfection into human hepatocytes. Moreover, the target characteristic of preS/liposome/DNA was analyzed in vivo. After preS/liposome/DNA VLP was injected into immunocompromised (Nude) mice via the tail vein, most of beta-galactosidase was expressed in the liver; however, no significant target expression was found with the injection of liposome/ DNA or naked DNA. Our results show that preS/liposome/DNA VLP can be used as a novel liver-specific gene delivery system. 相似文献
4.
阳离子脂质体转染人类骨骼肌原代细胞的初步研究 总被引:1,自引:0,他引:1
探讨不同脂质体介导基因转染人类骨骼肌原代细胞的转染效率和基因的表达.将含有β-半乳糖苷酶LacZ结构基因的质粒,用三种不同的阳离子脂质体导入人类骨骼肌原代细胞中,通过X-Gal染色观察不同的转染效率.结果发现,Fugene 6转染效率最高,蓝染细胞达10%,其脂质体与DNA的最佳比例为3∶ 2.Fugene 6可有效地将外源基因导入骨骼肌原代细胞,而且外源基因可以长效高效地表达,有望用来作为基因治疗的载体. 相似文献
5.
We describe the synthesis of a series of alpha-galacto-omega-polyamine double-chain bolaamphiphiles (Gal-CL) and report on the gene transfer mediated with lipoplexes they form either when used in conjunction with DOPE or with pcTG90:DOPE. Lipofection was investigated with human HepG2 and murine BNL-CL2 hepatocytes expressing the asialoglycoprotein (ASGP) receptor which displays a high affinity for galactosyl residues, and with A549 cells which do not express ASGP. Our results show that cationic N/P = 5 and 2.5 Gal-CL lipoplexes constitute very efficient nonspecific gene transfer systems. Lipofection experiments performed in the presence of asialofetuin (a high affinity ligand of ASGP) led us to evidence also the involvement of a specific receptor-mediated endocytosis pathway for the transfection of the ASGP(+) HepG2 or BNL-CL2 hepatocytes with some Gal-CL formulations. This work suggests that targetable lipopolyamines presenting a single galactose residue appear as promising synthetic vectors for specific gene delivery to ASGP(+) cells. 相似文献
6.
肝细胞表面去唾液酸糖蛋白受体的流式细胞分析 总被引:3,自引:0,他引:3
建立肝细胞表面去唾液酸糖蛋白受体(ASGPR)的流式细胞分析方法(FCM),对正常及损伤鼠肝细胞、肝癌细胞(BEL-7402)表面的ASGPR作同步比较分析.以异硫氰酸荧光素标记的新半乳糖白蛋白(FITC-NGA)为ASGPR的特异性配体,以培养的正常肝细胞(L-02)为靶细胞,建立肝细胞表面ASGPR的FCM.测定并计算正常及损伤鼠肝细胞,BEL-7402细胞与同一浓度的FITC-NGA同步反应后的平均荧光强度(MIF)值.FITC-NGA与L-02细胞表面ASGPR趋近饱和结合的浓度为0.4 mg/L,该浓度下正常及损伤鼠肝细胞,BEL-7402细胞的MIF值分别为228.7、5.81、1.13.该结合可以被至少50倍于FITC-NGA的NGA或10 mmol/L的EDTA完全抑制.FCM能够良好地揭示FITC-NGA同ASGPR之间的受配体结合特性.该方法证实BEL-7402细胞表面几乎没有ASGPR,损伤鼠肝细胞表面ASGPR的数量较正常鼠肝细胞显著减少. 相似文献
7.
Stearylated octaarginine and artificial virus-like particles for transfection of siRNA into primary rat neurons
下载免费PDF全文
![点击此处可从《RNA (New York, N.Y.)》网站下载免费的PDF全文](/ch/ext_images/free.gif)
RNA interference (RNAi) provides a powerful experimental tool for sequence-specific gene silencing, allowing efficient analysis of gene function in a multitude of cell types. However, application of RNAi in primary mammalian neurons has been limited by low-transfection efficiency and considerable toxicity of conventional transfection methods. In this study, we evaluated a peptide-mediated and a polymer/lipid-based cellular delivery method for siRNA into rat primary neurons and compared the results with a commonly used liposomal transfection reagent. Stearylated octaarginine (Stearyl-R8) was used as polypeptide and artificial virus-like particles (AVPs) were used as a combined liposomal-polymeric vector, since both reagents have been previously shown to successfully transfect DNA into cell lines. Stearyl-R8 and AVPs both promoted siRNA transfection into primary hippocampal neurons via the endosomal pathway. SiRNA-mediated gene silencing could be effectively induced in primary neuron cultures. In comparison with the commonly used cationic liposome transfection agent, both novel reagents were less detrimental to cell metabolic activity. We conclude that these novel transfection methods yield performances comparable to cationic liposome-mediated transfection for siRNA, while being less cytotoxic in primary neurons. Stearyl-R8 and AVPs may therefore represent novel and more cost-efficient alternatives to conventional siRNA-transfection reagents. 相似文献
8.
Cell-specific DNA delivery offers a great potential for targeted gene therapy. Toward this end, we have synthesized a series of compounds carrying galactose residues as a targeting ligand for asialoglycoprotein receptors of hepatocytes and primary amine groups as a functional domain for DNA binding. Biological activity of these galactosyl compounds in DNA delivery was evaluated in HepG2 and BL-6 cells and compared with respect to the number of galactose residues as well as primary amine groups in each molecule. Transfection experiments using a firefly luciferase gene as a reporter revealed that compounds with multivalent binding properties were more active in DNA delivery. An optimal transfection activity in HepG2 cells requires seven primary amine groups and a minimum of two galactose residues in each molecule. The transfection activity of compounds carrying multi-galactose residues can be inhibited by asialofetuin, a natural substrate for asialoglycoprotein receptors of hepatocytes, suggesting that gene transfer by these galactosyl compounds is asialoglycoprotein receptor-mediated. These results provide direct evidence in support of our new strategy for the use of small and synthetic compounds for cell specific and targeted gene delivery. 相似文献
9.
Lysosomes can fuse with a late endosomal compartment in a cell-free system from rat liver 总被引:8,自引:6,他引:2
下载免费PDF全文
![点击此处可从《The Journal of cell biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
《The Journal of cell biology》1994,126(5):1173-1182
The passage of pulse doses of asialoglycoproteins through the endosomal compartments of rat liver hepatocytes was studied by subcellular fractionation and EM. The kinetics of disappearance of radiolabeled asialofetuin from light endosomes prepared on Ficoll gradients were the same as the kinetics of disappearance of asialoorosomucoid-horse radish peroxidase reaction products from intracellular membrane-bound structures in the blood sinusoidal regions of hepatocytes. The light endosomes were therefore identifiable as being derived from the peripheral early endosome compartment. In contrast, the labeling of dense endosomes from the middle of the Ficoll gradient correlated with EM showing large numbers of reaction product-containing structures in the nonsinusoidal parts of the hepatocyte. In cell-free, postmitochondrial supernatants, we have previously observed that dense endosomes, but not light endosomes, interact with lysosomes. Cell-free interaction between isolated dense endosomes and lysosomes has now been reconstituted and analyzed in three ways: by transfer of radiolabeled ligand from endosomal to lysosomal densities, by a fluorescence dequenching assay which can indicate membrane fusion, and by measurement of content mixing. Maximum transfer of radiolabel to lysosomal densities required ATP and GTP plus cytosolic components, including N-ethylmaleimide-sensitive factor(s). Dense endosomes incubated in the absence of added lysosomes did not mature into vesicles of lysosomal density. Content mixing, and hence fusion, between endosomes and lysosomes was maximal in the presence of cytosol and ATP and also showed inhibition by N-ethyl-maleimide. Thus, we have demonstrated that a fusion step is involved in the transfer of radiolabeled ligand from an isolated endosome fraction derived from the nonsinusoidal regions of the hepatocyte to preexisting lysosomes in a cell-free system. 相似文献
10.
《Journal of receptor and signal transduction research》2013,33(5-6):243-271
AbstractWe have purified woodchuck hepatic asialoglycoprotein receptor (ASGPR) by ligand affinity chromatography and have identified it as a heterooligomeric complex comprised of two subunits with molecular masses of 40 and 47 kD, designated as woodchuck hepatic lectin 1 and 2 (WHL1 and WHL2), respectively. With the help of antisera generated against the soluble, bioactive woodchuck and rabbit ASGPRs and anti-subunit monospecific antibodies, distinct antigenic specificity of each of the ASGPR polypeptide subunits and interspecies immunologic cross-reactivity of the receptor polypeptides displaying comparable molecular masses were documented. In contrast to the purified woodchuck receptor, WHL2 antigenic reactivity was not identifiable in woodchuck hepatocyte plasma membranes unless the intact membranes were exposed to an asialylated ligand or a soluble membrane fraction was incubated with anti-receptor antibody. These findings imply that both WHL1 and WHL2 are expressed on the hepatocyte surface and contribute to ligand binding, since antibody specific to either subunit blocks ligand attachment. Our results also indicate that ligand binding modifies antigenic properties of the membrane expressed ASGPR. 相似文献
11.
12.
肝细胞靶向pH敏脂质体的制备及性质分析 总被引:3,自引:0,他引:3
为了制备具有肝细胞特异靶向性和pH敏感性的脂质体,设计并合成了四种带有半乳糖残基的导向分子,与具有pH敏感性的DC-chol/DOPE混合制备脂质体,通过质粒转染实验、受体竞争抑制实验和红细胞溶血等实验选出最佳转染活性的十八醇-半乳糖甙(18-gal)脂质体,并证明其具有肝细胞特异受体介导的靶向性和pH敏感性,且细胞毒性较小,可以作为一种潜在的肝细胞靶向转运系统得到进一步发展. 相似文献
13.
Adenoviral transfection of hepatocytes with the thioredoxin gene confers protection against apoptosis and necrosis 总被引:3,自引:0,他引:3
Tsutsui T Koide H Fukahori H Isoda K Higashiyama S Maeda I Tashiro F Yamato E Miyazaki J Yodoi J Kawase M Yagi K 《Biochemical and biophysical research communications》2003,307(4):765-770
A recombinant adenovirus vector containing the human thioredoxin (TRX) gene was constructed using the Cre-loxP recombination system and used to transfect rat hepatocytes with very high efficiency. The TRX gene was expressed in a dose-dependent manner and significantly modulated rat cellular functions. The TRX gene conferred resistance to oxidative stress, such as hydrogen peroxide treatment, on the host hepatocytes. FACS analysis of DNA fragmentation showed that the TRX gene suppressed hepatocyte apoptosis. It also significantly extended the life span of hepatocytes cultured conventionally on polystyrene plates. Liver-specific functions were maintained in the viability-modulated hepatocytes. Moreover, TRX expression did not affect hepatocyte spheroid formation and it extensively suppressed necrosis in the internal cells. Thus, the transfection of hepatocytes with the TRX gene successfully confers global maintenance of liver functions. These findings provide important information for the development of bioartificial liver support systems and gene therapy for liver diseases. 相似文献
14.
The use of asialo GM1-containing small unilamellar liposome preparations in vivo caused a 2.8-fold increase in the uptake by the liver as compared with the control (neutral) preparations (without asialo GM1). The uptake of negatively charged dicetylphosphate and dipalmitoyl phosphatidic acid-containing small unilamellar liposomes was found to be 1.6-and 1.8-fold respectively higher than that of the neutral preparations. In studies with isolated liver cell types, inhibition of the galactosylated liposome uptake by asialofetuin indicated a possible involvement of hepatic galactose receptors in the recognition of asialo GM1 liposomes by the hepatic parenchymal cells, which in turn were found to be mainly responsible for the enhanced incorporation of these liposomes in the liver. Sub-cellular distribution studies with isolated liver cell types indicated lysosomal localization of the liposomes both in parenchymal and nonparenchymal cells, and it has been proposed that the asialo GM1 liposomes are cointernalized with asialofetuin through a common lysosomal route of ligand internalization. 相似文献
15.
A novel cholesterol-based cationic lipid containing a tri-2- hydroxyethylamine head group and ether linker (Chol- THEA) was synthesized and examined as a potent gene delivery vehicle. In the preparation of cationic liposome, the addition of DOPE as helper lipid significantly increased the transfection efficiency. To find the optimum transfection efficiency, we screened various weight ratios of DOPE and liposome/DNA (N/P). The best transfection efficiency was found at the Chol-THEA:DOPE weight ratio of 1:1 and N/P weight ratio of 10~15. Most of the plasmid DNA was retarded by this liposome at the optimum N/P weight ratio of 10. The transfection efficiency of Chol-THEA liposome was compared with DOTAP, Lipofectamine, and DMRIE-C using the luciferase assay and GFP expression. Chol-THEA liposome with low toxicity had better or similar potency of gene delivery compared with commercial liposomes in COS-7, Huh-7, and MCF-7 cells. Therefore, Chol-THEA could be a useful non-viral vector for gene delivery. 相似文献
16.
The handling of hepatocytes, a major cell population in the liver, is an important technique in both liver tissue engineering and hepatology. However, these cells are so fragile that it has been impossible to harvest hepatocytes with high viability from tissue culture dishes after a period of culture in vitro. In this study, we employed an artificial substrate for transfection of multilayer hepatocytes and harvested these cells with high viability after transfection. Hepatocytes cultured on an amphiphilic artificial substrate form multilayer aggregates (spheroids) in the presence of growth factors during gene transfection with cation liposomes. Compared to cells cultured on a collagen-coated plate, these spheroids are easily harvested with high viability by pipetting in EDTA solution. In addition, these spheroids rapidly spread on collagen after transfer from the artificial substrate, demonstrating that hepatocytes in the center of the spheroids were viable. Epidermal growth factor (EGF) increased the transfection efficiency into hepatocytes while hepatocyte growth factor (HGF) alone did not increase the efficiency. However, HGF synergestically increased the effect of EGF on transfection. Interestingly, this transfection required the process of spheroid formation because the gene was not transfected once the spheroid formation completed or under conditions where hepatocytes did not form spheroids. This method using spheroidal hepatocytes for in vitro transfection is promising for the development of ex vivo gene therapy. 相似文献
17.
Guillot C Coathalem H Chetritt J David A Lowenstein P Gilbert E Tesson L van Rooijen N Cuturi MC Soulillou JP Anegon I 《Journal of immunology (Baltimore, Md. : 1950)》2001,166(8):5225-5235
The putative role of IL-4 in human and animal models of hepatitis has not yet been directly determined. We now report that direct expression of IL-4 in the liver of rats or mice using recombinant adenoviruses coding for rat or mouse IL-4 (AdrIL-4 and AdmIL-4, respectively) results in a lethal, dose-dependent hepatitis. The hepatitis induced by IL-4 was characterized by hepatocyte apoptosis and a massive monocyte/macrophage infiltrate. IL-4-induced hepatitis was independent of T cell-mediated immune responses. Hepatitis occurred even after gene transfer of IL-4 into nude rats, CD8-depleted rats, cyclosporine A-treated rats, or recombinase-activating gene 2(-/-) immunodeficient mice. Peripheral depletion of leukocytes using high doses of cyclophosphamide, and/or the specific depletion of liver macrophages with liposome-encapsulated dichloromethylene diphosphonate in rats did not block lethal IL-4-induced hepatitis. Direct transduction of hepatocytes with adenoviruses was not essential, since injection of AdrIL-4 into the hind limb induced an identical hepatitis. Finally, primary rat hepatocytes in culture also showed apoptosis when cultured in the presence of rIL-4. IL-4-dependent hepatitis was associated with increases in the intrahepatic levels of IFN-gamma, TNF-alpha, and Fas ligand. Administration of AdmIL-4 to IFN-gamma, TNF-alpha receptor type I, or TNF-alpha receptor type II knockout mice also resulted in lethal hepatitis, whereas a moderate protection was observed in Fas-deficient lpr mice. IL-4-dependent hepatocyte apoptosis could be abolished by treatment with caspase inhibitory peptides. Our results thus demonstrate that IL-4 causes hepatocyte apoptosis, which is only partially dependent on the activation of Apo-1-Fas signaling and is largely independent of any immune cells in the liver. 相似文献
18.
去唾液酸糖蛋白受体(ASGPR)是肝细胞特异性表达的受体,且是一种高效的内吞型受体,去唾液酸糖蛋白、半乳糖、半乳糖胺、N-乙酰半乳糖胺等糖分子对其有高亲和性.该综述回顾了ASGPR的发现历程、结构特征、生物学功能、表达模式、胞吞特点.总结了影响ASGPR与其配体亲和、介导胞吞的影响因素(包括配体类型、触角数量、空间距离与颗粒粒径).概述了早期ASGPR与其特异性配体在药物递送中的应用.最后介绍了最近利用N-乙酰半乳糖胺的缀合或修饰来实现肝靶向核酸药物递送的研究进展. 相似文献
19.
Serikawa T Suzuki N Kikuchi H Tanaka K Kitagawa T 《Biochimica et biophysica acta》2000,1467(2):419-430
Cationic liposomes are useful to transfer genes into eukaryotic cells in vitro and in vivo. However, liposomes with good transfection efficiency are often cytotoxic, and also require serum-free conditions for optimal activity. In this report, we describe a new formulation of cationic liposome containing DC-6-14, O,O'-ditetradecanoyl-N-(alpha-trimethylammonioacetyl)diethan olamine chloride, dioleoylphosphatidylethanolamine and cholesterol for gene delivery into cultured human cells. This liposome, dispersed in 5% serum-containing growth medium, efficiently delivered a plasmid DNA for GFP (green fluorescent protein) into more than 80% of the cultured human cell hybrids derived from HeLa cells and normal fibroblasts. Flow cytometric analysis revealed that the efficiency of the GFP gene expression was 40-50% in a tumor-suppressed cell hybrid, while it was greatly reduced in the tumorigenic counterpart. The enhanced GFP expression in tumor-suppressed cell hybrids was quantitatively well correlated with a prolonged presence of the plasmid DNA, which had been labeled with another fluorescent probe, ethidium monoazide, within the cells. These results suggest that a newly developed cationic liposome is useful for gene delivery in serum-containing medium into human cells and the stability of the plasmid DNA inside the cell is a crucial step in this liposome-mediated gene expression. The mechanisms by which cationic liposome mediates gene transfer into eukaryotic cells are also discussed. 相似文献
20.
The asialoglycoprotein receptor (ASGPR) is a potential target in the search for hepatic cancer drugs. However, application of ASGPR targeting in the clinic is limited by inefficient synthetic methods for the ligand. In this study, we designed and synthesized a novel galactosylated lipid with a mono-galactoside moiety using a lipase. Then we investigated the optimal reaction conditions and analyzed the targeting ability of liposomes modified with the galactosylated lipid. In an organic phase system, different lipases were used as catalysts to synthesize (5-cholesten-3b-yl) [(4-O-β-D-galactopyranosyl)D-glucitol-6] sebacate (CHS-SE-LA). Variables in enzymatic esterification, such as the type of enzyme and solvent, were explored by single-factor experiments. Optimal reaction conditions were determined through response surface methodology. The (CHS-SE-LA)-incorporated galactosylated liposome containing fluorescent dye was then prepared by thin-film hydration and a HepG2 cell transfection test used to confirm the targeting efficiency of galactosylated liposomes to hepatic cancer cells. The structure of CHS-SE-LA was identified by electrospray ionization or ESI and nuclear magnetic resonance or NMR. Under optimal conditions, the predicted yield of CHS-SE-LA was 94.3%, and the actual experimental value was 95.6 ± 1.35%, n = 3. For HepG2 cells, the cellular fluorescence intensities of liposomes modified with CHS-SE-LA (galactosylated liposomes [GAL-FL]) were as much as 2.6-fold (P < 0.01) the control liposomes (FL). Moreover, the presence of excess galactose significantly inhibited the uptake of GAL-FL suggesting ASGPR mediated uptake. The novel galactosylated ligand was synthesized enzymatically with high efficiency under mild conditions. Liposomes containing CHS-SE-LA have great potential as drug delivery carriers for hepatocyte-selective targeting. 相似文献