脂质体介导C-erbB-2反义寡核苷酸转染小鼠乳腺癌TM40D细胞的效率及毒性

目的 探讨阳离子脂质体为载体的反义寡核苷酸（ODNs）转染小鼠乳腺癌TM40D细胞的最佳转染效率及对细胞的毒性作用。方法 分别将有／无脂质体介导的反义ODNs转染小鼠乳腺癌TM40D细胞,应用流式细胞仪观察不同时间的反义ODNs的转染效率,荧光显微镜观察反义ODNs在细胞内的分布,全自动生化分析仪检测转染培养上清液的乳酸脱氢酶（LDH）浓度。结果 无脂质体介导的反义ODNs转染细胞效率随着时间的延长增加,6h达到63．00％。脂质体介导的反义ODNs转染细胞4h时转染效率达到高峰为72．23％。细胞内荧光强度与细胞转染效率相关。无脂质体介导的FAM标记的反义ODNs分布在细胞浆,脂质体介导的反义ODNs在细胞浆及细胞核内均有分布。各组LDH浓度无统计学差异。结论 脂质体介导组反义ODNs转染效率高于无脂质体组,同时转染高峰提前出现。脂质体促进反义ODNs进入细胞核内。短时间内阳离子脂质体及反义ODNS对细胞的毒性不明显。     

脂质体转染bcl-2反义寡核苷酸G3139在HL-60细胞中的动力学模式

目的　应用阳离子脂质体介导或直接转染时 ,建立HL 6 0细胞摄入由 5 FITC标记的bcl 2反义寡核苷酸G3139的动力学模式及观察G 3139在细胞内的分布。方法　流式细胞仪测定细胞内的相对平均荧光强度 ,荧光显微镜观察细胞内的荧光分布。结果　①脂质体介导转染法显著提高HL 6 0细胞对G 3139的摄入 ,当DOSPER/G 3139(μg/μg)为 2 2 / 1时 ,细胞内相对平均荧光强度可达G 3139直接作用时的 40倍 ,而且HL 6 0细胞对G 3139的摄入跟G3139的浓度和作用时间有关。②细胞内的G 3139可向胞外转运 ,在DOSPER介导转染后 ,胞内荧光强度衰减缓慢 ,t1/ 2 约为 4h ,而G 3139直接作用后 ,t1/ 2 约为 0 5h ;③DOSPER介导转染 6h后 ,细胞内荧光物质主要以明亮的点状聚集在细胞核和部分细胞浆的细胞器中 ,而G 3139直接作用时 ,荧光物质则弥漫分布于胞浆中。结论　DOSPER可以增加G 3139的细胞摄入并改变其胞内分布 ,可能是阳离子脂质体增加bcl 2反义寡核苷酸生物活性的重要原因。     

脂质体转染bcl—2反义寡核苷酸G3139在HL—60细胞中的动力？…

目的 应用阳离子脂质体介导或直接转染时,建立ＨＬ－６０细胞摄入由５′－ＦＩＴＣ标记的ｂｃｌ－２反义寡核苷酸Ｇ３１３９的动力学模式及观察Ｇ３１３９在细胞内的分布。方法 流式细胞仪测定细胞内的相对平均荧光强度,荧光显微镜观察细胞内的荧光分布。结果 ①脂质体介导转染法显著提高ＨＬ－６０细胞对Ｇ３１３９的摄入,当ＤＯＳＰＥＲ／Ｇ３１３９（ｕｇ／ｕｇ）为２．２／１时,细胞内相对平均荧光强度可达Ｇ３１３９直接     

谷甾醇葡萄糖苷和卞泽双重修饰肝实质细胞靶向阳性脂质体介导的基因转染和抗乙肝病毒作用

目的研究载乙型肝炎病毒(HBV)反义寡核苷酸的双重表面修饰肝实质细胞靶向阳性脂质体的基因转染，抗乙肝病毒作用和其介导基因转染的机制。方法以3β-［N-(N′,N′-二甲氨基乙基)-氨甲酰基］胆固醇(DC-Chol)和二棕榈酰磷脂酰胆碱(DPPC)为脂材，分别以谷甾醇葡萄糖苷(sito-G)和卞泽(Brij 35)为膜表面修饰成分，制备载HBV反义寡核苷酸的阳性脂质体。采用大鼠原代肝实质细胞和人肝癌细胞HepG 2.2.15，通过流式细胞分析、荧光显微镜观察和酶联免疫吸附试验(ELISA)，考察脂质体对基因转染的促进作用及其病毒抑制作用；通过评价渥曼青霉素、尼日利亚菌素以及无涎胎球蛋白对其病毒抑制作用的影响，探讨其转染机制。结果以sito-G和Brij 35对脂质体进行双重表面修饰，显著提高了脂质体的转染率和病毒抑制作用；荧光显微镜下观察到较强转染，反义寡核苷酸的胞内分布以在细胞核中为主；渥曼青霉素、尼日利亚菌素和无涎胎球蛋白均不同程度地降低了载反义寡核苷酸脂质体的病毒抑制作用。结论Brij 35和sito-G双重修饰阳性脂质体显示出较高的基因转染效率和显著的病毒抑制作用，其基因转染过程以内吞和膜融合为主，并表现出肝实质细胞表面去唾液酸糖蛋白受体 (ASGPR)的靶向选择性。     

阳离子膜融合脂质体介导反义寡核苷酸在Hela细胞中的转染实验研究

目的研究阳离子膜融合脂质体(CFL)介导反义寡核苷酸(ASON)的细胞转染效率及影响因素。方法 逆相蒸发法制备3种不同阳离子含量的脂质体(CL),在CL上引入仙台病毒形成CFL,将制得的阳离子膜融合脂质体与反义寡核苷酸混合得到复合物,考察形态学及载药量,用MTT法考察该载体的细胞毒性,流式细胞仪测定阳性细胞百分率和平均荧光强度。结果制得的CFL形态均匀,粒径为(168±65) nm。载药量随着磷脂/ASON(+/-)电荷比增加而增加。CFL细胞毒性明显低于相同电荷比的CL,细胞转染效率是随阳离子含量、磷脂/ASON(+/-)电荷比增加而增加,血清和低温均对CFL的细胞转染有影响。结论阳离子膜融合脂质体作为载体在低电荷比条件下可降低细胞毒性并可提高细胞转染效率,可作为该ASON的给药系统而进一步研究。     

三种阳离子转染试剂对小鼠血管瘤内皮细胞转染效率及细胞毒性的影响

目的 从三种常用的阳离子转染试剂中筛选出对小鼠血管瘤内皮细胞(EOMA)有较高转染效率和较低细胞毒性的转染试剂.方法 以含有增强型绿色荧光蛋白(EGFP)报告基因的真核表达载体pEGFP-N1为报告基因,以阳离子脂质体LipofectamineTM2000、LipofectamineTM PLUS和阳离子聚合物JetPEITM为转染试剂,按照转染试剂盒的说明优化其转染条件,分别转染EOMA细胞,24 h后在荧光显微镜下计数阳性细胞率、MTT法检测各转染条件下对EOMA的细胞毒性.结果 经优化转染条件,LipofectamineTM PLUS和JetPEITM转染的细胞中阳性细胞的最高比例分别为45%和47%,LipofectamineTM2000转染的细胞中荧光蛋白表达的比例最高(＞80%),Lipofecta-mineTM2000试剂在最高转染效率的剂量下仍然保证了80%的细胞存活率.结论 阳离子脂质体LipofectamineTM2000对小鼠EOMA细胞有较高的转染效率和较低的细胞毒作用.     

反义寡核苷酸脂质体复合物性质对细胞摄入行为的影响

目的研究影响反义寡核苷酸脂质体复合物的性质和细胞摄取的因素。方法逆相蒸发法制备3种不同的空白脂质体,与反义寡核苷酸混合得到复合物,显微镜观察其形态,琼脂糖电泳分析载药量,流式细胞仪测定阳性细胞百分率和平均荧光强度。结果高电荷密度的脂质体和低离子强度介质可使复合物发生凝聚,载药量和细胞摄入量依赖于空白脂质体和药物的比例以及脂质体膜表面的电荷密度。结论阳离子脂质体可以提高载药量和细胞的摄入,其程度与复合物比例、脂质体膜表面电荷密度等有关。     

血管内皮生长因子反义核酸提高前列腺癌PC3细胞对化疗药物敏感性的研究

目的探讨血管内皮生长因子(VEGF)反义核酸对前列腺癌细胞PC3对常用化疗药物作用的影响,联合反义核酸和紫杉醇治疗裸鼠移植肿瘤。方法采用新型脂质体Oligofectamine携带VEGF反义核酸转染前列腺癌细胞PC3。定量反转录-聚合酶链反应(RT-PCR)和Western杂交的方法检测细胞VEGFmRNA和蛋白的表达,四甲基偶氮唑蓝法(MTT)检测转染反义核酸的细胞对化疗药物的敏感性,联合反义核酸和紫杉醇对裸鼠移植肿瘤进行治疗,观察肿瘤抑制率,计算药物相互作用指数(CDI)。结果新型脂质体携带VEGF反义核酸转染前列腺癌细胞PC3,与对照组和正义组比较,反义组细胞VEGFmRNA和蛋白的表达明显下降,对2种前列腺癌常用化疗药物紫杉醇和米托蒽醌的敏感性明显增强(P<0.05)。治疗裸鼠移植肿瘤28d后肿瘤生长抑制率分别为反义组58.5%,紫杉醇组60.2%,联合组84.1%,CDI=0.97。结论 VEGF反义寡核苷酸抑制VEGF的表达,提高细胞对化疗药物的敏感性,联合反义核酸和紫杉醇可以更有效地抑制肿瘤生长,并且2种药物具有协同作用。     

阳离子脂质材料和聚乙二醇对脂质体细胞转染率及膜流动性的影响阳离子脂质材料和聚乙二醇对脂质体细胞转染率及膜流动性的影响

目的制备包封荧光素钠（FS）的脂质体，考察阳离子脂质材料（DC-chol）和聚乙二醇（PEG）对脂质体包封率、细胞转染率及膜流动性的影响。方法以FS作为模型物质，制备并分离脂质体，测定脂质体包封率；通过观察荧光光谱的变化考察FS与脂质体膜之间的相互作用；以HepG₂ 2.2.15为细胞模型观察脂质体对FS细胞转染率的影响；通过荧光偏振技术考察阳离子脂质材料和PEG对脂质体膜流动性的影响。结果阳离子脂质材料和PEG能提高脂质体包封率（0.64％～86.57％）、细胞转染率（2.18％～48.46％）及脂质体膜流动性，PEG分子质量的增大有利于包封率、转染率的提高，并增加脂质体膜的流动性。结论在脂质体处方中加入阳离子脂质材料和高分子量的PEG有利于提高包封率、细胞转染率及增加脂质体膜的流动性。     

阴离子脂质体－阳离子脂质体复合物介导的基因转染

目的评价阴离子脂质体-阳离子脂质体复合物介导质粒转移至HepG2肝癌细胞中及其毒副作用。方法制备携载表达绿色荧光蛋白质粒的阳离子脂质体,与阴离子脂质体形成复合物。测定脂质体复合物的zeta电位,凝胶阻滞实验考察质粒包封情况,流式细胞仪测量各阴离子脂质体-阳离子脂质体复合物的转染效率,MTT法检测细胞毒性。结果复合物能完全包裹质粒,其zeta电位低于阳离子脂质体zeta电位;脂质体复合物介导的转染效率略低于阳离子脂质体,其细胞生存率高于阳离子脂质体。结论阴离子脂质体-阳离子脂质体复合物在降低细胞毒性的同时,可实现对HepG2细胞较高的转染效率。     

Effect of cationic liposomes on intracellular trafficking and efficacy of antisense oligonucleotides in mouse peritoneal macrophages

We have investigated the intracellular fate and antisense effect of oligonucleotide/cationic liposome complexes using phosphorothioate oligonucleotides (S-Oligo) targeted to inducible nitric oxide synthase in mouse peritoneal macrophages. Confocal laser microscopic analysis revealed that, after application of fluorescein isothiocyanate (FITC)-labeled S-Oligo alone, the intracellular localization of fluorescence exhibited a punctate pattern in the cytoplasm, suggesting that the oligonucleotides were mainly confined to the endosomal and/or lysosomal compartments. In the case of complexation with Lipofectin and DMRIE-C liposomes, cellular uptake of FITC-S-Oligo was not greatly enhanced and the fluorescence localization in the cells was similar to that of FITC-S-Oligo alone. LipofectAMINE slightly enhanced cellular uptake of FITC-S-Oligo; however, the intracellular localization profile of FITC-S-Oligo remained largely unchanged. The antisense effect was slightly enhanced by LipofectAMINE under only very limited experimental conditions. It was concluded that cationic liposomes are not a potential carrier for S-Oligo in peritoneal macrophages because of their inability to promote the release of S-Oligo from the endosomal compartments to the cytosol over a non-toxic concentration range.     

Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense oligonucleotides.

We have investigated the use of a cationic lipid preparation to enhance antisense oligonucleotide activity in human umbilical vein endothelial cells. A liposomal preparation containing the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) was found to increase by at least 1000-fold the potency of an antisense oligonucleotide (ISIS 1570) that hybridizes to the AUG translation initiation codon of human intercellular adhesion molecule-1. In the presence of 8 microM DOTMA, 6-15-fold more 35S-ISIS 1570 associated with cells, at oligonucleotide concentrations from 0.01 to 5 microM, than did in the absence of DOTMA. Both 35S-ISIS 1570 association with cells and antisense activity were increased as a function of DOTMA concentration and with increasing time of incubation with the cationic lipid. Fluorescein-labeled ISIS 1570 was used to assess the intracellular distribution of the oligonucleotide in the presence and absence of DOTMA. In the absence of DOTMA, the oligonucleotide localized to discrete structures in the cytoplasm of the cell, resulting in a punctate fluorescence pattern. In the presence of DOTMA, cellular fluorescence markedly increased and the oligonucleotide localized within the nucleus, as well as to discrete structures in the cytoplasm. Accumulation of the oligonucleotide in the nucleus in the presence of DOTMA was time and temperature dependent. Nuclear accumulation was inhibited by preincubation of the cells with monensin but not chloroquine, NH4Cl, nocodazole, colcemid, or brefeldin A. These data demonstrate that cationic lipids increase antisense activity by increasing the amount of oligonucleotide associated with cells and altering intracellular distribution of the oligonucleotide.     

Liposome-oligonucleotides interaction for in vitro uptake by COS I and HaCaT cells

Liposomes are considered very promising delivery systems for antisense therapeutic approach, offering drug protection and facilitating oligonucleotide cell internalization. The present study was aimed to investigate the influence of phospholipid composition of the liposomal systems both on the encapsulation and on the oligonucleotide carrier capacity in vitro. Liposomes composed of neutral (phosphatidylcholine, cholesterol and dioleoylphosphatidylethanolamine) and/or cationic lipids (N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride salt, DOTAP) with different molar ratios were complexed with 5' fluorescein conjugated 29-mer phosphorothioate oligonucleotide (PS-ODN). The interaction was evaluated using atomic force microscopy (AFM), gel electrophoresis and HPLC analysis. Cytofluorimetric analysis and fluorescence microscopy were applied to evaluate the uptake and intracellular distribution of fluorescently labelled PS-ODN after transfection in two cell lines, COS I (fibroblast cell) and HaCaT (immortalized keratinocyte cell). The AFM studies reveal that the liposome/PS-ODN interaction leads the formation of a new irregular structure that completely hides the PS-ODN. Gel electrophoresis experiments and HPLC analysis have clearly demonstrated that also neutral liposomes are able to keep a little amount of PS-ODN but without strain to the complexation; the interaction was weak and rapidly destabilized when the complex was added to the cells.Transfection experiments performed with different incubation times show that DOTAP liposomes increase the rate of cellular uptake of PS-ODN and seem to influence its intracellular distribution in COS I cells where the oligonucleotide looks localized in nucleoli. Similar behaviour, at a lesser extent, is exhibited in HaCaT cells.     

Monosialoganglioside containing cationic liposomes with a cationic cholesterol derivative promote the efficiency of gene transfection in mammalian culture cells

We have studied the effects of monosialoganglioside (GM1)-containing cationic liposomes with a cationic cholesterol on the liposome-mediated gene transfection into mammalian culture cells. The results showed that both cationic liposomes with either a cationic cholesterol derivative of a hydrophobic amino head group (I) and a hydrophilic amino head group (II) promoted the transfection of luciferase plasmids (pGL3) into HeLa and CHO-K1 cells more than the control cationic liposomes without GM1. In addition, we found that cationic liposomes with a cationic cholesterol derivative (II) were about ten times as effective as that by commercially available cationic liposome Lipofectin. Confocal fluorescence microscopy showed that the liposome/DNA complex was transferred more efficiently into the target cells by the GM1-containing liposomes than by the liposomes without GM1. In proportion to the above results, free antisense DNAs were also more efficiently transferred into the nucleus of the target cells by the GM1-containing liposomes. When there was 100 mM galactose in the transfection medium, the luciferase activity by the GM1-containing liposomes was reduced to the level of the control liposomes. The results suggest that GM1-containing cationic liposomes with a cationic cholesterol derivative of a hydrophobic amino head group or a hydrophilic amino head group should significantly increase the transfection efficiency of plasmid DNAs and antisense DNAs by galactose receptor-mediated endocytosis. This means that the GM1-containing liposomes described here should be very promising for gene transfection in vitro.     

Studies on uptake, sub-cellular trafficking and efflux of antisense oligodeoxynucleotides in glioma cells using self-assembling cationic lipoplexes as delivery systems

The cellular uptake of antisense oligodeoxynucleotides (ODNs) may be enhanced by the use of carriers such as cationic liposomes or lipoplexes, but little is known about the intracellular fate and subcellular trafficking of these systems in target cells. In this study, we report on the cellular uptake and biodistribution of ODNs in the presence and absence of optimised self-assembled cationic lipoplexes using the C6 glioma cell line as an in vitro model. Biotin or radiolabelled 15-mer phosphorothioate (PS) ODNs were synthesised and their cellular uptake and subcellular biodistribution characterised in the presence and absence of an optimised cationic lipoplex delivery system using studies ranging from cellular association, cellular efflux and transmission electron microscopy (TEM). Ultrastructural studies clearly showed PS ODNs in the absence of liposomal delivery to be sequestered within endosomal and lysosomal vesicular bodies indicative of endocytic uptake. ODNs were also visible, to a lesser extent, in the nucleus and cytoplasm. By employing DOSPA (2'-(1",2"-dioleoyloxypropyldimethyl-ammonium bromide)-N-ethyl-6-amidospermine tetra trifluoroacetic acid) and DOPE (dioleoylphosphatidylethanolamine) complex in a 3 : 1 ratio, as a delivery system for ODNs at a optimal lipid/DNA charge ratio of 1 : 1, the level of ODN cellular association was significantly increased by approximately 10-12 fold with a concomitant change in subcellular distribution of PS ODN. TEM studies indicated enhanced penetration of ODN within the cytosol and the cell nucleus with reduced presence in vesicular compartments. Efflux studies confirmed that cationic lipoplexes promoted entry of ODNs into 'deeper' cellular compartments, consistent with endosomal release. Optimised cationic lipoplexes improved cellular delivery of ODNs by enhancing cell association, uptake and by favourably modulating the intracellular trafficking and distribution of ODNs into non-vesicular compartments including the cytosol and nucleus.     

Efficient telomerase inhibition in human non-small cell lung cancer cells by liposomal delivery of 2'-O-methyl-RNA

The antisense oligonucleotide 2'-O-methyl-RNA is a selective telomerase inhibitor targeting the telomerase RNA component and represents a potential candidate for anticancer therapy. The poor cellular uptake of 2'-O-methyl-RNA is a limiting factor that may contribute to the lack of functional efficacy. To improve delivery of 2'-O-methyl-RNA and consequently antitumoral efficiency in human lung cancer cells, we have investigated several transfection reagents. The transfection reagents DOTAP, MegaFectin 60, SuperFect, FuGENE 6 and MATra-A were tested for intracellular delivery. A FAM-labeled 2'-O-methyl-RNA was used to assess the intracellular distribution by confocal laser scanning microscopy in A549 human non-small cell lung cancer cells. Telomerase activity was measured using the telomeric repeat amplification protocol. Cell viability after transfection was quantified by the MTT assay. All transfection reagents enhanced 2'-O-methyl-RNA uptake in A549 cells but the cationic lipid reagents DOTAP and MegaFectin 60 were most efficient in the delivery of 2'-O-methyl-RNA resulting in telomerase inhibition. Among both DOTAP exhibited the lowest cytotoxicity. Our experiments show that DOTAP is the most suitable transfection reagent for the delivery of 2'-O-methyl-RNA in human lung cancer cells according to its relatively low cytotoxicity and its ability to promote efficient uptake leading to the inhibition of telomerase.     

INCREASED APOPTOSIS,CHANGES IN INTRACELLULAR Ca2+, AND FUNCTIONAL ALTERATIONS IN LYMPHOCYTES AND MACROPHAGES AFTER IN VITRO EXPOSURE TO STATIC MAGNETIC FIELD

Electromagnetic-related alteration of cellular functions is well documented for extremely low-frequency low-energy pulsing electromagnetic fields (ELF-EMF). In this study we examined the in vitro effects of static magnetic fields (SMF) on the cellular immune parameters of the C57Bl/ 6 murine macrophages, spleen lymphocytes, and thymic cells. The cells were exposed in vitro for 24 h at 37 C, 5% CO2, to 250-1500 G SMF. Exposure to the SMF resulted in the decreased phagocytic uptake of fluorescent latex microspheres, which was accompanied by an increased intracellular Ca2+ level in macrophages. Exposure to SMF decreased mitogenic responses in lymphocytes, as determined by incorporation of \[3H]thymidine into the cells. This was associated with the increased Ca2+ influx in concanavalin A-stimulated lymphocytes. Furthermore, exposure to SMF produced markedly increased apoptosis of thymic cells, as determined by flow cytometry. Overall, in vitro exposure of immunocompetent cells to 250-1500 G SMF altered several functional parameters of C57Bl/6 murine macrophages, thymocytes, and spleen lymphocytes.