阳离子脂质体介导基因转染肿瘤细胞

使用基因转运载体运载肿瘤细胞进行转染是基因治疗的关键环节之一。Lipo-fectamine2000和DOTAP作为商品转染试剂,具有较高的转染效率。为了进一步发掘其作为基因转运载体的应用潜力,该文研究了Lipofectamine2000和DOTAP的粒径、Zeta电位及形态,并分别与绿色荧光蛋白基因（pGFP—N2）、荧光素酶基因（pGL3）结合,形成脂质体／DNA复合物,通过载入人喉癌细胞（Hep-2）和人肺癌细胞（NCI—H460）,考察了其转染效率和细胞毒性。结果表明,脂质体Lipofectamine2000与DOTAP都能有效压缩DNA,形成复合物。Lipofectamine2000与DOTAP井目比,转染效率高,与DNA最佳转染比例范围为2：1～4：1。毒性实验显示,在N／P大于3／l时,Lipofectamine2000与DOTAP对癌细胞具有一定的细胞毒性。细胞种类对脂质体的转染效率有很大影响,Lipo—fectamine2000对Hep－2细胞的转染效率比NcI—H460高。     

依赖RNA的RNA聚合酶介导RNA干扰的扩增效应

RNA干扰(RNA interference,RNAi)是一种非常高效的基因沉默效应,RNA依赖性RNA聚合酶(RNA—de—pendent RNA polymerase,RdRP)介导的扩增作用可能是RNAi具有高效性的一个主要原因。了解RdRP在生物体中存在的证据、RdRP及其复合体的结构、次级siRNA的产生及转移性RNAi的发生机制等问题,对深入理解RNAi的作用机制和促进RNAi的临床应用有重要意义。     

阳离子脂质体介导基因转染的最优化条件

阳离子脂质体介导基因转染的最优化条件王瓞林其谁（中国科学院上海生物化学研究所分子生物学国家重点实验室,上海２０００３１）关键词阳离子脂质体基因转染表达真核细胞阳离子脂质体转染的重要参数有：脂质体浓度和ＤＮＡ的浓度、细胞密度以及脂质体－ＤＮＡ复合物与细...     

基于RNA干扰CMTM7 对肺腺癌A549细胞凋亡的影响

目的:探讨基于RNA干扰CMTM7对肺腺癌A549细胞凋亡的影响。方法:选择肺腺癌A549细胞株分为si RNA组、阴性对照组与空白对照组,在对数生长的A549细胞中转染RNA干扰CMTM7载体、脂质体空载体和不进行转染,观察A549细胞的生长、凋亡与细胞周期状况。结果:MTT实验显示si RNA转染组的抑制率明显高于阴性对照组和空白对照组(P0.05),阴性对照组与空白对照组的对比无明显差异(P0.05)。流式细胞术实验表明si RNA转染组的细胞凋亡率明显高于阴性对照组和空白对照组(P0.05),阴性对照组与空白对照组的对比无明显差异(P0.05)。流式细胞术实验表明si RNA转染组的G0/G1期细胞数目较阴性对照组和空白对照组增多明显(P0.05),同时si RNA转染组的S、G2/M期细胞数目较阴性对照组和空白对照组明显减少(P0.05),阴性对照组和空白对照组对比差异无统计学意义(P0.05)。结论:RNA干扰CMTM7能够促进肺腺癌A549细胞凋亡,抑制肿瘤细胞的生长,其作用机制可能通过干扰细胞周期而实现。     

dsRNA介导的RNA干扰

在多种生物中 ,外源或内源性的双链ＲＮＡ(ｄｏｕｂｌｅ ｓｔｒａｎｄｅｄＲＮＡ ,ｄｓＲＮＡ)导入细胞中 ,与ｄｓＲＮＡ同源的ｍＲＮＡ则受到降解 ,因而其相应的基因受到抑制。这种转录后基因沉默 (ｐｏｓｔ ｔｒａｎｓｃｒｉｐｔｉｏｎａｌｇｅｎｅｓｉｌｅｎｃｉｎｇ ,ＰＴＧＳ)机制首先在线虫 (Ｃ .ｅｌｅｇａｎｓ)中得以证实。由于这是一种在ＲＮＡ水平的基因表达抑制 ,故也称为ＲＮＡ干扰 (ＲＮＡｉｎｔｅｒｆｅｒ ｅｎｃｅ) ,简称ＲＮＡｉ[1] 。随后发现 ,在各种生物 ,如果蝇[2 ] 、拟南芥菜[3 ] 、及小鼠[4] 等均存在ｄｓＲＮＡ介导…     

阳离子脂质体介导的基因转染大鼠BM-EPC的研究

目的:探讨阳离子脂质体法对体外分离培养的Sprague-Dawley(SD )大鼠骨髓来源-血管内皮祖细胞(Bone Marrow-Endothelial progenitorcells,BM-EPCs) 进行基因转染时脂质 体和DNA质粒安全有效的剂量组合.方法:体外分离、培养SD大鼠BM-EPCs ;免疫荧光技术测 定CD34、CD133、Dil-acLDL,对细胞进行鉴定;按照正交设计,用不同水平的脂质体和质粒 pEGFP转染细胞;荧光显微镜下计数阳性转染细胞,计算转染率.结果:SD 大鼠BM-EPCs细胞 表面抗原CD133、CD34呈阳性表达并具有吞噬Dil-acLDL的功能,形态学上两种细胞亚型-早 期及晚期外生EPCs共存于其中.Lipofectamine 2000和pEFGP不同剂量组合均可转染大鼠BM -EPCs,其中脂质体5ìl/质粒8ìg时的转染效率高于二者其它剂量组合(P＜0.05). 结论:优化条件后的阳离子脂质体Lipofectamine 2000可安全、有效转染体外分离培养的SD大鼠BM-EPCs.     

慢病毒载体介导的RNA干扰

RNA干扰(RNAinterference)是指由双链RNA分子抑制同源基因的表达。慢病毒载体(lentivirusvector)则是高效的基因转导工具,能将外源序列稳定导入分裂相和非分裂相细胞。将慢病毒载体和RNA干扰结合,能在哺乳动物各类细胞中,特异性抑制同源基因的表达;也是基因功能研究和基因治疗的有力手段。     

脂质体介导转染法的原理与应用

脂质体是磷脂分散在水中时形成的脂质双分子层 ,又称为人工生物膜。最初 ,人们只是运用脂质体模拟生物膜 ,研究膜的构造及功能 ,从而发现了膜的融合及内吞作用 ,因而可用作外源物质进入细胞的载体。相对于电穿孔法和磷酸钙共沉淀转染法 ,脂质体介导转染法简便易行 ,成本适中 ,具有较高的转染率和较小的细胞毒性。1 .脂质体的组成和制备1 .1　组成脂质体的脂类　　现有的商业化脂质体均为阳离子脂类与中性脂类的复合体 ,如ＬｉｐｏｆｅｃｔＡＭＩＮＥ、Ｌｉｐｏｆｅｃｔｉｎ等 ,中性脂类多为二油酰磷脂酰乙醇胺 (ＤＯＰＥ)。其中 ,阳离子脂类…     

RNA干扰抑制血管内皮生长因子介导结肠癌细胞凋亡

目的应用RNA干扰技术抑制血管内皮生长因子(VEGF)表达,并通过体外实验,观察VEGF受抑制后,凋亡情况的变化,为RNA干扰技术的临床应用奠定基础。方法通过Tunel和流式细胞计数的方法,观察VEGF受抑制后,肿瘤细胞凋亡发生率的变化。结果Tunel和流式细胞计数分析显示,当VEGF受抑制后,肿瘤细胞凋亡发生率明显增加。结论Tunel和流式细胞计数分析显示,当VEGF受抑制后,肿瘤细胞凋亡发生率明显增加。     

Optimization of cationic polymer-mediated transfection for RNA interference

Transfection efficiency was estimated to optimize the conditions for RNA interference (RNAi), including transfection time, validity, and nucleic acid concentration and type, using the EZ Trans Cell Reagent, a cationic polymer. An shRNA against GFP was designed and transfected into cells using the EZ transfection reagent. The shRNA significantly decreased the expression of GFP. In addition, pre-diluted transfection reagent at room temperature and small nucleic acids increased the transfection efficiency, which peaked at 24 h. Compared with circular nucleic acids, linear nucleic acids showed higher transfection efficiency and a higher genome integration rate. We optimized cationic polymer-mediated RNAi conditions, and our data will be useful for future RNAi studies.     

Small interfering RNA delivery into the liver by cationic cholesterol derivative-based liposomes

Purpose: Previously, we reported that the cationic liposomes composed of a cationic cholesterol derivative, cholesteryl (2-((2-hydroxyethyl)amino)ethyl)carbamate (OH-C-Chol) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (termed LP-C), could deliver small interfering RNAs (siRNAs) with high transfection efficiency into tumor cells. In this study, to develop a liposomal vector for siRNA delivery in vivo, we prepared the poly(ethyleneglycol) (PEG)-modified cationic liposomes (LP-C-PEG) and evaluated their transfection efficiency in vitro and in vivo.

Materials and methods: We prepared LP-C-PEG/siRNA complexes (LP-C-PEG lipoplexes) formed in water or 50?mM NaCl solution, and evaluated their siRNA biodistribution and gene silencing effect in mice after intravenous injection.

Results: LP-C-PEG lipoplexes strongly exhibited in vitro gene silencing effects in human breast tumor MCF-7 cells as well as LP-C lipoplexes. In particular, formation of LP-C and LP-C-PEG lipoplexes in the NaCl solution increased the cellular association. When LP-C-PEG lipoplexes with Cy5.5-labeled siRNA formed in water or NaCl solution were injected into mice, accumulation of the siRNA was observed in the liver. Furthermore, injection of LP-C-PEG lipoplexes with ApoB siRNA could suppress ApoB mRNA levels in the liver and reduce very-low-density lipoprotein/low-density lipoprotein levels in serum compared with that after Cont siRNA transfection, although the presence of NaCl solution in forming the lipoplexes did not affect gene silencing effects in vivo.

Conclusions: LP-C-PEG may have potential as a gene vector for siRNA delivery to the liver.     

Rescue of retroviral envelope fusion deficiencies by cationic liposomes

Background

Retroviral particles that are inappropriately enveloped can transduce target cells if pre‐associated with cationic liposomes. This study optimises and addresses the mechanism of liposome‐enhanced gene delivery, and explores the potential for such agents to compensate for fusion deficiency associated with chimaeric envelope proteins.

Methods

Particles bearing wild‐type, chimaeric or no envelope proteins were complexed with DOTAP or DC‐Chol/DOPE cationic liposomes and added to target cells for various times. Particle binding was determined by detection of cell‐associated capsid protein and infectivity was measured histochemically.

Results

Stable association of cationic liposomes with retrovirus particles significantly enhanced their binding rate to target cells in proportion to the increase of transduction kinetics for infectious virus. Binding of virus was equivalent with or without envelope protein and/or virus receptor, indicating that a non‐specific interaction precedes receptor recognition. Non‐infectious combinations were rescued by the intrinsic fusogenicity of the cationic liposomes, which enabled entry of the viral core, but left subsequent events unaltered. The optimised transduction rate with non‐enveloped particles and DOTAP approached that of amphotropic‐enveloped virus in some cases, although the effect was target‐cell‐dependent. DC‐Chol/DOPE was less potent at direct fusion but was able to enhance 600‐fold the receptor‐dependent action of chimaeric envelopes that were deficient in fusion by virtue of the addition of targeting domains.

Conclusions

These data have implications for the development of retroviral vector targeting strategies from the perspectives of the specificity of target cell interaction and compensating for chimaeric envelope fusion deficiency. Copyright © 2002 John Wiley & Sons, Ltd.

     

Polyethylenimine of various molecular weights as adjuvant for transfection mediated by cationic liposomes

Over the last years significant progress has been made in non-viral gene delivery mediated by cationic liposomes. However, the results obtained are still far from being satisfactory regarding transfection efficiency, particularly when compared to that achieved using viral vectors. We have previously demonstrated that association of transferrin with cationic liposomes significantly improves transfection in a large variety of cells, both in vitro and in vivo. In this work, several strategies have been explored in order to further improve transfection mediated by transferrin-associated lipoplexes. To this regard, the effect on transfection of pre-condensation of DNA with polyethylenimine of low MWs (2.7, 2.0 and 0.8 KDa) at various N/P ratios, lipid composition, cationic lipid/DNA (+/-) charge ratio and the presence of a surfactant in the lipoplexes was investigated. Two different modes for preparing the liposomes were tested and the extent of cell association of their complexes with DNA as well as their capacity to protect the carried DNA were evaluated. Our results show that complexes generated from cationic liposomes prepared by the ethanol injection method in which the carried DNA was pre-condensed with low MW polyethylenimine are highly efficient in mediating transfection. The differential modulating effect observed upon association of transferrin to various liposome formulations on transfection mediated by the polyethylenimine-complexes suggests that these complexes enter into the cells through different pathways (involving clathrin versus caveolin), most likely by taking advantage of their intrinsic biophysical properties to escape from the endosome to the cytosol.     

Involvement of intracellular caveolin‐1 distribution in the suppression of antigen‐induced mast cell activation by cationic liposomes

Cationic liposomes are commonly used as vectors to effectively introduce foreign genes into target cells. In another function, we recently showed that cationic liposomes bound to the mast cell surface suppress the degranulation induced by the cross‐linking of high‐affinity immunoglobulin E receptor in a time‐ and dose‐dependent manner. This suppression is mediated by the impairment of the sustained level of intracellular Ca²⁺ concentration ([Ca²⁺]_i) via the inhibition of store‐operated Ca²⁺ entry. Further, we revealed that the mechanism underlying an impaired [Ca²⁺]_i increase is the inhibition of the activation of the phosphatidylinositol 3‐kinase (PI3K)‐Akt pathway. Yet, how cationic liposomes inhibit the PI3K‐Akt pathway is still unclear. Here, we focused on caveolin‐1, a major component of caveolae, which is reported to be involved in the activation of the PI3K‐Akt pathway in various cell lines. In this study, we showed that caveolin‐1 translocated from the cytoplasm to the plasma membrane after the activation of mast cells and colocalized with the p85 subunit of PI3K, which seemed to be essential for PI3K activity. Meanwhile, cationic liposomes suppressed the translocation of caveolin‐1 to the plasma membrane and the colocalization of caveolin‐1 with PI3K p85 also at the plasma membrane. This finding provides new information for the development of therapies using cationic liposomes against allergies.     

Induction of apoptosis in macrophages by cationic liposomes

The effects of liposomes on apoptosis in macrophages were evaluated from DNA content and DNA fragmentation. Cationic liposomes composed of different kinds of cationic lipids induced apoptosis in mouse splenic macrophages and the macrophage-like cell line, RAW264.7 cells. Generation of reactive oxygen radicals from macrophages treated with cationic liposomes was detected using flow cytometry, and further apoptosis was inhibited by the addition of oxidant scavenger, N-acetylcysteine. From these findings, the production of reactive oxygen species may be important in the regulation of apoptosis induced by cationic liposomes.     

RNA干扰技术及其在细胞周期研究中的应用

RNA干扰(RNA interference,RNA i)是由双链RNA(doub le-stranded RNA,dsRNA)引发的转录后基因沉默(posttran-scridptional gene silenc ing,PTGS)。dsRNA经D icer酶降解成21-23nt的siRNA,并以其为模板,特定位点、特定间隔降解与之序列相应的mRNA。随着RNA i机制的深入研究与广泛应用,目前该技术已经普遍应用于细胞周期研究中,在阐明各种调控机制的同时也为基因治疗提供了新靶点。