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。     

Gene Transfection in High Serum Levels: Case Studies with New Cholesterol Based Cationic Gemini Lipids

Background

Six new cationic gemini lipids based on cholesterol possessing different positional combinations of hydroxyethyl (-CH₂CH₂OH) and oligo-oxyethylene -(CH₂CH₂O)_n- moieties were synthesized. For comparison the corresponding monomeric lipid was also prepared. Each new cationic lipid was found to form stable, clear suspensions in aqueous media.

Methodology/Principal Findings

To understand the nature of the individual lipid aggregates, we have studied the aggregation properties using transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential measurements and X-ray diffraction (XRD). We studied the lipid/DNA complex (lipoplex) formation and the release of the DNA from such lipoplexes using ethidium bromide. These gemini lipids in presence of a helper lipid, 1, 2-dioleoyl phophatidyl ethanol amine (DOPE) showed significant enhancements in the gene transfection compared to several commercially available transfection agents. Cholesterol based gemini having -CH₂-CH₂-OH groups at the head and one oxyethylene spacer was found to be the most effective lipid, which showed transfection activity even in presence of high serum levels (50%) greater than Effectene, one of the potent commercially available transfecting agents. Most of these geminis protected plasmid DNA remarkably against DNase I in serum, although the degree of stability was found to vary with their structural features.

Conclusions/Significance

-OH groups present on the cationic headgroups in combination with oxyethylene linkers on cholesterol based geminis, gave an optimized combination of new genera of gemini lipids possessing high transfection efficiency even in presence of very high percentage of serum. This property makes them preferential transfection reagents for possible in vivo studies.     

一种可降解的阳离子聚合物基因转染试剂

目的：合成一种高效、低毒的新型阳离子聚合物载体。方法：以低分子量的聚乙烯亚胺（PEI）为阳离子聚合物的基本单位,以可水解的2,4-戊二醇二丙烯酸盐（PODOA）为交联剂,合成高分子聚合物。用DNA凝胶迟滞实验验证聚合物与DNA的亲和力,以绿色荧光蛋白基因和萤光素酶检测其基因转染效率,用聚合物凝胶电泳实验鉴定其降解性,以MTT法检测其细胞毒性。结果：聚合物具有高的DNA结合亲和力、高基因转染效率,基因转染后的萤光素酶活性高达3×10^9RLU／mg,其基因转染效率相当于甚至优于目前市售的转染试剂,而细胞毒性明显低于其他试剂。该聚合物在中性环境下可降解,而在酸性条件下非常稳定。结论：合成的聚合物具有高转染效率和低细胞毒性,可能在转染和基因治疗研究中起重要作用。     

阳离子脂质体Lipofectamine2000对PC12细胞转染效率的研究

通过改变转染试剂及DNA用量和转染时间等影响转染效率的重要因素来实现阳离子脂质体lipofectamineTM2000(lipo2000)对PC12细胞的高效转染.结果表明,lipo2000转染PC12细胞的最佳转染条件:lipo2000用量为5μL,DNA 2μg,转染时间为6 h,这种条件下的PC12细胞转染率高达40%,且未影响细胞的正常分泌,这为应用PC12细胞进行神经生物学研究提供了基础资料.     

Gold Nanoparticle Mediated Laser Transfection for Efficient siRNA Mediated Gene Knock Down

Laser based transfection methods have proven to be an efficient and gentle alternative to established molecule delivery methods like lipofection or electroporation. Among the laser based methods, gold nanoparticle mediated laser transfection bears the major advantage of high throughput and easy usability. This approach uses plasmon resonances on gold nanoparticles unspecifically attached to the cell membrane to evoke transient and spatially defined cell membrane permeabilization. In this study, we explore the parameter regime for gold nanoparticle mediated laser transfection for the delivery of molecules into cell lines and prove its suitability for siRNA mediated gene knock down. The developed setup allows easy usage and safe laser operation in a normal lab environment. We applied a 532 nm Nd:YAG microchip laser emitting 850 ps pulses at a repetition rate of 20.25 kHz. Scanning velocities of the laser spot over the sample of up to 200 mm/s were tested without a decline in perforation efficiency. This velocity leads to a process speed of ∼8 s per well of a 96 well plate. The optimal particle density was determined to be ∼6 particles per cell using environmental scanning electron microscopy. Applying the optimized parameters transfection efficiencies of 88% were achieved in canine pleomorphic adenoma ZMTH3 cells using a fluorescent labeled siRNA while maintaining a high cell viability of >90%. Gene knock down of d2-EGFP was demonstrated and validated by fluorescence repression and western blot analysis. On basis of our findings and established mathematical models we suppose a mixed transfection mechanism consisting of thermal and multiphoton near field effects. Our findings emphasize that gold nanoparticle mediated laser transfection provides an excellent tool for molecular delivery for both, high throughput purposes and the transfection of sensitive cells types.     

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。     

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.     

Synthesis and Transfection Efficiencies of Divalent Ammonium Headgroup Cationic Lipids with Different Hydrophobic Tails

Russian Journal of Bioorganic Chemistry - Cationic lipids with amide or carbamate linker and divalent cationic headgroups were synthesized for a transfection study. Sixteen cationic lipids with...     

Calcium Movements Mediated by Proteolipid Protein and Nucleotides in Liposomes Prepared with the Endogenous Lipids from Brain White Matter

A lipid extract with a composition similar to that of myelin was used to prepare liposomes and proteoliposomes containing the Folch-Lees proteolipid apoprotein. Freeze-fracture replicas of the proteoliposomes were prepared to demonstrate the presence of intramembrane protein particles in the fracture faces of the lipid bilayer. Experiments with 45CaCl2 showed that a steady calcium movement occurs across liposomal membranes, approaching equilibrium between intra- and extravesicular spaces. The most significant finding was that Mg-ATP, ATP analogues, and other nucleotides depressed significantly the calcium fluxes in proteoliposomes, having no effect on liposomes that lacked the proteolipid protein. It is suggested that this intrinsic protein, interacting with nucleotides and endogenous lipids, could be involved in the regulation of calcium levels in myelin by means of a conformational change mechanism. These observations could lead to implications concerning the pathophysiology of myelin.     

PEG修饰的聚阳离子基因载体PEG-Polycarbam-SP在COS-7细胞中转染和毒性的研究

目的：寻找一种转染效率高,细胞毒性低的非病毒基因载体,研究以人体内源性精胺为单体,以乙二醇二氯甲酸酯作为连接剂,以聚乙二醇（PEG）作为亲水基团连接剂合成亲水修饰聚阳离子载体PEG-Polycarbam-SP的基因担载效率,以及对非洲绿猴肾癌细胞COS-7的转染活性和细胞毒性的影响。方法：琼脂糖凝胶电泳方法考察复合物的基因担载效率,检测基因复合物的粒径和电位,以荧光素酶质粒为报告基因,研究PEG-Polycarbam-SP/DNA的复合物在COS-7细胞的转染活性,用MTT方法研究PEG-Polycarbam-SP对COS-7细胞的毒性。结果：聚合物与质粒在质量比5以后形成的复合物粒径稳定在50nm左右,Zate电位在20mV左右。COS-7细胞实验显示PEG-Polycarbam-SP具有低于PEI 25kDa的细胞毒性,同时也具有高效输送DNA的能力。结论：PEG-Polycarbam-SP是一种新型的高效、低毒,在基因治疗领域有潜在应用价值的非病毒基因输送载体。     

Direct Gene Transfer by Liposomes

Abstract

Cationic liposomes are widely used for the delivery of genes both in vivo and in clinical trials. DC-chol liposome formulation was developed by us for relatively high activity of transfection and low level of toxicity for most cell types. Different strategies are described for achieving regulated transgene expression as well as expression for a prolonged period of time using DC-chol liposomes.     

Multilamellar Cationic Liposomes are Efficient Vectors for in Vitro Gene Transfer in Serum

Abstract

Multilamellar vesicles (MLVs) containing the cationic lipid DOTAP were used as vectors to lipofect a number of culture cell lines in the presence of serum. The lipofection efficiency of lipoplexes made of MLVs and the plasmid pSV-β galactosidase are much less sensitive to the lipofection-inhibitory effect of serum than the conventionally used lipoplexes made of sonicated small unilamellar vesicles (SUVs). In order to determine the factors favoring the lipofection efficiency of MLVs, we measured the size, as well as the cellular association and uptake of MLV and SUV lipoplexes containing DOTAP alone or DOTAP:DOPE (1:1). Electron microscope images of these complexes were taken to confirm their structure and size. The single most important factor that correlates with transfection efficiency in serum is the size of the lipoplex. SUV lipoplexes remain smaller than 300 nm in the presence of serum, and the lipofection efficiencies are low. MLV lipoplexes are larger (>300 nm) and the lipofection efficiency, as well as cellular association and uptake, are much higher than those of SUV lipoplexes. Exceptions are those lipoplexes made of MLVs of DOTAP and DOPE (1:1) combined with DNA at higher charge ratios, which form hexagonal structures and show poor lipofection as well as cellular association and uptake, even if their lipoplex size exceeds 300 nm. This finding lends credence to our theory of the serum inhibition effect upon lipofection, and suggests ways to improve the transfection efficiency in the presence of serum, by fabricating lipoplexes of defined sizes.     

Transfection of KB Cells by Liposomes Containing Adenovirus Type 2 DNA

Adenovirus type 2 DNA was entrapped in liposomes which were then used to transfect KB cells with an efficiency of ~4,000 plaques per μg of encapsulated DNA.     

SA脂质体介导DNA转染细胞的进一步研究

ＳＡ脂质体可高效介导ＤＮＡ转染ＣＶ－１细胞,本文进步研究表明,ＳＡ脂质体还可介导ＤＮＡ高效瞬时和稳定地转染ＣＨＯ和ＣＯＳ细胞。ＳＡ脂质体和ＤＮＡ形成复合物可保护ＤＡＮ不被核酸内切酶和ＤＮａｓｅＩ降解。荧光标记和细胞松驰素Ｂ抑制实验分别表明,ＳＡ脂质体易被细胞吸附,主要通过内吞传送ＤＮＡ进入细胞,而Ｌｉｐｏｆｅｃｔｉｎ主要通过融合传送ＤＮＡ进细胞。     

载脂蛋白基因多态性与血脂关系研究进展

探讨载脂蛋白(apolipoprotein,apo)基因多态性与血脂的相关性.apoA,apoB,apoH,apoA-V,apoM基因具有多态性,有研究发现其基因多态性与血脂具有相关性,但是也有一些研究所得的结果相互不一致,甚至相反,还有发现人群或种族不同所得的结果亦不同.通过对载脂蛋白基因多态性的研究,揭示其与血脂的关系,以利我们从基因水平进一步认识和预防脂代谢紊乱,具有重要意义和广阔的应用前景.     

脂质体介导法转染肿瘤细胞效率的优化

目的:研究优化影响脂质体转染效率的因素,以提高脂质体转染效率,为相关研究和应用提供参考.方法:以绿色荧光蛋白(GFP)作为报告基因,采用脂质体Lipofectamine 2000包裹pU6H1-GFP-FAK重组质粒转染Caco-2细胞,研究了细胞接种密度、DNA用量、脂质体与DNA的比例、脂质体-DNA复合物的形成时间、细胞与脂质体复合物的孵育时间、血清的有无及细胞的传代次数等因素对脂质体转染效率的影响.结果:2-5次细胞传代,2×105接种密度、4μg DNA用量、2.5:1的脂质体与DNA比例、30min脂质体-DNA复合物形成时间以及6h细胞与复合物孵育时间,转染效率最高.血清在本实验室条件下并不影响转染效率.结论:实验获得的优化条件可以明显提高脂质体对肿瘤细胞的转染效率,可作为有关研究或应用的参考.     

SA脂质体介导DNA转染昆虫细胞的研究

ＳＡ脂质体介导ＤＮＡ转染昆虫细胞的研究张传溪（浙江农业大学应用昆虫学研究所杭州３１００２９）吴祥甫（中国科学院上海生物化学研究所上海２０００３１）杆状病毒昆虫表达系统是８０年代发展起来的高效真核表达系统,具有表达量高,表达产物后加工较完全等优点,因...     

Complement-Dependent Phagocytosis of Liposomes: Suppression by “Stealth” Lipids

Abstract

Liposomes containing either ganglioside G_M1, phosphatidylinositol, sulfogalactosyl ceramide, certain other anionic phospholipids, prostaglandin E₂, or thromboxane B₂ have a reduced ability to undergo complement-dependent phagocytosis by cultured macrophages. We propose that this phenomenon is partially responsible for the prolonged circulation time that is observed after intravenous injection of certain liposomes that are said to have “stealth” properties.     

Improved Encapsulation of DNA in pH-Sensitive Liposomes for Transfection

Abstract

A simple method has been developed to prepare liposomes containing large amounts of DNA. The procedure consisted of three cycles of freeze-thawing a mixture of sonicated liposomes and DNA. The encapsulation efficiency depended on the size of DNA. For a small plasmid (2.7 kb), approximately 40% of input DNA was entrapped with an efficiency of 16 μgDNA/μmol lipid. For larger plasmids, the encapsulation efficiency decreased considerably. Transfection of cultured mouse L929 cells mediated by the DNA-containing liposomes was assayed with a plasmid containing the E. coli chloramphenicol acetyl transferase gene. The transfection activity of the liposome was primarily determined by its pH sensitivity. Acid-sensitive liposomes transfected cells efficiently, whereas pH-insensitive liposomes were much less active. The level of the expression of the exogenous gene in the treated cells could be further modulated by protein kinase C (PKC) activators that were incorporated into the liposomal membrane as a minor lipid component. Transfection conditions were optimized with respect to DNA, lipid, and PKC activator concentrations. The results of the current study may help the use of liposomal delivery system for applications in gene therapy.