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…     

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。     

阳离子脂质体的转染机制及转染效率影响因素

阳离子脂质体是一种非常具有发展前景的基因载体。简要介绍了阳离子脂质体的结构特点;着重讨论了阳离子脂质体作为基因载体时介导基因转移的机制以及在转染过程中对基因转染效率产生影响的主要因素。     

阳离子脂质体的转染机制及转染效率影响因素

阳离子脂质体是一种非常具有发展前景的基因载体。简要介绍了阳离子脂质体的结构特点;着重讨论了阳离子脂质体作为基因载体时介导基因转移的机制以及在转染过程中对基因转染效率产生影响的主要因素。     

人肾细胞癌细胞阳离子脂质体的转染效率

以ＭＴＳ染色法测定实验剂量的Ｌｉｐｏｆｅｃｔｉｎ对细胞的毒性作用,以β－半乳糖苷酶基因为报告基因,通过Ｌｉｐｏｆｅｃｔｉｎ而转染,用Ｘ－ｇａｌ染色法,测定转染效率,结果表明实验剂量（１０μｇ／ｍｌ）的Ｌｉｐｏｆｅｃｔｉｎ对细胞生长无明显毒性。Ｌｉｐｏｆｅｃｔｉｎ对多数肾细胞癌细胞的转染是有效的,且转染效率随Ｌｉｐｏｆｅｃｔｉｎ 度的增高（２．５－１０μｇ／ｍｌ）而增高,说明Ｌｉｐｏｆｅｃｔｉｎ可安     

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...     

影响小鼠体细胞脂质体法转染效率的因素

We studied the effects of the amount of liposome and plasmid, exposure time of cells to the liposome-plasmid complexes, number of cell passages and cell types on GFP gene transfection of mouse somatic cells. The maximal GFP transgene expression (30.7%) was achieved when mouse fetal fibroblast cells (MFFC) at 70%-90% confluence of passage 3 were exposed for 6 h to the complexes of 4 microg liposome (LipofectAMINE) and 0.3 microg plasmid (pEGFP-N1). Under these conditions, we compared the effect of the number (from primary to 15) of passages on the transfection efficiency of MFFC. The transfection efficiency of MFFC was 10.0%, 28.9% and 7.2% at the primary, 3rd and 15th passage, respectively, which indicated that the transfection efficiency decreased with passaging. When MFFC, mouse oviductal epithelial cells (MOEC) and mouse granulosa cells (MGC) were transfected at passage 3, the transfection efficiency was 27.8%, 13.7% and 14.2%, respectively, under the described transfection conditions. When the cell cycle stages of different cell types at transfection were examined, it was found that 17.2% of MFFC, 8.7% of MOEC and 9.9% of MGC were at M phases of the cell cycle. Examination of the cell cycle stages of MFFC at different passages showed that MFFC at the third passage had the highest percentage of M cells and the percentage decreased afterwards. This suggested that the transfection efficiency was correlated with the percentages of cells at M phase, and provided essential data for improvement of the transfection efficiency.     

影响非洲猴肾细胞脂质体转染效率的因素

已有实验表明,细胞转染效率可能决定于ＤＮＡ－脂质体复合物的形成以及所转染的细胞种类。利用脂质体ＬｉｐｏｆｅｃｔＡＭＩＮＥ,研究了影响Ｖｅｒｏ细胞转染效率的参数如ＤＮＡ和脂质体的用量,转染的细胞数量以及细胞暴露子ＤＮＡ－脂质体复合物的时间长度。通过检测报告基因β－半乳糖苷酶（β－ｇａｌ）的表达,发现最高转染率在一较窄范围内获得。β－ｇａｌ的表达随脂质体量增加而显著增加。在标准转染条件下,增加ＤＮＡ用     

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。     

脂质体介导外源基因体外转染牛胎儿成纤维细胞条件的优化

通过脂质体（FuGENE-6）介导,将真核表达载体pEGFP-C1成功导入体外培养的牛胎儿成纤维细胞,探讨影响外源基因转染效率的参数,如DNA和脂质体的用量、转染的细胞数量以及细胞暴露于DNA与脂质体复合物的时间长度。通过实验发现,绿色荧光蛋白（green fluorescent protein,GFP）基因的表达随DNA、脂质体量的增加而增加,延长细胞暴露时间反而使转染效率下降,转染细胞数适当才能得到较高的转化率。 Optimization of Parameters of Exogene Transfection of Bovine Fetal Fibroblasts in vitro Mediated by Liposome LI Yang,WU Kai-feng,GUO Xu-dong,GUO Ji-tong,BOU Shor-gan The Research Center for Laboratory Animal Science of Inner Mongolia University,The Key Laboratory of Ministry of Education of China for Mammal Reproduction Biology and Biotechnology,Huhhot 010021,China Abstract:pEGFP-C1 eucaryon expression vector was successfully transfected by liposome into bovine fetal fibroblasts.We investigated the effect of parameter such as the dose of DNA and liposome,number of cell transfected and exposure time of the cell to the DNA-liposome complexes.It was indicated that GFP（green fluorescent protein）expression was enhanced as the dose of DNA and liposome increased and on decline as the exposure time was prolonged.The improvement of transfection efficiency depent on the suitable cell number. Key words：liposome; GFP; bovine fetal fibroblasts; transfection     

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.     

适配体介导脂质体靶向递送siRNA的研究

为探讨适配体介导的脂质体靶向递送siRNA的可行性,采用前列腺癌细胞膜表面抗原(PSMA)的适配体A10-3.2与脂质体结合,构建适配体-脂质体靶向递送体系(Apt-LP),并利用Apt-LP递送p EGFP-N1质粒和Bcl2 siRNA到前列腺癌细胞LNCa P(PSMA+)和PC-3(PSMA-),转染48h后,用荧光显微镜检测绿色荧光蛋白的表达,q PCR检测Bcl2 mRNA表达,蛋白印记法检测Bcl2蛋白表达,Hoechst 33258核染法分析体外抗肿瘤活性。结果显示:与脂质体递送体系相比,Apt-LP显著提高递送p EGFP-N1质粒和Bcl2 siRNA到靶细胞LNCa P(PSMA+)的效率;显著提高Bcl2 siRNA诱导的靶细胞LNCa P(PSMA+)Bcl2基因沉默效应,更有效的诱导靶细胞LNCa P(PSMA+)凋亡。结果表明:Apt-LP是一种有效的siRNA靶向递送体系,具有潜在的临床应用价值。     

CHO-K1细胞中基因瞬时转染的条件优化

目的:以CHO-K1细胞为宿主基因瞬时转染条件的优化.方法:以GFP(Green Fluorescence Protein)为报告基因,考察了DNA∶PEI比例、DNA用量及血清的加入对CHO-K1细胞的转染效率和细胞数目的影响.结果:DNA∶PEI=1∶2(w/w)、2 gDNA/106 cells时,转染结果最优;血清的加入可降低细胞转染效率.结论:在CHO-K1细胞中进行瞬时转染的最佳条件为DNA∶PEI=1∶2(w/w)、2 gDNA/106 cells,及血清的加入抑制细胞转染.     

Resistance to Tumor Necrosis Factor-Induced Cell Death Mediated by PMCA4 Deficiency

We used retrovirus insertion-mediated random mutagenesis to generate tumor necrosis factor (TNF)-resistant lines from L929 cells. Using this approach, we discovered that the plasma membrane calcium ATPase 4 (PMCA4) is required for TNF-induced cell death in L929 cells. Under basal conditions, PMCA4-deficient (PMCA(mut)) cells have a normal phenotype. However, stimulation with TNF induces an abnormal increase in the intracellular calcium concentration ([Ca(2+)](i)). The substantially elevated [Ca(2+)](i) caused resistance to TNF-induced cell death. We found that an increase in the total volume of acidic compartments (VAC), mainly constituted by lysosomes, is a common event in cell death caused by a variety of agonists. The increased [Ca(2+)](i) in PMCA(mut) cells promoted lysosome exocytosis, which, at least in part, accounted for the inhibition of TNF-induced increase in VAC and cell death. Promoting lysosome exocytosis by calcium inhibited TNF-induced cell death in wild-type L929 cells, while inhibition of lysosome exocytosis or increase of VAC by sucrose restored the sensitivity of PMCA(mut) cells to TNF-induced cell death. Thus, increase of the volume of acidic compartment is a part of the cell death process, and the antideath effect of calcium is mediated, at least in part, by inhibition of the TNF-induced increase in VAC.