核糖体基因区打靶载体电转染肝细胞的孵育条件的优化

目的:提高核糖体基因区打靶载体的转染效率,优化其电转染肝细胞的孵育条件.方法:在其它电转染参数一致的前提下,设计了6组不同的孵育条件,通过分析转染后细胞活率以及48h后目的基因GFP的表达效率,来比较多组不同的孵育条件对电转染效率的影响.结果:6组条件中,转染前21℃,1min,转染后21℃,1min孵育,得到的转染效率最优,比其它的条件得到的转染效率高50%.结论:孵育条件的优化能提高为核糖体基因区靶向表达我体在体外电转染肝细胞的转染效率.     

人核糖体DNA打靶载体介导mda-7基因对肝癌的体外基因治疗研究

人核糖体DNA打靶载体pHr是一种由中南大学医学遗传学国家重点实验室开发构建的针对人类基因组的同源重组质粒载体．利用pHr构建了一种mda-7/GFP融合基因的人源基因表达载体pHr-CMG,并研究了其在肝癌细胞系Bel-7402中的作用．利用荧光显微镜、RT-PCR和Western blotting检测了mda-7/GFP融合基因的表达;利用细胞周期分析、MTT和Hoechst33258染色研究了其在细胞中的作用．结果显示,pHr-CMG载体能在Bel-7402细胞中有效表达MDA-7/GFP融合蛋白,进而抑制细胞增殖和诱导细胞凋亡,推测其可能是由载体表达了mda-7基因引起细胞在G2/M期累积所导致的．同时,实验结果证实了人核糖体DNA打靶载体系统以及pHr-CMG表达载体的有效性,为其在进一步基因治疗研究中的应用提供了理论和实验基础．     

非病毒载体聚乙烯亚胺介导DNA转染的研究

目的 研究新型非病毒类载体聚乙烯亚胺（PEI）的最适转染条件.方法 分析各种因素对25ku线性PEI转染效率的影响,优化实验条件.结果 PEI与DNA的质量比≥2能保证DNA与PEI完全结合,最佳混合时间为10 min,但血清的存在将降低其结合效率.在一定范围内提高PEI与DNA的质量比有利于提高转染效率.293T细胞最适转染密度为培养面积的80%.结论 确定了PEI转染细胞的最优条件.     

纳米羟基磷灰石表面修饰及其转染细胞的研究

目的:探讨羟基磷灰石-聚乙烯亚胺(nHA-PEI 10KD)纳米颗粒的癌细胞基因转染效率.方法:通过透射电子显微镜(TEM)观察HA-PEI(10KD)纳米颗粒的形态及粒径,Zeta电位仪测定nHA-PEI和HA在酸、碱、中性环境中的电位,用琼脂糖凝胶电泳检测nHAP-PEI(10KD)与DNA结合的能力,MTT比色法检测nHAP-PEI(10KD)对nepG2细胞的毒性,选用增强型绿色荧光蛋白质粒pEGFP1与nHA-PEI结合后,分别转染真核细胞HepG2、Hela、SW620,计算其转染率.结果:nHA-PEI(10KD)分散程度好,粒径60-80nm,在PH7.2时,Zeta电位42.87mV,能转染实验中的细胞,转然效果最好的是HepG2细胞,其次Hela、SW620,转染率高于PEI(10KD)、nHA,但低于脂质体.结论:通过阳离子PEI修饰HA,可有效将增强型绿色荧光蛋白质粒转入HepG2细胞,HA-PEI(10KD)纳米颗粒复合物有望成为基因传递的有效栽体.     

交联聚乙烯亚胺衍生物的构建及其转染COS-7 细胞的研究

目的:优化构建交联聚乙烯亚胺(Polyethylenemine,PEI)衍生物PEI-Bu,研究其对非洲绿猴肾成纤维细胞系(COS-7)的转染活性和细胞毒性。方法:以PEI 800Da为骨架,1,4-丁二醇二氯甲酸酯为连接剂制备聚合物PEI-Bu,琼脂糖凝胶电泳考察其复合质粒DNA的能力,MTT法检测PEI-Bu对COS-7的毒性,以荧光素酶质粒作为报告基因,测定PEI-Bu/DNA复合物在COS-7细胞的转染活性。结果:凝胶电泳表明PEI-Bu/DNA在质量比大于1时即具有复合DNA的能力,PEI-Bu的细胞毒性随浓度增大而增大,在同一浓度下PEI-Bu的细胞毒性小于PEI 25kDa,(P<0.05),PEI-Bu/DNA在质量比为5时达到最高转染活性,高于PEI 25kDa(P<0.01),并与Lipofectamine2000相当(P>0.05)。结论:PEI-Bu在COS-7细胞中是一种低细胞毒性、高转染活性的非病毒基因载体(与商业化的PEI 25kDa比较),其在基因治疗领域中具有潜在的应用前景。     

肿瘤细胞对腺病毒载体转染的敏感性研究

带有基因LacZ的5型缺陷性腺病毒感染肿瘤细胞,X－gal染色,检测腺病毒对肿瘤细胞的转染率。结果显示,随着病毒感染复数量（multiplicityofinfection,m.o．i）的增加,转染率也增高,且无细胞毒作用。各种肿瘤达到100％转染所需病毒量是不同的,人类肿瘤细胞系100％转染所需的m.o．i分别是：Hela细胞为100,GRC肾癌细胞为200,Hep-2喉癌细胞为200,鼠肿瘤所需病毒量较大,Lewis肺癌细胞为500,BST膀胱癌细胞为600。说明不同肿瘤细胞对腺病毒载体转染的敏感性是不同的。     

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。     

核糖体蛋白L6/Taxreb107的核定位信号的分析

核糖体蛋白L6（RpL6,Taxreb107）含有三个具有核定位信号特征的基序.用作者构建的核定位信号捕获系统分析了这些核定位信号是否具有介导蛋白质进行核转位的功能.将RpL6/Taxreb107分段插入核定位信号捕获载体的克隆位点后转化宿主酵母,发现其前两个核定位信号可以介导融合蛋白进入细胞核,而第三个核定位信号无此作用.将RpL6/Taxreb107分段与绿色荧光蛋白融合后转染培养的哺乳类细胞,证实了以上在酵母中所得的结果.进一步发现RpL6/Taxreb107的前两个核定位信号同时具有核仁定位的功能.当在细胞中表达的早期,进入核内的融合蛋白优先定位于核仁.这些结果一方面有助于理解RpL6/Taxreb107核转位的机理,同时说明作者构建的核定位信号捕获系统也可用在蛋白质中寻找核定位信号.     

植物核基因组核糖体基因间隔区序列的结构特点及其在系统发育研究中的应用

目前,在植物系统发育研究中应用较多的核基因组的核糖体DNA基因间隔区序列主要有5S rDNA基因间隔区、内转录间隔区ITS和基因间间隔区IGS.虽然这些间隔区序列在长度、结构等方面各不相同,但都具有进化速率较快的特点,在植物属及属下分类水平的系统发育关系研究中非常有用.本文重点就核基因组的5S rDNA基因间隔区以及IGS在植物中的特点以及各自在植物系统发育研究中的应用进行了综述.     

杜氏盐藻核基质结合区结合蛋白的细胞定位

为研究核基质结合区结合蛋白的功能及调控机制,PCR扩增杜氏盐藻MBP的cDNA全长序列及N端和C端序列,与绿色荧光蛋白基因融合构建真核表达载体,脂质体转染CHO细胞,Western blotting和荧光显微镜检测基因表达情况和细胞定位。结果显示:MBP及N端和C端融合蛋白成功在CHO细胞表达,MBP和C端部分定位于细胞核且聚集于核仁,N端部分分布整个细胞,说明MBP定位于细胞核且细胞定位信号位于C端,MBP可能与rRNA前体结合发挥作用。     

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。     

聚乙烯亚胺转基因影响因素的测定及其优化

聚乙烯亚胺 (PEI)为阳离子多聚物 ,可浓缩DNA形成纳米级颗粒 ,作为基因释放载体转染真核细胞 .选用Mr2 5 0 0 0 ,分枝状的聚乙烯亚胺转染质粒 ,比较多种转基因效率的影响因素 .通过MTT法测定PEI对COS 7细胞的细胞毒性 .利用电泳阻滞实验测定PEI与DNA形成复合物时所需的比例 .通过PEI转染增强型绿色荧光蛋白的pEGFP质粒、编码β 半乳糖苷酶的pSVβ质粒 ,探索氯喹、白蛋白、血清、盐离子浓度、质粒剂量、细胞数量等对聚乙烯亚胺转基因效率的影响 .实验发现 ,PEI对细胞的毒性作用与剂量相关 .PEI DNA的N P比在 3 0以上方可完全结合DNA .溶酶体抑制剂氯喹可增加转染效率 .培养液中的白蛋白、血清会降低转染效率 .生理盐溶液作为配制PEI DNA复合物的溶媒 ,转染效率高于 5 %葡萄糖作为溶媒 .随着转染质粒剂量的增加 ,转染效率呈剂量依赖正效应 .聚乙烯亚胺是有效的体外真核细胞转染剂 ,可用于合成更复杂的基因释放载体 .     

可磷酸化短肽偶联壳聚糖促进CS/DNA转染效率

合成基序为LLLRRRDNEY*FY*VRRLL的短肽(pSP),其中含有两个可被JaK2蛋白激酶磷酸化的酪氨酸残基.将此短肽与壳聚糖(CS)相偶联,体外磷酸化及DNA释放实验检测哺乳动物细胞裂解液对短肽的磷酸化及pSP-CS/DNA复合物中DNA释放的影响.放射性标记DNA转移实验验证pSP-CS/DNA复合物的入胞能力后,将荷荧光素酶或GFP报告基因的质粒与pSP-CS制成pSP-CS/DNA复合物,转染体外培养的C2C12小鼠成肌细胞,观察GFP的分布及细胞裂解液中的荧光素酶活性以表征转染效率.继而进行多种细胞系的转染,衡量pSP偶联的壳聚糖对不同种属细胞的转染效率.结果表明,哺乳动物细胞裂解液可有效地使短肽发生磷酸化,并藉此促进DNA与壳聚糖载体的解离.以pSP修饰的壳聚糖进行转染时,细胞裂解液的荧光素酶活性可达普通壳聚糖转染的两倍,细胞中GFP的含量也明显增加.据此推论,短肽被磷酸化后产生电荷属性的改变,促进DNA与壳聚糖载体的解离从而显著提高壳聚糖的转染效率.     

An NLS peptide covalently linked to linear DNA does not enhance transfection efficiency of cationic polymer based gene delivery systems

BACKGROUND: Transfection with non-viral gene delivery vectors, such as cationic polymers, generally results in low transgene expression in vivo. This is likely due to poor cytoplasmic transport and intra-nuclear DNA delivery. METHODS: In this study two strategies to improve nuclear import were investigated. Linear DNA constructs with or without an NLS peptide were prepared by PCR. Alternatively, linear DNA obtained by enzymatic cleavage followed by capping of both ends with DNA-hairpins was used. An NLS peptide was attached to one of the capped ends of the linear DNA. Both biodegradable (pDMAEAppz) and non-degradable polymers (PEI or pDMAEMA) were used to complex the DNA. Several cell types, dividing and non-dividing, were transfected with the linear DNA constructs containing a SV40-derived NLS peptide. Nuclear import of the DNA constructs was studied using digitonin-permeabilized cells. RESULTS: Linear DNA prepared by PCR proved not useful as it was degraded from the 3'end. Linear DNA capped with hairpins was more successful with regard to stability. However, Cells transfected with linear DNA constructs by electroporation or by using cationic polymers with linear DNA containing a NLS peptide, failed to show significantly higher luciferase expression levels when compared to cells transfected with plasmid DNA or linear DNA without an NLS peptide attached. No nuclear localization was observed in digitonin-permeabilized cells. CONCLUSION: Taken together, these data demonstrate that this nuclear localisation signal when attached to DNA is neither able to improve transfection efficiency of cationic polymers nor the nuclear import of the DNA constructs.     

Human-derived NLS enhance the gene transfer efficiency of chitosan

Nuclear import is considered as one of the major limitations for non-viral gene delivery systems and the incorporation of nuclear localization signals (NLS) that mediate nuclear intake can be used as a strategy to enhance internalization of exogenous DNA.In this work, human-derived endogenous NLS peptides based on insulin growth factor binding proteins (IGFBP), namely IGFBP-3 and IGFBP-5, were tested for their ability to improve nuclear translocation of genetic material by non-viral vectors. Several strategies were tested to determine their effect on chitosan mediated transfection efficiency: co-administration with polyplexes, co-complexation at the time of polyplex formation, and covalent ligation to chitosan. Our results show that co-complexation and covalent ligation of the NLS peptide derived from IGFBP-3 to chitosan polyplexes yields a 2-fold increase in transfection efficiency, which was not observed for NLS peptide derived from IGFBP-5.These results indicate that the integration of IGFBP-NLS-3 peptides into polyplexes has potential as a strategy to enhance the efficiency of non-viral vectors.     

Enhancement of gene delivery to human airway epithelial cells in vitro using a peptide from the polyoma virus protein VP1

BACKGROUND: Current liposome-based gene delivery methods for therapeutic benefit are limited by their low efficiency. One possible way to improve gene expression is to include a peptide with a nuclear localization signal (NLS) to enhance the movement of the transfection complex from the cytoplasm to the nuclei of target cells. We have tested a synthetic peptide based on the amino terminal region of the polyoma virus VP1 protein. This region has non-overlapping motifs for DNA binding and nuclear localization. METHODS: Luciferase gene transfer efficiency was evaluated using this peptide and a control peptide with a mutated NLS in subconfluent, confluent and polarized human bronchial epithelial (16HBE) cells compared to lipoplex alone. RESULTS: Gene transfer efficiency with a lipopolyplex containing the VP1 peptide enhanced gene delivery compared to lipoplex. Transfection with a lipopolyplex containing the control peptide failed to enhance gene delivery. The VP1 peptide increased the amount of plasmid associated with the nucleus while the mutant VP1 peptide did not. The order of lipopolyplex formation was important, with greatest enhancement when peptide was added to the plasmid before addition of the liposome. A bipartite peptide with the VP1 sequence and an integrin-binding motif (RGD) resulted in a reduction in gene transfer efficiency compared to lipoplex. Cell adhesion studies showed that the integrin binding associated with the RGD motif was lost when it was attached to the VP1 sequence. The combination of the two peptide sequences in cis may have compromised the function of both. CONCLUSIONS: Our results indicate that the VP1 peptide represents a strategy to enhance liposome-mediated gene delivery to airway epithelia in vitro. Comparison of transfection efficiencies between the VP1 and the mutant VP1 peptides and the direct measurement of plasmid associated with the nucleus suggests that this enhancement is caused by the NLS signal sequence in the peptide.     

信号肽与蛋白质的分选转运

蛋白质一般在位于细胞质中的核糖体内合成,但是它们发挥生理功能的地点却分布在细胞的不同区域,这些区域通常由蛋白质所不能自由透过的脂膜所包裹。因此,细胞质中新合成的蛋白质必须进行准确的定向运输才能保证各项生命活动的正常运行。研究发现,一般情况下,新生蛋白通常在位于其N端的信号肽的指引下到达细胞特定区域,并由其介导跨膜转运。本文重点介绍信号肽的结构、功能及作用机制等的研究成果。