线性与分枝状聚乙烯亚胺介导基因体外转染的比较与研究

聚乙烯亚胺(PEI)是一类新兴的阳离子多聚物非病毒载体,可缩聚DNA分子形成颗粒并转入真核细胞进行表达.本文选用分子量750 ku分枝状PEI与分子量25 ku线性PEI转染增强型绿色荧光蛋白(EGFP)报告基因于HeLa细胞,并对这两种PEI的转染效率进行比较.利用MTT法检测比较线性PEI与分枝状PEI的细胞毒性.通过凝胶阻滞实验比较两种PEI结合DNA能力.最后利用肝素介导释放实验比较两者在形成PEI/DNA复合物后释放DNA的能力.研究结果表明:线性PEI转染效率优于分枝状PEI,且随PEI/DNA质量比的增加而升高,而分枝状PEI则相反;两种PEI细胞毒性在一定范围内相近;分枝状PEI结合DNA能力略强于线性PEI;但是分枝状PEI释放DNA的能力远小于线性PEI.这可能导致DNA在细胞内无法从PEI/DNA复合体上释放出来,从而影响转录的正常进行,最终导致分枝状PEI转染效率下降.     

基于生育酚修饰的低分子量聚乙烯亚胺基因载体

制备了基于生育酚交联的低分子量聚乙烯亚胺(PEI)可降解脂质体聚合物基因载体。凝胶阻滞实验结果表明它能很好地包裹质粒DNA,使其免受核酸酶的降解。通过在体外He La细胞和HEK 293细胞中的绿色荧光蛋白转染实验结果表明,这种生育酚修饰的聚合物材料在无血清存在下表现出比"黄金标准"PEI高的转染效率。因此,推测这种材料具备了作为非病毒基因载体的潜在性。     

聚乙烯亚胺在锰锌铁氧体纳米粒子表面定量吸附

以可自动恒温控温的锰锌铁氧体磁性纳米粒子(MZF-NPs)为核心,在其表面修饰聚乙烯亚胺(PEI)以制备一种新型纳米基因载体.利用扫描电镜、红外光谱仪、Zeta电位仪对其形貌、表面包覆功能团、电位等进行表征;UV/vis光谱仪及DNA体外结合、释放、转染实验研究了PEI在MZF-NPs上的定量吸附与修饰效果.电镜下修饰后粒子之间的聚集明显减轻、等电点由pH 7.0移至pH 11.5.PEI质量、介质的pH值以及离子强度均可影响PEI在锰锌铁氧体上的吸附.不同的吸附量也影响了纳米粒子的DNA结合、释放与转染能力.     

组氨酸修饰的聚乙二醇-聚谷氨酸-聚乙烯亚胺作为基因载体的体外性能研究

为了对组氨酸修饰的阳离子聚合物载体进行研究,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。采用自由基聚合法合成聚乙二醇-聚谷氨酸-聚乙烯亚胺-组氨酸(PEG-b-PLG-g-PEIsHISs,GGIS)聚阳离子载体,用1H-NMR表征载体材料结构;通过凝胶电泳实验研究载体对质粒DNA的压缩能力,粒径分析仪测定载体结合DNA后在不同N/P比条件下的粒径及表面电荷;用MTT法测定载体在HEK293T,Hela,BEL7402和A549等细胞株上的细胞毒性,考察GGIS体外细胞转染效率。结果显示在N/P≤30时,载体材料的平均粒径在100~200 nm,表面电荷在10~30 m V,适合细胞吞噬。体外细胞毒性表明,GGIS的细胞毒性较PEI 25K低。GGIS具有较强的DNA缩合能力,且有较好的体外基因转染能力。因此,GGIS有较好的体外基因转染能力,能够携带报告基因在体外有效表达,是具有应用前景的非病毒性基因药物载体。     

一种新颖的阳离子非病毒基因载体 Chitosan-g-PEI-g-PEG-OH 的性能研究

研究了新型阳离子聚合物Chitosan-g-PEI-g-PEG-OH的性能，重点考察其粒度，基因转染效率与细胞毒性，探讨了其作为基因载体的可能性.通过动态光散射仪（DSL）、透射电镜（TEM）观察了Chitosan-g-PEI-g-PEG-OH与DNA自组装形成的颗粒形态及粒径，Chitosan-g-PEI-g-PEG-OH可复合DNA形成粒径160～210 nm的纳米复合物，适合进入细胞.使用MTT比色法分析Chitosan-g-PEI-g-PEG-OH的毒性并与PEI，PEI-g-PEG-OH比较.选用增强型绿色荧光蛋白(EGFP) 转染Hela细胞，应用流式细胞术检测转染效率.新型阳离子多聚物Chitosan-g-PEI-g-PEG-OH在提高基因转染效率的同时降低了其细胞毒性，有望成为基因转移的有效载体.     

构建HMGB1/PEI非病毒载体介导高效基因传递的研究

 研究了以聚乙烯亚胺（PEI）为骨架复合高迁移率族蛋白 B1 (HMGB1 )的复合型载体HMGB1/PEI的性能,以期提高非病毒基因载体的转染效率。透射电镜观察pDNA/HMGB1/PEI复合物粒子形态呈球形;动态光散射法测定粒径与表面电位,结果显示复合HMGB1后,复合物粒径降低,且随HMGB1加入量的增大表面电位有增大的趋势;凝胶电泳阻滞试验表明HMGB1可协助PEI与pDNA结合;MTT试验结果显示HMGB1/PEI复合载体的细胞毒性低于PEI;HMGB1/PEI复合载体的转染率较PEI的转染率增大2.9~4.0倍,且HMGB1可以弱化血清对转染的阻碍作用。所以HMGB1被证实能有效提高PEI的体外转染效率。     

以芳香亚胺为连接剂的聚乙烯亚胺衍生物的合成及体外活性研究

通过有机合成的方法合成新型聚阳离子载体聚五乙烯六胺对苯二甲醛-1,4-二亚胺(PEHA-TPAI),产物结构经核磁共振氢谱(1H NMR)和傅里叶红外光谱(FT-IR)确定系目标产物。采用琼脂糖凝胶电泳实验观察PEHATPAI包裹小干扰RNA(siRNA)的能力,并用粒度仪和Zeta电位仪对PEHA-TPAI/siRNA复合物进行表征。结果表明:PEHA-TPAI和siRNA复合后可形成稳定且粒径均一的纳米颗粒,在PEHA-TPAI/siRNA复合物质量比为10∶1的条件下,颗粒粒径为(129.90±1.02)nm,Zeta电位为(21.1±0.54)mV。体外细胞毒性实验和转染实验的结果表明:PEHA-TPAI的细胞毒性显著低于商业化转染试剂聚乙烯亚胺(PEI,分子量为25 000)(P0.001)且具有良好的转染效率,有作为低毒性、高转染效率的基因输送载体的潜力。     

聚乙烯亚胺介导生存素反义核酸体外投递肝癌SMMC-7721细胞的条件优化

目的:探讨聚乙烯亚胺(polyethylene im ine,PEI)作为生存素(survivin)反义核酸(anti-sense oligodeoxynuc leotides,ASODN)的载体投递肝癌SMMC-7721细胞的条件。方法:凝胶阻滞实验筛选PEI与survivin ASODN形成静电复合物的最佳质量比;W ST-8法检测PEI对肝癌细胞的毒性;流式细胞仪及W ST-8法筛选荧光标记的PEI与ASODN在不同质量比时的瞬时转染效率及48 h转染效率;流式细胞仪及荧光显微镜分别检测荧光标记的ASODN以及PEI-ASODN复合物进入细胞的情况;W ST-8法检测血清对PEI转染效率的影响。结果:m(PEI)∶m(ASODN)为0.625∶1~2.5∶1时可以形成电中性状态的静电复合物;PEI终质量浓度≤4μg/mL对肝癌细胞的毒性较小;m(PEI)∶m(ASODN)为0.75∶1时转染效率最高;血清存在对PEI转染效率无明显影响。结论:PEI是一种低毒的ASODN投递载体,其终质量浓度≤4μg/mL可作为肝癌细胞的转染载体,血清条件下m(PEI)∶m(ASODN)为0.75∶1时,PEI携带sur...     

环氧开环聚合制备的基因载体研究

缺乏安全高效的基因载体制约了基因治疗的发展。该综合实验通过环氧开环聚合反应设计合成三种阳离子聚合物基因载体。环氧开环反应生成的羟基均匀分布在聚合物骨架上,聚合物骨架中还含有脂肪链或芳香环,用于调节聚合物刚性,并协同羟基共同调节亲水/疏水平衡。采用核磁共振氢谱和凝胶渗透色谱表征聚合物结构,然后研究聚合物与DNA结合能力、细胞毒性、蛋白吸附能力和转染效率,进一步得到环氧开环聚合物作为基因载体的初步构效关系。     

复合环境离子强度对BDCP/DNA的粒径与转染效率的影响

为了探讨组装环境对基因载体/DN复合物的粒径和转染效果的影响,在三种不同离子浓度溶液体系(PBS, 5% 葡萄糖溶液, H₂O)中测量BDCP（biodegradable cationic polymer,一种生物可降解的阳离子聚合物）/DNA复合物粒径和结合力,进行了体外转染试验和毒性试验.结果显示,PBS最适合组装转染复合物,可取得更好的稳定性、最高的转染效率和较低细胞毒性、低溶血率;在5%葡萄糖溶液和水中组装的BDCP/质粒复合物结合力较弱,转染效率比较低.得出结论,BDCP/DNA粒径、结合力和基因转染效率受组装体系的离子强度影响.     

一种非病毒载体的组成与体外转基因能力的研究

目的：合成羟丙基β-环糊精偶联小分子量聚乙烯亚胺的共聚物,探讨不同聚乙烯亚胺偶联率的共聚物缩合DNA的能力,探讨不同偶联率的阳离子共聚物组成与转基因效率的关系。方法：通过NMR表征,凝胶阻滞DNA试验考察其缩合DNA能力,以MTT法检测不同偶联率共聚物的细胞毒性。以不同的偶联率共聚物为载体,在不同的N/P比值条件下,将荧光素酶基因导入非洲绿猴肾细胞COS-7,通过荧光素酶的表达检测比较不同偶联率的共聚物转染效率。结果：合成的不同偶联率的共聚物缩合DNA能力不同,转染效率也不同。结论：聚乙烯亚胺偶联率较高的阳离子共聚物缩合DNA的能力较强,转染效率较高。     

纳米谷氨酸壳聚糖制备及其体外质粒转染研究

采用表面修饰方法制备出谷氨酸修饰的壳聚糖纳米基因载体。对样品进行红外分析、粒度分析、zeta电位分析、生物相容性、凝胶阻滞分析、DNA保护性试验、体外细胞转染研究。结果显示所制得的谷氨酸修饰的壳聚糖纳米颗粒平均粒径为170nm,其zeta电位为 4.7mV。红外分析显示谷氨酸已通过酰胺键结合在壳聚糖上。MTT实验结果显示纳米颗粒与细胞有良好的生物相容性。凝胶阻滞分析和DNA保护试验结果表明纳米载体可与DNA通过电性结合作用而结合,并可以有效保护DNA,防止核酸酶对其的降解作用。而体外细胞转染的结果表明,谷氨酸修饰的纳米粒能介导pEGFP-N1质粒转染HepG2细胞并在细胞中表达绿色荧光蛋白。因此,谷氨酸修饰的壳聚糖纳米颗粒可作为一种新型非病毒基因载体介导核酸类生物大分子进入细胞内。     

Enhancement of transfection efficiency for HeLa cells via incorporating arginine moiety into chitosan

Arginine-rich peptides have attracted considerable attention due to their distinct internalization mechanism. It was reported that arginine and guanidino moieties were able to translocate through cell membranes and played a critical role in the process of membrane permeation. In this work, arginine was conjugated to the backbone of chitosan to form a novel chitosan derivative, arginine modified chitosan (Arg-CS). Arg-CS/DNA complexes were prepared according to the method of coacervation process. The physicochemical properties of Arg-CS and Arg-CS/DNA complexes were characterized and the transfection activity and efficiency mediated by Arg-CS/DNA complexes were investigated taking HeLa cells as target cells. Arg-CS was characterized by FTIR and 13C NMR. Arg-CS/DNA polye- lectrolyte complexes were investigated by agarose gel retardation, dynamic light scattering (DLS) and atomic force microscopy (AFM). The results revealed that the Arg-CS/DNA complexes started to form at N/P ratio of 2:1, and the size of particles varied from 100 to 180 nm. The cytotoxicity of Arg-CS and their complexes with plasmid DNA were determined by MTT assay for HeLa cells, and the results suggested that Arg-CS/DNA complexes were slightly less toxic than Arg-CS. Moreover, the derivative alone and their complexes showed significantly lower toxicity than PEI and PEI/DNA complexes, respectively. Taking HeLa cells as target cells and using pGL3-control as reporter gene, the luciferase expression mediated by Arg-CS was greatly enhanced to about 100 folds compared with the luciferase expression mediated by chitosan at different pH media. These results suggest that Arg-CS is a promising candi- date as a safe and efficient vector for gene delivery and transfection.     

Enhancement of transfection efficiency for HeLa cells via incorporating arsinine moiety into chitosan

Arginine-rich peptides have attracted considerable attention due to their distinct internalization mechanism. It was reported that arginine and guanidino moieties were able to translocate through cell membranes and played a critical role in the process of membrane permeation. In this work, arginine was conjugated to the backbone of chitosan to form a novel chitosan derivative, arginine modified chitosan （Arg-CS）. Arg-CS/DNA complexes were prepared according to the method of coacervation process. The physicochemical properties of Arg-CS and Arg-CS/DNA complexes were characterized and the transfection activity and efficiency mediated by Arg-CS/DNA complexes were investigated taking HeLa cells as target cells. Arg-CS was characterized by FTIR and ^13C NMR. Arg-CS/DNA polyelectrolyte complexes were investigated by agarose gel retardation, dynamic light scattering （DLS） and atomic force microscopy （AFM）. The results revealed that the Arg-CS/DNA complexes started to form at N/P ratio of 2：1, and the size of particles varied from 100 to 180 nm. The cytotoxicity of Arg-CS and their complexes with plasmid DNA were determined by MTT assay for HeLa cells, and the results suggested that Arg-CS/DNA complexes were slightly less toxic than Arg-CS. Moreover, the derivative alone and their complexes showed significantly lower toxicity than PEI and PEI/DNA complexes, respectively. Taking HeLa cells as target cells and using pGL3-control as reporter gene, the luciferase expression mediated by Arg-CS was greatly enhanced to about 100 folds compared with the luciferase expression mediated by chitosan at different pH media. These results suggest that Arg-CS is a promising candidate as a safe and efficient vector for gene delivery and transfection.     

抗人PD-L1单克隆抗体的瞬转表达

利用HEK293 6E细胞瞬转表达抗人PD L1单克隆抗体. 将HEK293 6E细胞无血清悬浮培养, 筛选最佳生长培养基; 考察HEK293 6E细胞的筛选表达培养基、 DNA和PEI质量比及转染时细胞密度等转染条件, 并对收获的抗体进行SDS PAGE和ELISA鉴定. 结果表明: 在A无血清表达培养基中, 以2×106个/mL的细胞密度和m(DNA)∶m(PEI)=1的条件转染, 第4天收获抗体的产量最高, 为170.9 μg/mL; 收获PD-L1抗体的重链分子量约为50 000, 轻链分子量约为25 000.     

Enhancement of the efficiency of PEI/liposome transfection by magnetofectins formed via electrostatic self-assembly

One of the major challenges for successful gene therapy is improving the transfection efficiency of non-viral vectors. Magnetic nanoparticles (MNPs) have been developed as enhancers of non-viral vehicles. We prepared MNPs and modified them with polyethyleneimine (PEI), citric acid (CA) or carboxylmethyl-dextran (CMD). Both positively charged MNPs (MNPs@PEI) and negatively charged MNPs (MNPs@CA, MNPs@CMD) could spontaneously form transfection complexes (magnetofectins) with plasmid DNA and PEI/liposome via electrostatic self-assembly. Our results showed as-prepared magnetofectins apparently enhanced PEI/liposome transfection efficiency and/or gene expression level into COS-7 cells with reduced transfection time from 4 h to 15 min under a magnetic field in vitro. Meanwhile, the effect of magnetofection was cell line-dependant. These results suggest that charged MNPs could improve transfection efficiency for non-viral vectors by simply mixing with them and by exerting a magnetic force. Thus such MNPs provide a convenient platform for further applications of gene delivery.     

Adsorption of polyethyleneimine characterized by spectroscopic ellipsometry

Polyethyleneimine (PEI) is a well known gene transfection reagent that has been extensively studied in the pass two decades. In this work, we have explored its physiochemical properties by examining its interfacial adsorption at the hydrophilic silicon oxide/water interface and its ability to immobilize bioactive DNA under physiological conditions. Its surface excess was found to show a gradual increase with pH between pH 3 and pH 10. At pH 7 the surface adsorbed amount increased with increasing salt concentration. The amount of DNA immobilized was found to be controlled by the charge ratio between PEI and DNA under these conditions. Thus, the amount of bioactive DNA immobilized can be tuned by adjusting the amount of PEI preadsorbed. This study provides useful information for fabrication of biosensors and gene chips via interfacial adsorption.     

不同分子质量软骨多糖对K562细胞的增殖抑制作用

研究不同分子质量的软骨多糖对慢性髓系红白血病K562细胞的增殖抑制作用。用不同浓度的酒精对软骨提取物进行分级沉淀，得到不同分子质量的软骨多糖，并用聚丙烯酰胺凝胶电泳（SDS-PAGE）检测所得软骨多糖的纯度及分子质量；采用噻唑蓝（MTF）比色法测定细胞的增殖活性；用琼脂糖凝胶电泳法检测DNA电泳特征。结果显示，短链软骨多糖（分子质量约为30ku）对K562细胞具有明显的增殖抑制作用，这种抑制作用呈现明显的时间依赖性，且琼脂糖凝胶电泳出现明显的DNA梯状条带。     

Non-viral gene carrier mediated short hairpin RNA interference for inhibition of tumor cells growth

A tumor-targeting gene vector G250mAb-PEI-PEG has been prepared by modification of polyethylenimine （PEI） with polyethyleneglycol （PEG） and G250, a monoclonal antibody against the G250 antigen on tumor cell surface. The transfection efficiency was as high as 70% in G250 positive HeLa cells, whereas the transfection efficiency was relatively low （30%） in normal NIH3T3 cells. A plasmid encoding the short hairpin RNA （shRNA） specific for nucleostemin gene （NS） was efficiently transfected into the HeLa cells with this nonviral gene vector. RNA interference down-regulated the expression of NS gene in HeLa cells, inhibited cells proliferation and induced apoptosis. However, the growth and activity of the NIH3T3 cells were not affected under the same treatment. These results indicate that the reported nonviral gene vector, G250mAb-PEI-PEG, can target and efficiently deliver genes into HeLa cells, and has the potential for the cervical cancer treatment.