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.     

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.     

山羊精子电穿孔转染外源DNA影响因素及最适条件的研究

本文以山羊精子为材料用电穿孔导入法转染外源基因,为生产转基因山羊奠定基础。用血细胞计数器在显微镜下记数死亡精子数检测精子活力,并用原位杂交实验检测电击精子消化前、后阳性率,以探讨山羊精子电穿孔转染外源DNA的方法及影响因素,摸索并优化电穿孔转染程序和条件。结果显示:电穿孔导入法可用于山羊精子介导转染外源DNA;电场强度、电击时间和电击次数同时影响精子活力,其中电击时间显著影响外源DNA的内化转运(P<0.05),山羊精子最适电穿孔条件为400V、200μs、电击1次,该条件下电击后精子活力和消化前、后阳性率分别为0.45和27.6%、22.5%;最适电穿孔条件下电击洗涤精子比未洗涤精子的消化后阳性率提高14%(P<0.01),将洗涤精子与外源DNA共孵育后再电击比不孵育处理组的消化后阳性率提高5.8%(P>0.05);电穿孔处理消化后阳性率在个体间无显著差异,且电击转染的精子被内化转运到精细胞内的外源DNA分布不规则。结果表明,山羊精子电穿孔转染外源DNA的最适转染条件为对精子充分离心洗涤并与外源DNA共孵育后,在400V/200μs条件下电击1次。     

氧化剂含量对聚苯胺／磁流体纳米复合粒子性能影响

用IR,UV,XRD分析了聚苯胺／磁流体纳米复合粒子性能与氧化剂过硫酸铵（APS）的定量关系。对制备的系列不同氧化剂含量的导电聚苯胺（PANI）／磁流体（Ni0.5Zn0.5Fe2O4氧体）纳米复合粒子样品的电性能和磁性能进行测试。结果表明,聚苯胺／磁流体纳米复合粒子的导电性随着APS用量的增加而降低。聚苯胺／磁流体纳米复合粒子UV谱线随着APS用量增加的峰逐渐红移,饱和磁化强度和矫顽力变化较小。     

无畸变极化转移增强技术（DEPT）在Ac－80核磁共振仪中的应用

无畸变极化转移增强技术是近几年来广泛应用在确定化合物，特别是对一些结构非常复杂，含不同数目氢原子的碳信号的指认和识别具有简单准确的特点．本文应用这一技术在Ａｃ—８０型核磁共振仪上（Ｂｒｕｋｅｒ），进行了一些实验性的摸索并取得满意的结果．     

nm23-H1基因对乳腺癌细胞MCF-7S增殖及移动特性的影响

目的：探讨nm23—H1基因转染乳腺癌细胞后对其增殖和转移能力的影响。方法：用人nm23—H1基因与真核表达质粒pcDNA3．1(—)构建成重组表达质粒pcDNA3．1—NMHl,转染人乳腺癌细胞MCF—7S,经G418筛选4周后,筛选出稳定表达nm23—H1的细胞株,绘制其生长曲线,检测其增殖、移动能力,用SPSS统计软件处理实验数据,分析nm23—H1基因对乳腺癌细胞的作用。结果：与对照组相比,转染nm23—H1基因的MCF—7S细胞的对数生长期延长,细胞增殖速度减慢,移动距离缩短,克隆形成率降低。结论：nm23—H1基因在乳腺癌细胞的体外实验中有抑制癌细胞生长、增殖、转移能力的作用。     

Preparation of Fe3O4@Au nano-composites by self-assembly technique for immobilization of glucose oxidase

Superparamagnetism amination nanocrystals Fe₃O₄ with 3-aminopropyltriethyloxy silane (APTES) were prepared by modified co-precipitation method. Next, 4-5 nm gold nanoparticles, prepared by classical Frens procedure, were coated on the surface of the amination Fe₃O₄ by self-assembly technology. The prepared Fe₃O₄@Au nanocomposite particles were investigated by transmission electron microscopy (TEM), UV-vis, infrared spectrum (FT-IR), and vibrating sample magnetometer (VSM) in order to elucidate the morphology, optics and magnetic properties of the nanocomposites. Their uniform distribution of particle size, which is about 15 nm, and good magnetic responsiveness were observed. In view of the fact that Fe₃O₄ owns superparamagnetism and that nano-gold can readily combine with biological molecules, glucose oxidase (GO _x ) was chosen as a model to penetrate the condition of immobilizing enzyme, and enzymatic properties of resultant immobilized enzyme were studied as well. By systematic optimization, we established that at 28°C, and pH (5.5) and when mole ratio of Fe₃O₄:HAuCl₄ was 0.5:1, the immobilization provided the best results. Finally, we are glad to find that the immobilized enzyme exhibits excellent thermostability in addition to its better stability than free enzyme. Thus, herein described immobilized enzyme could be used repeatedly with the assistance of an external magnetic field. Supported by the Science Foundation of Sichuan Province, China (Grant No. 2005A033) and Science Foundation of Sichuan Agricultural University for Distinguished Young Teachers (Grant No. 007202)     

基因优化网络技术在磁悬浮轴承控制中的研究

针对普通BP算法收敛速度慢，容易陷入局部极小值以及网络初始状态对网络性能的影响等问题，建立了单自由度磁悬浮控制系统的数学模型，设计了一种基因算法优化神经网络的智能控制方法，该方法提出用基因算法离线优化网络。并设计了一种双链异或交叉基因算子，改善了基因算法的效率，在较大程度上改善了单自由度磁悬浮系统的性能。     

硬磁CoFe2O4/软磁CoFe2复合物的磁性研究

采用金属有机盐热分解方法制备出均匀、分散的硬磁CoFe_2O_4纳米颗粒,在H2环境、低温条件下进行还原,合成硬磁CoFe_2O_4/软磁CoFe_2复合磁体.采用低温还原,目的在于尽可能减少粒子的聚集.制备出的样品具有较好的磁性能:有较高的剩磁比Mr/Ms,其值为0.64;随着还原时间延长,饱和磁化强度增加至124emu·g~(-1),同时保持较高的矫顽力.分析认为,可以通过减弱粒子间的偶极相互作用来有效提高硬磁/软磁复合磁体的矫顽力和剩磁比.     

一次氧气对氢氧焰水解制备纳米TiO_2颗粒的影响

在氢氧焰燃烧合成细米TiO2时,一次氧气浓度直接影响燃烧过程性质、火焰的特征。通过调节一次氧气浓度,可以得到大小、形貌和晶型不同的纳米颗粒。在未达到化学计量比以前,φO2增加,二氧化钛一次粒子的粒度减小,粒度分布变窄,颗粒中的金红石含量显著下降。当四氯化钛进料浓度为0.47mol/m3,φO2由0增加到0.10时,纳米TiO2颗粒平均粒径由70nm降到32nm,金红石的质量分数由0.93降到0.03。水解制备的TiO2颗粒为球形。     

RF-PCVD法制备纳米TiO_2粒径分布的数值模拟及进料浓度的影响

射频等离子体反应器适用于气相法快速合成高纯纳米微晶.针对小流量TiCl4氧化合成TiO2纳米微晶反应体系,在Matlab软件中采用有限差分法,以求解温度场的二阶偏微分方程模拟出反应器内的温度分布.通过将温度场与长大过程动力学方程偶联,沿每条气体流线计算,模拟出最终TiO2纳米微晶粒径及其粒度分布.不同进料浓度条件下的数值解与实验结果基本吻合.由于流动状态和浓度分布过于理想以及未考虑长大过程中传质因素的影响等,数值解与实验结果还存在一定的差距.     

用FORC研究纳米双相Fe3B/Nd2Fe14B的微观磁学特征

通过熔融快淬法制备具有非晶结构的Nd4.5Fe77B18.5合金,在氩气保护下660℃、10 min热处理获得了最佳磁性能的纳米双相复合永磁材料.由于材料具有双相复合纳米结构,磁体内部的微观磁化行为显示出复杂的交互作用.引入一阶回转曲线图谱法(FORC)研究材料的磁化机制和表征内部的交互作用.该材料的FORC图谱显示:纳米双相材料中存在明显的可逆磁化与不可逆磁化,同时两者相互耦合,耦合作用体现在图谱中的负值区域.不可逆磁化磁矩之间存在强烈的交互作用,体现在不可逆磁化峰的向下偏移和不对称性,整体表现出退磁特性,同时在δM曲线中得到证实.     

稻瘟病抗性基因 Pi63 启动子4 个构建体的表达及抗性分析

利用农杆菌介导法获得带有不同长度启动子区域的稻瘟病抗性基因Pi63全长基因的转基因水稻,并采用实时荧光定量PCR方法鉴定了T1代阳性植株的基因拷贝数,进一步测定了单拷贝转基因植株在接种稻瘟病菌前后Pi63基因的表达量变化.结果表明:接种后4个分别带有不同长度启动子的Pi63转基因植物T1代中Pi63基因的表达量均升高,其抗病相关的顺式作用元件处于P1与P2中间的区域,P0到P1的区域存在负调控元件.此结果可为进一步揭示水稻稻瘟病抗性基因Pi63的分子机理奠定基础.