基于二氧化硅微颗粒促细胞增殖效应的基因转染新方法

报道了二氧化硅微颗粒(SiMPs)的促细胞增殖效应, 并基于该效应发展了一种以二氧化硅微颗粒为转染伴侣的基因转染新方法, 采用MTT实验证明了二氧化硅微颗粒具有促细胞增殖效应, 并以绿色荧光蛋白表达载体质粒pEGFP为报告基因, COS-7细胞为靶细胞, 在多聚-L-赖氨酸介导的基因转染中, 利用二氧化硅微颗粒的促细胞增殖效应, 加入二氧化硅微颗粒作为转染伴侣开展基因转染实验, 获得了显著增强的基因转染效率, 相比于未加入二氧化硅微颗粒为转染伴侣的基因转染方法, 该方法的转染效率提高到5倍多. 利用二氧化硅微颗粒的促细胞增殖效应, 以其作为转染伴侣的基因转染新方法不仅为相关研究提供了一种高效简便的基因转染新方法, 而且也为基因转染效率的提高提供了新的思路.     

电击法磁性纳米颗粒作为水稻转基因载体的研究

建立了以磁性纳米颗粒为载体,由电击法介导基因导入植物细胞的优化方法。制备了粒径小于10nm的磁性纳米颗粒,与绿色荧光蛋白(Green fluorescent protein,GFP)基因融合制备基因载体,同时,制备水稻悬浮细胞,加入电极杯中,调节电击条件为:电压分别是800,1000和1200V,电容25μF,电阻200Ω,直径10mm,电击数次。利用倒置荧光显微镜观察转化后的悬浮细胞,可以明显看到绿色荧光蛋白在水稻细胞内表达。说明纳米颗粒基因载体在电击作用下能有效进入细胞,利用磁性纳米颗粒作为基因载体电击法转化植物细胞初步成功。     

基于超声波下淀粉纳米颗粒作载体的基因转导

报道了多聚赖氨酸淀粉纳米颗粒(PLL-StNP)在超声波介导下作基因载体的研究,实验发现DNA-PLL-StNPs复合物经过超声波处理不同的时间后的电泳分析显示,结合的DNA受到保护,其生物学性质没有改变;将pIRGFP质粒DNA-PLL-StNPs复合物与COS-7细胞混合,在120W和40kHz的超声波强度下处理2min,细胞表达绿色荧光蛋白的比率最大,达到70.这种基于超声波下淀粉纳米颗粒作载体的基因转导方法可被广泛应用于动物转基因技术和人类基因治疗,同样可被广泛应用于植物转基因技术.     

纤维素磁性微球的制备及其生物活化研究

通过基因重组技术将增强型绿色荧光蛋白(EGFP)基因插入含有纤维素结合域(CBD)标签的载体pET-35b(+),使EGFP与CBD进行融合表达,表达的融合蛋白分子量约为53kDa,该蛋白既具有与纤维素结合的功能,又能够作为示踪信号.此外,通过反向悬浮包埋技术制备纤维素磁性微球(MCMS),并对其性能进行了考察,其平均粒径为(158.7±14.7)μm,磁含量为(3.83±0.29)%,磁响应时间为(66±2.9)s,磁漏损为(0.139±0.06)‰,蛋白的偶联量和漏损量分别为(12.27±0.82)mg/mL和(77±7.35)ng/mL.经由EGFP的示踪信号观察制备的MCMS与CBD具有良好的结合能力,吸附后微球表面具有明显的绿色荧光信号,本研究建立的CBD-MCMS将为后继的MCMS的生物活化和随后的分离、纯化等工作奠定基础.     

包裹RuBpy二氧化硅荧光纳米探针的制备及其用于肝癌细胞的识别

制备了两种不同官能团修饰的偶联亲和素的包裹钌联吡啶(RuBpy)二氧化硅荧光纳米探针A和探针B,并分别用于肝癌细胞的识别。通过反相微乳液法制备得到表面修饰不同官能团的纳米颗粒,然后通过亲和素与羧基化包裹RuBpy二氧化硅纳米颗粒相互连接而制备得到探针A;通过亲和素与PEG修饰的荧光二氧化硅纳米颗粒相互作用而制备得到探针B。与探针A不同的是,探针B通过一个长链PEG分子将亲和素与荧光二氧化硅纳米颗粒偶联在一起。利用免疫荧光成像法将这两种探针分别用于人肝癌细胞的识别,结果表明,含有长链PEG分子的探针B更能够有效地识别肝癌细胞表面肿瘤标志物癌胚抗原(CEA)。     

新型双荧光二氧化硅纳米颗粒的制备

本文采用改良的反相微乳液法制备得到双染料掺杂的二氧化硅荧光纳米颗粒。扫描电镜结果显示,荧光纳米颗粒呈球形,平均粒径约为90nm。利用荧光光谱及共聚焦荧光显微镜对该纳米颗粒的光学性能进行表征,结果发现其可同时发出红、绿双色荧光,这为进一步制备具有更多种荧光信号的硅纳米颗粒提供基础。同时,由于硅纳米颗粒生物相容性好,易功能化的特点,使得该新型硅纳米荧光颗粒在生物标记及生物成像的多组分分析中具有潜在的应用。     

信号肽功能化二氧化硅纳米颗粒的线粒体靶向作用研究

以N-(p-Maleimidophenyl)isocyanate(PMPI)为交联剂, 将线粒体信号肽分子共价修饰到二氧化硅荧光纳米颗粒表面, 构建线粒体信号肽功能化二氧化硅荧光纳米颗粒. 采用荧光分光光度计、Zeta电位仪以及透射电子显微镜对修饰前后的二氧化硅纳米颗粒进行了表征. 结果表明, 信号肽可被成功修饰在纳米颗粒表面, 并且纳米颗粒粒径在信号肽分子修饰前后没有发生明显变化. 以分离纯化的细胞核作为对照, 采用流式细胞术考察了信号肽功能化二氧化硅荧光纳米颗粒与分离纯化后的线粒体的相互作用. 结果表明, 线粒体信号肽修饰到二氧化硅纳米颗粒表面后依然保持良好的生物活性, 能够介导二氧化硅纳米颗粒特异性识别及结合分离纯化的线粒体, 从而为线粒体监测及其功能调控研究提供了新的思路.     

载有量子点的介孔二氧化硅微球的制备与性能研究

采用自组装方法制备了一种磁核/介孔二氧化硅壳的微球, 调节体系中C₁₈TMS的加入量可控制介孔硅球的比表面积; 并通过化学修饰的方法对介孔微球表面进行巯基功能化修饰. 利用巯基与量子点之间的相互作用可将一定尺寸的量子点吸附于介孔二氧化硅球的孔中, 令介孔微球具有荧光效应; 同时可以利用吸附不同粒径的量子点的荧光光谱对介孔二氧化硅微球孔径的大小进行近似考察.     

氨基化单分散量子点/二氧化硅核壳纳米粒子的制备及其细胞标记

通过反向微乳液法, 在油溶性量子点表面包裹二氧化硅外壳, 使油溶性量子点水溶性化, 再利用3-氨丙基三乙氧基硅烷(APTES)在已形成的二氧化硅纳米颗粒表面进行氨基化改性, 制备富含氨基的二氧化硅包裹的量子点荧光纳米球. 通过透射电子显微镜(TEM)、粒径分析、zeta电位检测、紫外-可见分光光度、荧光分光光度和红外光谱等手段对产品进行了表征. 结果表明, 所制备的二氧化硅量子点纳米球(45 nm)具有单分散性、水溶性好及光化学稳定性强等优点. 通过静电作用, 所制备的单分散氨基化二氧化硅量子点对肿瘤细胞表面膜电荷进行了初步标记显像.     

主客体组装构建环糊精修饰基因微载体的研究

合成了二茂铁接枝聚乙烯亚胺( PEI-Fc),利用二茂铁与β-环糊精的主客体嵌套作用制备了环糊精修饰聚乙烯亚胺,核磁测定结果显示,每条PEI-Fc链上通过主客体作用嵌套的CD平均为26个.这种基于弱相互作用力的β-环糊精修饰聚乙烯亚胺能有效诱导DNA分子的缔合,在N/P值达到3以上时,可形成表面为正电荷、粒径为150 ～ 250 nm的球形粒子.在含10％胎牛血清的DMEM体外细胞培养基中,由于培养基中的蛋白质能够在粒子表面发生静电吸附,PEI-Fc/CD/DNA基因微载体显示出良好的稳定性.HEK293细胞培养结果显示,以表达绿色荧光蛋白的质粒pEGFP为模型,以N/P值为10的PEI/DNA组装体作为对照,N/P值为3、5和10的PEI-Fc/CD/DNA组装体的转染效率均达到对照组的2～3倍,这种基于主客体组装构建的环糊精修饰基因微载体显著提高了基因转染效率.     

Delivering DNA into Plant Cell by Gene Carriers of ZnS Nanoparticles

The development of nanotechnology provides a new method for genetic engineering. However, the nanoparticles as gene carriers have been mainly used in the mammalian cells so far. We observed that ZnS nanoparticles modified with positively charged poly-L-lysine(PLL) successfully delivered GUS-encoding plasmid DNA into tobacco cells by means of ultrasound-assisted method. Polymerase chain reaction(PCR) detection, Southern blot analysis and GUS histochemical staining were carried out for the regenerated plants. The stable genetic modified plants mediated by ZnS nanoparticles can be obtained. This article demonstrates the great potential of nanoparticles as gene carrier in plant transformation and proves a novel approach for plant genetic decoration.     

Study of catalytic action of micro-particles and synthesized nanoparticles of CuS on cellulose pyrolysis

The catalytic action of copper sulfide (CuS) micro-particles and as-synthesized nanoparticles was studied on cellulose pyrolysis. The market procured CuS powder was used as micro-particles without any treatment. The CuS nanoparticles were synthesized at ambient temperature by simple wet chemical technique. Before using the micro-particles and nanoparticles for catalytic study, they were comprehensively characterized. The thermal analysis including catalytic properties of both the micro-particles and nanoparticles of CuS on cellulose pyrolysis was studied employing thermogravimetric (TG), differential thermogravimetric, and differential thermal analysis techniques. Prior to the study as catalyst in cellulose pyrolysis, the CuS micro- and nanoparticles were characterized by thermal analysis in inert atmosphere. The TG curves showed two steps and five steps decomposition having total mass loss of 29 and 42 % in case of CuS micro- and as-synthesized nanoparticles, respectively. The catalytic study in cellulose pyrolysis showed that the decomposition commences at temperature 295 °C for pure cellulose, 270 °C for cellulose mixed with 3 % CuS micro-particles and 205 °C for cellulose mixed with 3 % CuS nanoparticles. It clearly showed that the decomposition starting temperature decreased by 65 °C in case of cellulose mixed with CuS nanoparticles compared to cellulose mixed with CuS micro-particles. Thus, CuS nanoparticles act as better catalyst then CuS micro-particles in cellulose pyrolysis. The obtained results are deliberated in details.     

Migration analysis of micro-particles in liquids using microscopically designed external fields.

The recent development of new migration methods of micro-particles in liquids using various external fields is reviewed. The combination of a laser scattering force and a photothermal effect produced photothermal-conversion laser-photophoresis. A dielectric field generated in a planer or a capillary quadrupole electrode realized dielectrophoresis. Using a micrometer-scaled magnetic field gradient, the "Magnetophoretic velocimetry" of micro-particles was invented. Furthermore, the Lorentz force generated by combining an electric field and a magnetic field was utilized for electromagnetophoresis. These new methods were overlooked and the advantages in analytical use were discussed.     

纺锤形β-FeOOH纳米结构和α-氧化铁亚微米/微米粒子的合成与转变

使用一种简易的无表面活性剂辅助的水热合成方法,在温度为140 ℃时实现了纺锤形β-FeOOH纳米结构向α-氧化铁亚微米/微米粒子的转变。研究表明,通过实验参数的简单调控,实现了单晶α-氧化铁亚微米粒子与β-FeOOH的纺锤形纳米结构和纳米棒的控制制备。基于实验结果,提出了该过程中的相转变机理。     

Biodegradable starburst carbon chain polymer-protein and lysine dendrite conjugates: adjustment of immune properties,DNA bonding and delivery

In the past decade, the development of gene therapy technology has focused on the design of new nonviral carriers for gene delivery. Proteins modified with polyethyleneimine^[1] or polylysine^[2] as well as dendrites^[3] have shown to be perspective carriers for DNA targeted delivery. The usage of protein conjugates as carriers of biologically active compounds will depend on the adjustment of their immune properties. To investigate this we have prepared starburst carbon chain polymer/protein conjugates containing low molecular weight biologically active compounds, salsolinol and bradykinin, in the polymer moieties and studied their immune properties. We have shown that chemical structure of the polymer moiety determines the conjugate biodegradation as well as their immune properties. The starburst poly(N-vinylimidazole) transferring poly(N-vinylimidazole) and polylysine 3G lysine dendrite conjugates have been prepared. The study of their ability to bind DNA and to guarantee its targeted delivery have shown that they are perspective DNA carriers.     

Recent progress in cationic polymeric gene carriers for cancer therapy

In recent years,various carriers for gene delivery nave been developed for biomedical applications.Among all kinds of gene carriers,cationic polymeric carriers for delivery therapeutic gene as non-viral carriers have received growing interests due to their improved high transfection efficiency with the relative safety.In particular,the advancement of novel polymeric gene carriers has gained much progress in the development of effective anticancer therapy.Herein,this review focused on the development of cationic polymeric carriers for cancer therapy,including polyethylenimine(PEI),polyamidoamine(PAMAM) dendrimers,polylysine(PLL),chitosan and modified cationic polymers.And recent progresses in the development of novel polymeric carriers for gene delivery,such as targeted gene carriers,responsive gene carriers and multifunctional gene carriers,were summarized.Finally,the future perspectives in the development of novel polymeric carriers for delivery gene were presented.     

A method to enhance porosity of micro-particles

The nuclear track technique (NTT) is used to enhance the porosity of silica micro-particles. The enhanced porosity is a result of the formation of surface and interior pores or tracks in the silica by the action of external and internal fission fragments. The fission tracks produced at the surface and within the interior of the micro-particles are a result of coating the particles with trace quantitities of uranium, instead of having trace quantities of uranium incorporated within the silica matrix.     

Sphere to disk transformation of micro-particle composed of azobenzene-containing amphiphilic diblock copolymers under irradiation at 436 nm

Diblock copolymer composed of azobenzene-containing polyacrylate and poly(acrylic acid) (PAzoM-b-PAA) was prepared using reversible addition-fragmentation chain transfer (RAFT) radical polymerization, which was found to form micro-particles through self-assembly. Under irradiation of light at 436 nm sphere to disk transformation of the micron-particle was observed in situ through an optical microscope. Detailed spectral analysis showed that a disruption of H-aggregate of azobenzene units had taken place during the transformation, from which it is realized that possible driving force of the transformation comes from the photoinduced disruption of H-aggregates within the micro-particles.     

Instrument for Manipulating and Transporting Micro-particles in Liquid Phase

More and more peoples begin to care for the ability to manipulate and transport single micro-particles such as cells, subcellular organelles and macromolecules for the purpose of study in biochemistry, biology, molecular medicine and molecular assemblies etc. According to the reports, the main methods for manipulating micro-particles in liquid phase are optical tweezers^[1], electrophoresis^[2] and suction syringe^[3]. However, they are many shortcomings by using these methods mentioned above,for example, optical tweezers would lead to optical matter, electrophoresis method would lead to electric matter when the particles placed in the field, and the method of suction is difficult to reach automatization. Here, according to the theory of electroosmosis pump, a novel instrument so-called electro-micromanipulator developed in our laboratory for manipulating or transporting single micro-particles in liquid-phase is introduced. This electro-micromanipulator isolated the sample from electric field so that it could avoid the shortcomings of methods mentioned above. Finally, this instrument has been demonstrated for manipulate and transport different size single alga cells successfully,more research works of manipulating and transporting DNA macromolecules for study in biochemistry, biology, molecular assemblies etc are going on.     

Cationic liposomes modified with non-ionic surfactants as effective non-viral carrier for gene transfer

A defined change in formulation components affects the physical and chemical characteristics of cationic liposomes (CLs) carriers in many ways. Therefore, a great degree of control can be exercised over the structure by modifying the CLs with various materials, leading to new innovations for carrier improvement. In the present study, surface modifications of cationic liposomes with non-ionic surfactants—sorbitan monoesters serials (Span 85, 80, 40 and 20) were carried out for developing a new gene transfer carrier. Span modified cationic liposomes (Sp-CLs) were prepared by reverse phase evaporation method (RPV) and self-assemble complexes of antisense oligonucleotides/surfactant modifying cationic liposomes were prepared by auto-coacervation through electrostatic effect. Characterization of Sp-CLs and the self-assembled complex was performed by electron microscope, particle size, zeta potential, turbidity and agarose electrophoresis. Furthermore, in vitro cellular uptake experiment showed that Span plays a role in enhancing the cellular uptake of encapsulated oligonucleotides mediated by Sp-CLs by the endocytosis-dependent route. CLs modified with Span 40 significantly facilitated the cellular uptake by COS-7 cells and HeLa cells; also showed some positive effect on gene expression. That suggests it is a potential non-viral carrier for efficient gene transfer.