TIMP-1特异性小干扰RNA真核表达载体转染肝星状细胞效率的检测

目的 提高TIMP-1小干扰RNA真核表达载体pGCsi-U6/TIMP-1 shRNA在大鼠肝星状细胞HSC-T6中的转染效率.方法 构建携带绿色荧光蛋白(GFP)基因的小干扰RNA真核表达载体pGCsi-U6/TIMP-1 shRNA,采用阳离子脂质体介导法将pGCsi-U6/TIMP-1 shRNA质粒转染入HSC-T6细胞,实验根据质粒DNA和阳离子脂质体LipofectamineTM 2000的不同比例分为5组:2 μg/0μL、2 μg/4 μL、2 μg/6 μL、2 μg/8 μL、2 μg/10 μL,同时设空白对照组.转染48 h后,应用激光共聚焦显微镜观察各组转染情况,同时用流式细胞仪计算转染效率.结果 激光共聚焦显微镜观察和流式细胞仪检测均说明质粒与脂质体比例为2 μg/8 μL组转染效率显著高于其余各比例组,差异具有统计学意义.转染效率与脂质体和质粒的比例有关.结论 按照合适的质粒和脂质体比例,质粒pGCsi-U6/TIMP-1 shRNA转染HSC-T6可达较高转染效率,是转染HSC-T6较为理想的瞬时表达载体.     

阳离子脂质体介导日本血吸虫组织蛋白酶L1基因的真核表达

目的真核表达日本血吸虫组织蛋白酶L1(SjCL1)以研究其生物学功能。方法采用阳离子脂质体复合质粒SjCL1/AD1-1 DNA并转染HeLa细胞,通过RT-PCR检测SjCL1基因在HeLa细胞中的转录,SDS-PAGE电泳法分析目的基因的蛋白表达产物,并进一步通过Western Blot法证实。结果RT-PCR检测到转染的细胞中有与目的基因相符大小约1000bp转录条带,SDS-PAGE电泳发现表达质粒SjCL1/AD1-1DNA转染的细胞上清液中存在大小约为31kDa的表达蛋白带,Western Blot法证实该表达蛋白产物可和日本血吸虫兔血清发生特异的反应。结论阳离子脂质体将SjCL1/AD1-1转染入HeLa细胞后可特异地表达一个大小约为31kDa的可分泌的日本血吸虫蛋白产物。     

介入途径下中药白芨提取物作为基因递送载体的可行性

目的:探讨经介入途径中药白芨提取物作为基因递送载体的可行性.方法:从中药白芨中提取其有效成分-白芨多糖, 采用胺化还原法制备阳离子型白芨多糖,体外实验检测该阳离子型多糖对质粒DNA的结合与保护作用, 以及阳离子白芨多糖载基因复合物对肝癌细胞系HepG2的转染, 进一步通过介入途径经肝动脉给予该复合物, 以GFP作为报告基因检测复合物在体内对活体兔肝细胞的转染.结果:所制备的阳离子型白芨多糖载基因复合物可以结合并保护质粒DNA免受DNA酶的降解; 体外实验证实该复合物可以转染入体外培养肝癌细胞, 以阳离子白芨多糖作为转染载体时转染效率要低于脂质体组, 差异具有统计学意义(28.87%±3.27% vs 36.64%±6.87%,P <0.05). 采用介入方法经肝动脉给药时复合物能靶向转染入活体兔肝细胞内并实现表达.结论:采用介入途径经肝动脉给药时阳离子白芨多糖载基因复合物可以实现活体肝细胞靶向转染, 有望作为一种新型的多聚阳离子型的基因载体在基因治疗中发挥作用.     

磁性纳米粒子在肝细胞癌磁共振成像及靶向治疗方面的研究进展

超顺磁性纳米粒子作为新型材料在临床领域应用广泛,如医学成像及诊断、药物靶向治疗、磁热疗等。它不仅可以在肝细胞癌（HCC）的早期作出特异性诊断,更是一种理想的靶向药物纳米载体。介绍了磁性纳米粒子作为靶向药物载体的性质特点、磁共振成像原理、靶向HCC的作用机制等,重点介绍了磁性纳米粒子的表面修饰及功能化,及其在主动靶向药物输送系统中的应用。认为磁性纳米粒子的应用必将在HCC的诊治中发挥更大的作用。     

CXCR4基因shRNA表达质粒的构建及筛选鉴定

目的 构建编码CXCR4mRNA的短发卡样RNA(shRNA)表达质粒用以筛选出基因沉默效果最明显的shRNA质粒表达载体.方法 根据GenBank中登录的CXCR4基因的核苷酸序列,应用shRNA设计软件设计并合成4条短发卡shRNA的oligo DNA,构建shRNA重组质粒.采用酶切电泳和测序方法进行鉴定.将鉴定后的CXCR4基因shRNA重组质粒经脂质体介导转染胶质瘤U251细胞,24 h后经RT-PCR筛选有效的shRNA重组质粒.结果 构建的质粒表达载体PCR鉴定均可扩增出目的条带,DNA测序结果与理论序列完全一致.RT-PCR检测显示,转染pG-PU6/GFP/Rac 1-421的U251细胞CXCR4基因mRNA的转录水平下降.结论 成功构建CXCR4基因shRNA表达质粒,并筛选出效果最为明显的shRNA质粒表达载体为pGPU6/GFP/Rac1-421.     

以乙型肝炎病毒为载体的基因治疗研究

目的 探讨乙型肝炎病毒(HBV)作为肝靶向性基因治疗载体的可能性。方法 用外源报告基因绿色荧光蛋白(GFP)取代HBV S基因读码框构建重组HBV载体,通过脂质体转染HepG2细胞,荧光显微镜下观察外源基因的表达,半巢式聚合酶链反应(PCR)检测细胞核内HBV 共价闭合环状DNA构型,常规PCR和Southern杂交检测上清液重组病毒DNA。结累 携带外源基因GFP的重组HBV载体能够在肝细胞内表达外源蛋白,此重组载体为复制缺损型,单独转染后不能在肝细胞包装与复制,在缺失包装信号ε的辅助HBV质粒下可被包装成携带外源基因的成熟重组HBV颗粒并分泌到胞外。结论 HBV可被改造成肝靶向性基因治疗载体。     

靶向Midkine基因的siRNA真核表达载体的构建及鉴定

目的 构建靶向midkine基因的siRNA 的表达载体,为研究midkine在肿瘤中作用提供一个新的方向.方法 根据基因库中midkine cDNA 序列设计和合成针对人midkine 基因的siRNA 寡核苷酸,定向克隆入质粒载体PGCsiU6,对该重组表达载体进行酶切鉴定及DNA 测序.结果 酶切鉴定、DNA 测序证实表达质粒构建成功,无碱基突变.结论 成功构建了靶向midkine 基因表达的siRNA 干扰质粒载体PGCsiU6/midkine,为进一步运用RNA干扰技术进行midkine 基因功能研究奠定了基础.     

超声介导载基因微泡靶向传输血管内皮生长因子促心肌血管新生

目的　探讨超声介导载血管内皮生长因子 16 5 (VEGF165)基因靶向微泡促梗死心肌血管新生的可行性。方法　构建真核表达质粒 pcDNA3 1-/VEGFcDNA165,将其包载于脂质泡中 ,超声辐射下向鼠心肌传输。采用RT PCR、WesternBlot检测mRNA、蛋白表达 ,观察微血管密度评价血管新生效果。结果　 (1)成功构建VEGF165基因 ;(2 )脂质体微泡可包载VEGF165基因 ;(3)超声介导辐射下向心肌靶向传输VEGF165基因 ,实验组基因表达及血管密度高于空白对照组 ,但低于VEGF165基因心肌直接注射。结论　具有心肌显影功能的脂质体载VEGF165基因微泡在超声辐射下可向大鼠梗死心肌靶向传输 ,并产生促血管新生效应。     

日本血吸虫病新型树枝状载体-DNA疫苗的构建及其评价

[摘要] 目的构建并评价一种新型的日本血吸虫病聚酰胺-胺（PAMAM）型树枝状载体DNA疫苗。方法采用赖氨酸对4.0 G PAMAM进行表面修饰,合成端基改性产物PAMAM-Lys。用电泳阻滞实验确定质粒DNA与PAMAM-Lys复合的比例,用透射电镜测试复合物微观结构变化,并通过电泳分析复合物的稳定性。同时采用MTT法对PAMAM-Lys进行体外细胞活性评价。分别用纯化质粒pJW4303、pJW4303-SjC23、树枝状载体PAMAM-Lys和复合物PAMAM-Lys/pJW4303-SjC23免疫50只小鼠,检测各组小鼠的特异性抗体水平以评价其免疫反应性。结果 琼脂糖凝胶电泳结果显示,电荷比在2～4时,PAMAM-Lys与DNA正负电荷完全中和,DNA电泳被完全阻滞,两者能完全结合。透射电镜测试结果表明,树枝状高分子载体与DNA的复合导致DNA结构收缩,粒径减小,分布均匀。树枝状高分子与DNA复合物具有良好的稳定性。噻唑蓝法检测结果表明,改性后的树枝状高分子载体及其DNA复合物作用于293T细胞较未改性树枝状载体产生的细胞毒性低;经PAMAM-Lys/pJW4303-Sj23免疫的小鼠产生的特异性抗体水平显著高于注射裸DNA疫苗组（P<0.05）。结论 氨基酸修饰的树枝状高分子PAMAM-Lys是DNA转染的优良载体,具有良好的生物相容性,赖氨酸修饰可以显著降低PAMAM树枝状高分子的细胞毒性,并能增强DNA疫苗的免疫反应性。     

重组结缔组织生长因子shRNA质粒的构建及其对肝星状细胞基因表达的影响

[目的]构建同时干扰大鼠结缔组织生长因子(CTGF)2个不同基因位点的shRNA质粒,并将其转染肝星状细胞(HSC),研究其对CTGF基因表达的影响。[方法]针对大鼠CTGF mRNA靶向序列设计并合成2对DNA模版序列,用PCR的方法将2条CTGF靶向特异性的shRNA分别链接到质粒pGenesil1.2载体的U6启动子和H1启动子。将构建的干扰质粒以阳离子脂质体法转染HSC-T6细胞,应用RT-PCR及Western Blot检测CTGF的表达。[结果]成功构建CTGF shRNA重组质粒;通过RT-PCR和Western Blot分析发现干扰质粒组的CTGF mRNA与蛋白表达水平明显下降(P0.05)。[结论]重组CTGF shRNA能有效抑制HSC中CTGF的表达。     

Magnetic Properties of Collagen–Chitosan Hybrid Materials with Immobilized Superparamagnetic Iron Oxide Nanoparticles (SPIONs)

The paper presents results of our studies on hybrid materials based on polymers of natural origin containing superparamagnetic iron oxide nanoparticles (SPIONs). Such nanoparticles, coated with the chitosan derivative, were immobilized in a chitosan-collagen hydrogel matrix by crosslinking with genipin. Three types of biopolymer matrices of different collagen-to-chitosan ratios were studied. A thorough magnetic characterization was performed, including magnetic susceptibility, magnetization, and hysteresis loop measurements in a temperature range of 4 K to 300 K and a magnetic field induction up to 8 Tesla. The effect of SPION immobilization and material composition on the magnetic properties of the hybrids was investigated. The results showed that hybrid materials with covalently bounded SPIONs preserved the superparamagnetic character of SPIONs and exhibited promising magnetic properties, which are important for their potential applications.     

Therapeutic angiogenesis using genetic transfection. An in vitro quantitative and functional study after gene code transfer for vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) gene transfer is a new therapeutic approach in clinical situations where insufficient angiogenesis may impair processes that require vessel formation, such as myocardial ischemia or peripheral vascular disease. The feasibility of this strategy was addressed in vitro by (i) optimisation of DNA transfection into cultured cells using a cationic liposome, (ii) study of the transgene production and it's angiogenic properties. An expression plasmid encoding VEGF165 was used to transfect endothelial cells (EAhy-926) in vitro. Different amounts of the cationic liposome were complexed to the plasmid DNA in order to achieve increasing ratios (1/1, 2/1, 3/1, 4/1, 5/1). Maximal gene expression was obtained when the cationic liposome was mixed to plasmid DNA in a 3/1 ratio. Using this optimised liposome/plasmid DNA formulation, VEGF expression measured by ELISA, reached a maximum of 60 ng/mL 24 hours after gene transfer then rapidly decreased. Conditioned media from transfected cells (CMVEGF) significantly increased the proliferation rate of endothelial cells. The maximal mitogenic effect was observed when the cell media was supplemented with 25% of CMVEGF, corresponding to 10 ng/mL of recombinant VEGF. Addition of the VEGF165 peptide into the cell media showed a similar mitogenic effect. The angiogenic property of the transgene was assessed by the demonstration of its ability to stimulate capillary tubes formation in a tridimensional biologic gel.     

Mechanism of oligonucleotide release from cationic liposomes.

We propose a mechanism for oligonucleotide (ODN) release from cationic lipid complexes in cells that accounts for various observations on cationic lipid-nucleic acid-cell interactions. Fluorescent confocal microscopy of cells treated with rhodamine-labeled cationic liposome/ fluorescein-labeled ODN (F-ODN) complexes show the F-ODN separates from the lipid after internalization and enters the nucleus leaving the fluorescent lipid in cytoplasmic structures. ODN displacement from the complex was studied by fluorescent resonance energy transfer. Anionic liposome compositions (e.g., phosphatidylserine) that mimic the cytoplasmic facing monolayer of the cell membrane released ODN from the complex at about a 1:1 (-/+) charge ratio. Release was independent of ionic strength and pH. Physical separation of the F-ODN from monovalent and multivalent cationic lipids was confirmed by gel electrophoresis. Fluid but not solid phase anionic liposomes are required, whereas the physical state of the cationic lipids does not effect the release. Water soluble molecules with a high negative linear charge density, dextran sulfate, or heparin also release ODN. However, ATP, spermidine, spermine, tRNA, DNA, polyglutamic acid, polylysine, bovine serum albumin, or histone did not release ODN, even at 100-fold charge excess (-/+). Based upon these results, we propose that the complex, after internalization by endocytosis, induces flip-flop of anionic lipids from the cytoplasmic facing monolayer. Anionic lipids laterally diffuse into the complex and form a charged neutralized ion-pair with the cationic lipids. This leads to displacement of the ODN from the cationic lipid and its release into the cytoplasm.     

Iron Oxide Silica Derived from Sol-Gel Synthesis

In this work we investigate the effect of iron oxide embedded in silica matrices as a function of Fe/Si molar ratio and sol pH. To achieve homogeneous dispersion of iron oxide particles, iron nitrate nonahydrate was dissolved in hydrogen peroxide and was mixed with tetraethyl orthosilicate and ethanol in a sol-gel synthesis method. Increasing the calcination temperature led to a reduction in surface area, although the average pore radius remained almost constant at about 10 Å, independent of the Fe/Si molar ratio or sol pH. Hence, the densification of the matrix was accompanied by similar reduction in pore volume. However, calcination at 700 °C resulted in samples with similar surface area though the iron oxide content increased from 5% to 50% Fe/Si molar ratio. As metal oxide particles have lower surface area than polymeric silica structures, these results strongly suggest that the iron oxides opposed the silica structure collapse. The effect of sol pH was found to be less significant than the Fe/Si molar ratio in the formation of molecular sieve structures derived from iron oxide silica.     

Preparation of Cationic Amphiphilic Nanoparticles with Modified Chitosan Derivatives for Doxorubicin Delivery

Polymeric micelle-like nanoparticles have demonstrated effectiveness for the delivery of some poorly soluble or hydrophobic anticancer drugs. In this study, a hydrophobic moiety, deoxycholic acid (DCA) was first bonded on a polysaccharide, chitosan (CS), for the preparation of amphiphilic chitosan (CS-DCA), which was further modified with a cationic glycidyltrimethylammounium chloride (GTMAC) to form a novel soluble chitosan derivative (HT-CS-DCA). The cationic amphiphilic HT-CS-DCA was easily self-assembled to micelle-like nanoparticles about 200 nm with narrow size distribution (PDI 0.08–0.18). The zeta potential of nanoparticles was in the range of 14 to 24 mV, indicating higher positive charges. Then, doxorubicin (DOX), an anticancer drug with poor solubility, was entrapped into HT-CS-DCA nanoparticles. The DOX release test was performed in PBS (pH 7.4) at 37 °C, and the results showed that there was no significant burst release in the first two hours, and the cumulative release increased steadily and slowly in the following hours. HT-CS-DCA nanoparticles loaded with DOX could easily enter into MCF-7 cells, as observed by a confocal microscope. As a result, DOX-loaded HT-CS-DCA nanoparticles demonstrated a significant inhibition activity on MCF-7 growth without obvious cellular toxicity in comparison with blank nanoparticles. Therefore, the anticancer efficacy of these cationic HT-CS-DCA nanoparticles showed great promise for the delivery of DOX in cancer therapy.     

Optimization of cationic liposome-mediated gene transfer to human bronchial epithelial cells expressing wild-type or abnormal cystic fibrosis transmembrane conductance regulator (CFTR).

We determined optimum conditions for delivering DNA to transformed human bronchial epithelial cells expressing wild-type (BEAS) or abnormal (2CF) cystic fibrosis transmembrane conductance regulator (CFTR) using cationic liposomes (Lipofectin, [N-(N,N-dimethylaminoethane)carbamyl] cholesterol[DC-Chol]/dioleoylphosphatidylethanolamine[DOPE], or LipofectAMINE) and reporter genes which measured overall transgene expression (luciferase) or the fraction of cells transfected (heat-stable alkaline phosphatase). All liposomes showed dose-related toxicity. Optimal liposome and lipid: DNA ratios were different for BEAS than for 2CF cells. For all 3 liposome preparations, small particle size and net cationic charge related to transfection efficiency. Both LipofectAMINE and DC-Chol/DOPE transfected a maximum of 3% of BEAS cells, but luciferase expression could be increased without increasing the fraction of cells transfected. LipofectAMINE transfected a maximum of 6% of 2CF cells, and luciferase expression could be increased with no further increase in fraction of transfected cells. DC-Chol/DOPE transfected over 12% of 2CF cells with relatively small increases in luciferase expression. We conclude that an optimal cationic liposome and lipid: DNA ratio for transfecting bronchial epithelial cells depends on: (1) small particle size and net cationic charge, (2) whether the cells have the cystic fibrosis defect, and (3) whether the desired outcome is transfection of the maximum fraction of the cells or maximum total expression of the transgene.     

Application of Magnetic Nanoparticles Coated with Crosslinked Zwitterionic Poly(ionic liquid)s for the Extraction of Oligonucleotides

Magnetic nanoparticles coated with zwitterionic poly(ionic liquid)s were applied for dispersive solid-phase extraction of oligonucleotides. The materials were synthesized by miniemulsion copolymerization of ionic liquids and divinylbenzene on magnetic nanoparticles. The functional monomers contain a positively charged imidazolium ring and one of the anionic groups: derivatives of acetate, malonate, or butyl sulfonate ions. Adsorption of unmodified DNA oligonucleotide on obtained materials was possible in ion-exchange (IE) and hydrophilic interactions (HI) mode. The adsorption in IE was possible at low pH and was almost complete. The adsorption in HI mode required the usage of appropriate addition of organic solvent but did not provide full adsorption. Studies on the desorption of the analytes included determining the impact of ammonium acetate concentration and pH and organic solvents addition on the recovery. The material containing acetic fragments as an anionic group was selected for the final procedure with the use of 10 mM ammonium acetate (pH = 9.5)/methanol (50/50, v/v) as an elution solution. The magnetic dispersive solid-phase extraction procedure was tested for the oligonucleotides with various modifications and lengths. Moreover, it was applied to extract DNA oligonucleotide and its synthetic metabolites from enriched human plasma without any pre-purification, with recoveries greater than 80%.     

Surface tunability of nanoparticles in modulating platelet functions

Metallic nanoparticles are attractive candidates as MRI contrast agents and mediators for drug delivery, diagnostics, and therapy. Direct contact and exposure to blood circulation is common in many such applications. The consequent thrombotic response may therefore be important to study. The main objective of the present work was to study how platelet functions were changed in the presence of different nano-surface or surface capping, which may provide a measure for the safety of a nanoparticle, and also assess the use of such nanoparticles in platelet modulation. Aggregometry, ATP release reaction, flow cytometry and immune-blotting studies were performed to study platelet response to different nano-particles (iron oxide, gold and silver). For each nanoparticle surface conjugation (capping) was varied. It was found that citric acid functionalized iron oxide nanoparticles have anti-platelet activity, with a decrease in aggregation, tyrosine phosphorylation level, and granule release. On the other hand in other cases (e.g. gold nanoparticles) pro-aggregatory response was observed in the presence of nanoparticles and, in some cases, the nanoparticles behaved neutrally (e.g. for starch-coated iron oxide nanoparticles). Therefore, nanoparticles can induce antiplatelet or a pro-aggregatory response, or remain neutral depending on surface capping. A related observation is that antiplatelet drugs can be made more potent by nanoparticle conjugation.     

Top-down方法制备氯硝柳胺纳米剂的研究

目的研制氯硝柳胺纳米剂。方法将氯硝柳胺与各种助剂和水按一定的比例混合,选择适当的球磨条件,用行星式球磨机进行研磨,用光学显微镜和激光粒度仪评价所得到的纳米剂。结果使用行星式球磨方法,选用大球料比、高转速、长时间的高能量球磨条件,可提高球磨效果;在表面活性物质、消泡剂和助磨剂存在的条件下,加入亲水高分子物质可对氯硝柳胺的颗粒起到保护作用。实验配方为氯硝柳胺25%(质量比,下同)、吐温-8010%、甲基硅油5%、二氧化硅10%、聚氧乙烯10%时,在转速为600r/min、球料比为20∶1的球磨条件下,预磨20h后用1mm锆球研磨至100h,可得到粒径分布最大值(DMax)为71nm、50%粒子的粒径(D50)为81nm、90%粒子的粒径(D90)为164nm的纳米粒子,形成稳定、易于分散的氯硝柳胺纳米液。结论用行星式球磨方法,在亲水高分子的保护下,选用高能量球磨条件,可得到稳定的氯硝柳胺纳米剂。     

An Innovative Method of Converting Ferrous Mill Scale Wastes into Superparamagnetic Nanoadsorbents for Water Decontamination

The need to recycle and develop nanomaterials from waste, and use them in environmental applications has become increasingly imperative in recent decades. A new method to convert the mill scale, a waste of the steel industry that contains large quantity of iron and low impurities into a nanoadsorbent that has the necessary properties to be used for water purification is presented. The mill scale waste was used as raw material for iron oxide nanopowder. A thorough characterization was performed in each stage of the conversion process from the mill scale powder to magnetic nanopowder including XRD (X-ray diffraction), SEM (scanning electron microscopy), TEM (transmission electron microscopy), BET (Brunauer, Emmett and Teller) and magnetization properties. Iron oxide nanoparticles were approximately 5–6 nm with high specific surface area and good magnetic properties. These are the necessary properties that a magnetic nanopowder must have in order to be used as nanoadsorbents in the heavy metal removal from waters. The iron oxide nanoparticles were evaluated as adsorbents for the removal of Cu, Cd and Ni ions.