Method for reverse transfection using gold colloid as a nano-scaffold

DNA microarray of non-viral reverse transfection in cell engineering allows drastic downsizing of large-scale functional screening of genes and siRNAs. However the control of localizability and efficiency of the microarray is still considered as a critical barrier in practical use. One of the major breakthrough to increase the transfection efficiency may be control in the condition of DNA/transfection reagent complex on the microarray surface. In this paper, we showed that negatively charged gold colloid (GC) is successfully used to control the DNA/reagent complex on a glass surface. The conjugation of gold nanoparticles (20 nm in diameter) to the pEGFP-N1/Jet-PEI complex resulted in a more than 2.5-fold increase in the intensity of fluorescence of enhanced green fluorescent protein (EGFP) (based on the efficiency of transfection) from human mesenchymal stem cells (hMSCs), as compared to the control without GC. Our method for reverse transfection should be useful not only for cell array-based analyses but also as a novel gene-delivery method for gene therapy in regenerative medicine.     

Reverse transfection using antibodies against a cell surface antigen in mammalian adherent cell lines

Reverse transfection from a solid surface has the potential to deliver genes to various cells more efficiently than conventional methods. However, the effective gene delivery from a solid surface requires an optimized extracellular matrix (ECM) for the coating of glass slides, dependent on the nature of the cells. In a search for an appropriate substrate for the universal application to multiple types of cell, we focused on cell surface antigens and examined the effects of antibodies raised against them on gene transfer from an antibody-coated surface. We found that a coating of CD29-specific antibody allowed the most effective delivery of genes by reverse transfection in every type of cell that we examined. Our results suggest that reverse transfection with antibodies against CD29 might provide a universal tool for gene delivery and cell array-based analyses.     

Integrase-mediated nonviral gene transfection with enhanced integration efficiency

Retroviruses efficiently integrate their genome into the host chromosome. Two elements of the retrovirus genome are needed for the integration: long terminal repeats (LTRs) and integrase protein. We attempted to incorporate the retrovirus integration machinery in lipid vesicle-mediated gene transfection with the aim of achieving efficient stable transfection in a nonviral gene transfection system. A DNA fragment, in which a neomycin-resistant gene was flanked between partial LTR sequences derived from the Rous sarcoma virus (RSV), was constructed. This DNA fragment was transfected together with purified recombinant RSV integrase or integrase expression vectors by means of lipid vesicle-mediated gene transfection. The integrase-mediated transfection enhanced the stable transfection efficiency. The length and the end structure of the LTR sequences were important in achieving high efficiency. Under optimal conditions, the stable transfection efficiency showed a 16-fold improvement over that without integrase.     

RNAi技术在药物研究中的应用

RNA干扰(RNAi)是生物界中一种既古老且在进化上又高度保守的现象,是基因转录后沉默的重要机制之一。它主要通过双链RNA(dsRNA)被核酸酶切割成21-25nt的小RNA(siRNA)发挥作用,由siRNA介导识别并靶向切割同源性靶mRNA分子而实现,需要有多种蛋白因子以及ATP参与,而且具有生物催化反应特征。RNAi是新发现的一种通过siRNA介导的特异性高效抑制基因表达途径,在后基因组时代的基因功能研究和药物开发中具有广阔的应用前景。现将近年RNAi的研究进展作一综述。     

Determination of high molecular mass polycyclic aromatic hydrocarbons in refined olive pomace and other vegetable oils

A new and simple method has been developed for determining high molecular mass polycyclic aromatic hydrocarbons (PAHs) in refined olive pomace, and other vegetable oils from an initial sample of 0.25 g. The hydrocarbon fraction is isolated by solid‐phase extraction (SPE) on silica gel using hexane as eluent. The fraction is evaporated to reduced volume and cleaned‐up by SPE on an amino phase eluting with toluene. The evaporated residue, dissolved in acetonitrile, is analyzed by reverse‐phase high‐performance liquid chromatography with fluorescence detector using programmed excitation and emission wavelengths. The benzo(e)pyrene is determined together with other usual heavy PAHs. Interferences due to squalene and other hydrocarbons are minimized. Recoveries were greater than 80% and detection limits ranged from 0.01 to 0.2 µg kg^?1. The method was validated using certified oil samples and was applied to various vegetable oils. Copyright © 2004 Society of Chemical Industry     

红景天苷纳米脂质体的制备及若干工艺参数的优化

本实验首先比较了薄膜法、超声法、冻融法和反相蒸发法对红景天苷脂质体包封率的影响,结果表明红景天苷低浓度下,不同方法制得的脂质体包封率影响依次为冻融法>反相蒸发法>超声法>薄膜法,高浓度下的包封率影响不大。粒径测定表明粒子均处于纳米尺度。其次,对影响红景天苷纳米脂质体制备的反相蒸发法工艺参数进行了优化。结果表明,随着载量的降低包封率呈现上升后趋于平缓的变化。胆固醇/卵磷脂质量比1:2的嵌入量得到的脂质体包封率最高。吐温80对脂质的增溶具有十分明显的作用,2%质量分数可使得脂质体的包封率达到约80%。水合介质的离子强度对包封率的影响较大,脂质体在0.05mol/L、pH7.0的磷酸盐缓冲液体系中包封率较高。     

Polyethyleneimine/chitosan hexamer-mediated gene transfection into intestinal epithelial cell cultured in serum-containing medium

In search of an efficient nonviral vector, polyethyleneimine (PEI)-based vectors were examined. In general, the transfection efficiency of nonviral vectors is suppressed by serum. Here we show that PEI based vectors, particularly, the chitosan hexamer-PEI vector, could perform efficient gene transfection into intestinal epithelial cells (IEC-6) in the presence of serum. The conjugation order of the two polymers with a plasmid (first, chitosan hexamer; second, PEI) was found to be an important factor in enhancing transfection efficiency.     

Determination of ochratoxin A in wines by capillary liquid chromatography with laser induced fluorescence detection using dispersive liquid-liquid microextraction

A method based on reverse phase capillary high performance liquid chromatography (capillary HPLC) coupled to laser-induced fluorescence detection (LIF) has been proposed for the determination of ochratoxin A (OTA) in wine samples. An anionic micellar medium was added to the mobile phase for increasing the fluorescence intensity and peak efficiency. Dispersive liquid-liquid microextraction (DLLME) has been used as a simple and efficient sample pretreatment method for the analysis of OTA in wines, being optimised by means of experimental design. The limit of detection was 5.5 ng L(-1) (3 × S/N) and recoveries for different wines ranged from 91.7 to 98.1%. The proposed methodology could be classified as a green analytical chemistry alternative, combining the low organic solvent volumes required in the DLLME with the reduced consumption of mobile phase in capillary HPLC. The use of LIF as detector provided an extremely sensitive method for the determination of OTA in wines.     

A novel transfection method for mammalian cells using calcium alginate microbeads

The direct transfer of genetic materials into mammalian cells is an indispensable technique. We have developed calcium alginate (CA) microbeads which can deliver plasmid DNAs and yeast artificial chromosomes into plant and yeast cells. In this paper, we demonstrate the effective transfection of mammalian cells by CA microbeads immobilizing plasmid DNAs. The transfection was performed using the pEGFP-C1 plasmid containing the cytomegalovirus (CMV) promoter and enhanced green fluorescent protein (EGFP) gene. The transient expression of EGFP was observed 24 h after transfection. The expression efficiency was maximum when the concentration of sodium alginate was 1% and the amount of plasmid DNA was increased to 100 microg. The expression efficiency of our method using CA microbeads is 2-10 times higher than that of the polyethylene glycol (PEG) method. Our results suggest that the CA microbead mediated transfection of mammalian cells effectively delivers genetic materials into mammalian suspension cells.     

高效液相色谱法测定葡萄酒中溶菌酶的含量

目的建立高效液相色谱法检测葡萄酒中溶菌酶的含量。方法葡萄酒样品经5%盐酸溶液酸化后,采用流动相A乙腈∶三氟乙酸∶水(50:0.2:49.8,V:V:V)和流动相B乙腈∶三氟乙酸∶水(1:0.2:98.8,V:V:V)进行梯度洗脱,经反相苯基柱分离后,用高效液相色谱仪荧光检测器或紫外检测器进行测定,通过保留时间定性,外标法定量,并优化流动相的配比、梯度洗脱程序和样品的酸化条件。结果本方法在50、200、500和750 mg/L添加水平下的回收率为93.5%~99.2%,相对标准偏差为1.62~5.74%(n=6),方法定量限为50 mg/L。结论本方法具有分离效能高、重现性好、准确可靠,灵敏度高等优点,测定结果与酶联免疫法结果一致。该方法可以满足葡萄酒中溶菌酶的检测需求。     

叶酸-壳聚糖siRNA纳米复合物抗乙酰肝素酶的研究

目的：探讨叶酸-壳聚糖siRNA纳米复合物输送抗肿瘤细胞乙酰肝素酶的siRNA的效率和对乙酰肝素酶基因表达和肿瘤细胞侵袭力的影响。以期提高siRNA的输送效率,实现安全有效的靶向性基因治疗,并为研发抗肿瘤侵袭转移药物提供理论基础。方法：制备FA-CS-siRNA纳米复合物并测定其特征;分别以control组、FA-CS组、FA-CS-siRNA纳米复合物组、CS-siRNA组和lipo2000-siRNA组转染乳腺癌MCF-7细胞,RT-PCR和Western Blot检测乙酰肝素酶表达情况;肿瘤侵袭实验检测MCF-7细胞侵袭转移能力的变化。结果：红外光谱确认叶酸已成功与壳聚糖偶联;FA-CS-siRNA纳米复合物形态为圆形,分布较均匀,平均粒径为197.7nm;FA-CS-siRNA纳米复合物组能有效地干扰MCF-7细胞中乙酰肝素酶的表达;肿瘤细胞侵袭实验显示lipo2000-siRNA组和FA-CS-siRNA纳米复合物组能有效抑制MCF-7细胞的侵袭和转移。结论：FA-CS-siRNA纳米复合物能有效传递siRNA进入MCF-7细胞。     

Functions and potential applications of glycolipid biosurfactants--from energy-saving materials to gene delivery carriers

Biosurfactants (BS) produced by various microorganisms show unique properties (e.g., mild production conditions, lower toxicity, higher biodegradability and environmental compatibility) compared to their chemical counterparts. The numerous advantages of BS have prompted applications not only in the food, cosmetic, and pharmaceutical industries but in environmental protection and energy-saving technology as well. Glycolipid BS are the most promising, due to high productivity from renewable resources and versatile biochemical properties. Mannosylerythritol lipids (MEL), which are glycolipid BS produced by a yeast Candida antarctrica, exhibit not only excellent interfacial properties but also remarkable differentiation-inducing activities against human leukemia cells. MEL also show a potential anti-agglomeration effect on ice particles in ice slurry used for cold thermal storage. Recently, the cationic liposome bearing MEL has been demonstrated to increase dramatically the efficiency of gene transfection into mammalian cells. These features of BS should broaden its applications in new advanced technologies. The current status of research and development on glycolipid BS, especially their function and potential applications, is discussed.     

高效液相色谱法测定红茶菌中的功能因子

建立了一种利用高效液相色谱法同时分析红茶菌中2种主要功能因子(D-葡萄糖二酸1,4内酯和D-葡萄糖二酸)的方法。采用AgilentSB-Aq色谱柱(4.6mm×250mm,5μm),色谱条件:流动相为0.025mol/LKH2PO4(pH2.5),流速0.5mL/min,柱温30℃,进样量10μL,紫外检测波长为210nm。在此检测条件下,红茶菌中的2种主要功能因子能够得到较好分离,回收率为99.7%～102.3%,RSD<3%,精密度较高,重现性较好。     

Lactoferrin decreases primary bovine mammary epithelial cell viability and casein expression

The concentration of lactoferrin (LTF) in milk varies during lactation, rising sharply during involution. We proposed that LTF might have a regulatory role in involution and investigated its effects in vitro on the viability of bovine mammary epithelial cells (BMEC) and on casein expression in bovine mammospheres. Mammospheres capable of milk protein expression were formed by culturing primary BMEC on extracellular matrix in the presence of lactogenic hormones. Exogenously added LTF decreased beta-casein and kappa-casein mRNA expression in mammospheres while transfection of a short interfering RNA (siRNA) to suppress LTF expression resulted in increased casein mRNA expression. We believe that LTF exerts its effect on casein gene expression by up-regulating interleukin-1beta (IL-1beta) as IL-Ibeta gene expression was elevated in mammospheres treated with LTF. LTF also decreased viability of BMEC grown as monolayers and as mammospheres. Interestingly, LTF was only effective in reducing casein mRNA expression and viability in mammospheres when added at concentrations found during early involution but was inactive when used at concentrations found in milk. We suggest that LTF has a regulatory role during early involution, decreasing casein expression and reducing BMEC viability.     

A novel gene delivery system in plants with calcium alginate micro-beads

We have produced micrometer-sized calcium alginate beads referred to as "bio-beads" that encapsulate plasmid DNA molecules carrying a reporter gene. In order to evaluate the efficiency of the bio-beads in mediating genetic transfection, protoplasts isolated from cultured tobacco cells (BY-2) were transfected with bio-beads containing a plasmid that carries the modified green fluorescent protein gene CaMV35S-sGFP. With the bio-beads treatment, approximately ten-fold higher GFP expression was observed after 24 h incubation compared to that with the conventional method using a naked plasmid solution. Transfection was up to 0.22% efficient. These results indicate that bio-beads have a possibility for efficient transformation in plants.     

混合反胶团提取α-淀粉酶

以十六烷基三甲基溴化铵(CTAB)和脱水山梨醇单硬脂酸酯聚氧乙烯醚(Tween-60)为混合表面活性剂溶于正丁醇-异辛烷中构成反胶团系统,萃取纯化α-淀粉酶。研究不同萃取条件下,α-淀粉酶的萃取率。其中反胶团相组成为：ρ(CTAB+Tween-60)＝4g/L;n(CTAB):n(Tween-60)＝2.0:1.0;V(正丁醇):V(异辛烷)＝1.0:1.0。水相组成为：α-淀粉酶配制的粗酶液,此时c(NaCl)＝0.04mol/L,水相pH11.04。结果表明：萃取温度40℃、V(有机相):V(水相) ＝2.0:1.0、振荡时间10min时,α-淀粉酶萃取率可达91%;反萃取水相组成为c(NaCl)＝2.5mol/L、pH4.5、V(水相):V(有机相)＝1.0:2.0,反萃取振荡时间10min、温度50℃时,α-淀粉酶反萃取率可达65%。反胶团相可重复使用,当V(水相):V(有机相)＝1.0:2.75时,反胶团第2次α-淀粉酶萃取率达到71%。     

Combination of poly(ethylenimine) and chitosan induces high gene transfection efficiency and low cytotoxicity

In this study, the cytotoxicity and transfection efficiency of using chitosan/DNA complex combined with poly(ethylenimine) (PEI) were investigated. The combination of PEI with the chitosan/DNA complex markedly enhanced the gene expression of HeLa cells to 1000-fold of that induced by chitosan alone. PEI's cytotoxicity was considerably decreased upon combination with the chitosan/DNA complex. Furthermore, the PEI/chitosan/DNA could maintain the gene expression efficiency in the presence of serum.     

大豆磷脂克拉霉素脂质体制备及性质研究

采用薄膜冷冻法制备了克拉霉素脂质体,以高效液相色谱法为分析手段,采用反透析法测定克拉霉素脂质体的包封率,并通过正交实验确定了最佳制备条件为:水浴温度为45℃,m(克拉霉素):m(磷脂)=1:35,m(磷脂):m(胆固醇)=4:1,m(有机相):m(水相)=4:1,水合介质为pH6.8的磷酸盐缓冲溶液。在此条件下,克拉霉素脂质体的包封率达到81%,并从形态、粒径、渗漏率等方面对克拉霉素脂质体进行了质量评价。     

One-step method for isolation and purification of native β-lactoglobulin from bovine whey

BACKGROUND: The major whey protein β‐lactoglobulin (BLG) has been widely studied for its functional properties. The aim of this study was to develop an efficient, inexpensive and rapid one‐step method for the isolation and purification of BLG while preserving its native structure. RESULTS: BLG was purified from defatted whey obtained from raw cow's milk by anion exchange chromatography. Protein purity and identity were determined using reverse phase high‐performance liquid chromatography and mass spectrometry. Total BLG yield was 80% with protein purity from 97 to 99%. BLG isoforms A and B were separated into fractions of 91 and 99% purity respectively. The structure and native conformation of the isolated BLG were compared with those of standard commercial BLG by circular dichroism spectrometry, susceptibility to various crosslinking enzymes and enzyme‐linked immunosorbent assay inhibition. CONCLUSION: The proposed method is very useful for the rapid preparation of BLG suitable for studying antigenic and molecular characteristics of this protein, as well as the effect of food processing on these properties. The procedure requires only 1 day for the purification of about 300 mg of BLG from a single run using a small column (2.5 cm × 20 cm) of diethylaminoethyl Sephadex and has potential for scaling up. Copyright © 2011 Society of Chemical Industry