大鼠血管内皮细胞siRNA转染条件的优化

目的:旨在优化siRNA转染大鼠血管内皮细胞的转染条件.方法:将0.75μL和1.5μL的脂质体LipofectamineTM3000分别与20、40、60、80nmol带荧光标记的FAMsiRNA组合,转染6、12、18、24h后,用荧光显微镜计数阳性细胞率、MTT法检测各浓度条件下内皮细胞的存活率,筛选最优转染条件.结果:(1)转染12h后,用荧光显微镜检测20、40、60、80nmol各组,均可观察到绿色荧光(2)siRNA浓度为60nmol/L,脂质体为1.5μL的组合转染效率最高,继续增加siRNA的浓度,转染效率提高不明显.(3)转染时间超过24h,各组细胞荧光减弱,细胞死亡率显著增加.结论:结果表明,以1.5μL LipofectamineTM3000与60nmol/L的siRNA浓度组成转染混合物转染12h可以实现对大鼠血管内皮细胞高效转染并保持较高的细胞活性.     

国际RNA的基础和应用研究近况

RNA分子将有望在不久的将来成为制服病毒和癌症的强大工具.20世纪80年代核酶(ribozyme)的发现,使RNA的研究进入了一个崭新的时代,利用RNA分子作为防病治病的工具已成为可能.最近,siRNA的发现,使RNA的研究推向顶峰.文中对国际上RNA研究的现状进行了综述.     

Development of New Targeted Inulin Complex Nanoaggregates for siRNA Delivery in Antitumor Therapy

Here, a novel strategy of formulating efficient polymeric carriers based on the already described INU-IMI-DETA for gene material whose structural, functional, and biological properties can be modulated and improved was successfully investigated. In particular, two novel derivatives of INU-IMI-DETA graft copolymer were synthesized by chemical functionalisation with epidermal growth factor (EGF) or polyethylenglycol (PEG), named INU-IMI-DETA-EGF and INU-IMI-DETA-PEG, respectively, in order to improve the performance of already described “inulin complex nanoaggregates” (ICONs). The latter were thus prepared by appropriately mixing the two copolymers, by varying each component from 0 to 100 wt% on the total mixture, named EP-ICONs. It was seen that the ability of the INU-IMI-DETA-EGF/INU-IMI-DETA-PEG polymeric mixture to complex siGL3 increases with the increase in the EGF-based component in the EP-ICONs and, for each sample, with the increase in the copolymer:siRNA weight ratio (R). On the other hand, the susceptibility of loaded siRNA towards RNase decreases with the increase in the pegylated component in the polymeric mixture. At all R values, the average size and the zeta potential values are suitable for escaping from the RES system and suitable for prolonged intravenous circulation. By means of biological characterisation, it was shown that MCF-7 cells are able to internalize mainly the siRNA-loaded into EGF-decorated complexes, with a significant difference from ICONs, confirming its targeting function. The targeting effect of EGF on EP-ICONs was further demonstrated by a competitive cell uptake study, i.e., after cell pre-treatment with EGF. Finally, it was shown that the complexes containing both EGF and PEG are capable of promoting the internalisation and therefore the transfection of siSUR, a siRNA acting against surviving mRNA, and to increase the sensitivity to an anticancer agent, such as doxorubicin.     

A Versatile Solid-Phase Approach to the Synthesis of Oligonucleotide Conjugates with Biodegradable Hydrazone Linker

One of the ways to efficiently deliver various drugs, including therapeutic nucleic acids, into the cells is conjugating them with different transport ligands via labile or stable bonds. A convenient solid-phase approach for the synthesis of 5′-conjugates of oligonucleotides with biodegradable pH-sensitive hydrazone covalent bonds is proposed in this article. The approach relies on introducing a hydrazide of the ligand under aqueous/organic media to a fully protected support-bound oligonucleotide containing aldehyde function at the 5′-end. We demonstrated the proof-of-principle of this approach by synthesizing 5′-lipophilic (e.g., cholesterol and α-tocopherol) conjugates of modified siRNA and non-coding RNAs imported into mitochondria (antireplicative RNAs and guide RNAs for Mito-CRISPR/system). The developed method has the potential to be extended for the synthesis of pH-sensitive conjugates of oligonucleotides of different types (ribo-, deoxyribo-, 2′-O-methylribo-, and others) with ligands of different nature.     

Synthesis of Site‐Specifically Phosphate‐Caged siRNAs and Evaluation of Their RNAi Activity and Stability

A complete set of new photolabile nucleoside phosphoramidites were synthesized, then site‐specifically incorporated into sense or antisense strands of siRNA for phosphate caging. Single caging modification was made along siRNA strands and their photomodulation of gene silencing were examined by using the firefly luciferase reporter gene. Several key phosphate positions were then identified. Furthermore, multiple caging modifications at these key positions led to significantly enhanced photomodulation of gene silencing activity, suggesting a synergistic effect. The caging group on both the terminally phosphate‐caged siRNA and the single‐stranded caged RNA has comparatively high stability, whereas hydrolysis of the caged group from the internally caged siRNA was observed, irrespective of the presence of Mg²⁺. Molecular dynamic simulations demonstrated that enhanced hydrolysis of the caging group on internally phosphate‐caged siRNAs was due to easy fragmentation of the caging group upon formation of the pentavalent intermediate of the phosphotriester with attack by water. The caging group in the terminally phosphate‐caged siRNA or single‐stranded caged RNA prefers to form π–π stacks with nearby nucleobases. In addition to providing explanations for previous observations, this study sheds further light on the design of caged oligonucleotides and indicates the direction of future development of nucleic acid drugs with phosphate modifications.     

Cationic Vesicles Based on Amphiphilic Pillar[5]arene Capped with Ferrocenium: A Redox‐Responsive System for Drug/siRNA Co‐Delivery

A novel ferrocenium capped amphiphilic pillar[5]arene (FCAP) was synthesized and self‐assembled to cationic vesicles in aqueous solution. The cationic vesicles, displaying low cytotoxicity and significant redox‐responsive behavior due to the redox equilibrium between ferrocenium cations and ferrocenyl groups, allow building an ideal glutathione (GSH)‐responsive drug/siRNA co‐delivery system for rapid drug release and gene transfection in cancer cells in which higher GSH concentration exists. This is the first report of redox‐responsive vesicles assembled from pillararenes for drug/siRNA co‐delivery; besides enhancing the bioavailability of drugs for cancer cells and reducing the adverse side effects for normal cells, these systems can also overcome the drug resistance of cancer cells. This work presents a good example of rational design for an effective stimuli‐responsive drug/siRNA co‐delivery system.     

用siRNA抑制胃癌细胞AGS中COX-2表达及对AKT磷酸化的抑制

构建了可在真核细胞中表达针对COX-2基因的siRNA表达载体,并用胃腺癌细胞系AGS建立了COX-2 siRNA的稳定转染细胞系。用Western-Blot检测稳定转染细胞系中COX-2的表达以及AKT的磷酸化水平。结果表明稳定转染C0X-2 siRNA的细胞中COX-2的表达较转染空载体的细胞明显降低,且发现COX-2表达的降低可抑制AKT—Ser^473和Thr-^308的磷酸化水平。为进-步探索抑制COX-2对胃癌细胞生物学特性的影响,及其在肿瘤治疗中的作用奠定了基础。     

Effect of Cationic Lipid Nanoparticle Loaded siRNA with Stearylamine against Chikungunya Virus

Chikungunya is an infectious disease caused by mosquito-transmitted chikungunya virus (CHIKV). It was reported that NS1 and E2 siRNAs administration demonstrated CHIKV inhibition in in vitro as well as in vivo systems. Cationic lipids are promising for designing safe non-viral vectors and are beneficial in treating chikungunya. In this study, nanodelivery systems (hybrid polymeric/solid lipid nanoparticles) using cationic lipids (stearylamine, C9 lipid, and dioctadecylamine) and polymers (branched PEI-g-PEG -PEG) were prepared, characterized, and complexed with siRNA. The four developed delivery systems (F1, F2, F3, and F4) were assessed for stability and potential toxicities against CHIKV. In comparison to the other nanodelivery systems, F4 containing stearylamine (Octadecylamine; ODA), with an induced optimum cationic charge of 45.7 mV in the range of 152.1 nm, allowed maximum siRNA complexation, better stability, and higher transfection, with strong inhibition against the E2 and NS1 genes of CHIKV. The study concludes that cationic lipid-like ODA with ease of synthesis and characterization showed maximum complexation by structural condensation of siRNA owing to high transfection alone. Synergistic inhibition of CHIKV along with siRNA was demonstrated in both in vitro and in vivo models. Therefore, ODA-based cationic lipid nanoparticles can be explored as safe, potent, and efficient nonviral vectors overcoming siRNA in vivo complexities against chikungunya.     

在Hep G2细胞中利用siRNA沉默GFP基因的实验探讨

目的:通过探讨小分子双链RNA(siRNA)通过RNA干扰(RNAi)机制沉默肝癌细胞株Hep G2荧光素报告基因GFP的各项生物学指标,为进一步实验和临床研究提供依据.方法:通过体外实验,对siRNA和siRNA脂质体的稳定性和细胞毒性进行评估;通过荧光标记技术,研究不同时间siRNA脂质体的转染效率;通过荧光显微镜下观察转染细胞和RT-PCR检测靶基因mRNA表达,确定不同种类、形式、剂量的siRNA对靶基因的沉默效能.结果:在空白培养液中,siRNA和siRNA脂质体可稳定至4h,在5%血清中2～4h后明显降解;100nM的siRNA和siRNA脂质体无明显细胞毒性;siRNA脂质体转染细胞株的效率随时间不同而不同,4h可达90%;非特异性siRNA/脂质体无沉默靶基因表达作用,特异性siRNA/脂质体对靶基因的沉默作用在一定范围内随剂量升高(20nM、50nM、100nM)而升高.结论:siRNA的稳定性可满足实验需要,且无明显细胞毒性,特异性siRNA转染肝癌细胞株能有效抑制靶基因表达,用脂质体转染可提高转染效率.siRNA通过RNAi机制沉默靶基因表达,为肿瘤的治疗和基础研究提供了新的工具和思路.