脂质体介导RNAi的研究

本文选用阳离子脂质体Lipofectamine 2000为基因载体,使用沉默荧光素酶基因的小干扰RNA(siRNA)进行体外RNA干扰(RNAi),对阳离子脂质体转运siRNA的效率进行研究。首先以阳离子脂质体运载质粒DNA转染细胞,并以延滞实验对阳离子脂质体与siRNA的结合能力进行检测;然后通过基因载体将荧光素酶基因载入细胞,转运不同浓度的siRNA对其进行沉默,应用酶标仪对荧光素酶基因的表达进行分析,并用MTT法对转染后细胞存活率进行检测。结果表明:阳离子脂质体Lipofectamine 2000可以高效转运质粒DNA,并可与siRNA很好地结合。在较低的siRNA浓度下,对荧光素酶基因的沉默率达到较高的水平,转运siRNA转染细胞对细胞活性的影响很小,但细胞存活率随着siRNA浓度的增加而逐渐降低。通过优化实验条件,阳离子脂质体转运较低浓度的siRNA即可高效沉默目的基因。     

脂质体RNAiMAX介导siRNA转染沉默星形胶质细胞AQP4基因

目的明确脂质体RNAiMAX(lipofectamine RNAiMAX)是否可以介导小干扰RNA(siRNA)转染入原代培养星形胶质细胞并实现水通道蛋白4(AQP4)基因沉默。方法利用原代培养的Wistar大鼠大脑皮层星形胶质细胞,通过倒置荧光显微镜和Tecan酶标仪,观察lipofectamine RNAiMAX是否能介导Cy3标记的siRNA转染入细胞内,以及RNAiMAX浓度和siRNA浓度对转染效率的影响;利用Real-time PCR方法检测AQP4 siRNA转染的基因沉默效果。结果倒置荧光显微镜和Tecan酶标仪检测发现,随着siRNA和RNAiMAX浓度的增加,转染细胞荧光强度随之增加(n=6);Real-time PCR检测结果显示,3ml/L RNAiMAX和30nmol/L AQP4 siRNA以及6ml/L RNAiMAX和60nmol/L AQP4 siRNA转染星形胶质细胞24h、48h、72h时,AQP4 mRNA水平均降低80%以上(n=6)。结论 Lipofectamine RNAiMAX可以介导siRNA转染入原代培养星形胶质细胞中并实现AQP4基因的沉默,3ml/L RNAiMAX和30nmol/L AQP4 siRNA即可达到理想的沉默效果。     

阳离子脂质体介导的转染：血清对于表达及转染效率的影响

阳离子脂质体介导的转染：血清对于表达及转染效率的影响阳离子脂质体可以有效地对多种真核细胞进行ＤＮＡ和其它核酸的转染。大多数情况下，细胞在无血清培养基中用ＤＮＡ－脂质复合物处理２～２４小时。大多数细胞在处于无血清环境的有限时间内还可存活且无不良影响。但...     

聚乙烯亚胺-聚乙二醇-siRNA纳米复合物的制备和理化性质的研究

目的 探索聚乙烯亚胺-聚乙二醇(PEI-PEG)-siRNA纳米复合物的制备方法及其理化性质,以提高siRNA的细胞转染率.方法 设计合成了PEI-PEG共聚物基因载体,使其与针对细胞表面受体CD44v6的siRNA形成纳米复合物.通过粒径与电位测定、凝胶阻滞电泳、扫描电镜、流式细胞仪测定等方法,观察不同N/P比的纳米复合物的复合效果、表面形态和大小、基因转染率等.结果 电镜下纳米复合物呈近球形、大小较一致、分散良好的纳米颗粒.N/P=5、10时复合物粒径分别为(174.6±1.2)nm,(267.7±1.8)nm.当N/P超过10时纳米复合物粒径减小,zeta电位为正值且增大.此时,siRNA被PEI-PEG完全复合,产生一种荧光淬灭作用.流式细胞仪结果表明纳米复合物的基因转染率随着N/P的增加而增大.当N/P比为30时,其转染率为(75.6±9.2)%.结论 PEI-PEG是一种有潜力的阳离子基因载体,它的制备为下一步体外实验及动物实验提供了条件.     

聚乙烯亚胺纳米粒递送siRNA的抗肿瘤生长作用

聚乙烯亚胺(polyethylenimine,PEI)是一种阳离子聚合物,能够结合并压缩siRNA,形成稳定的PEI/siRNA纳米复合物。PEI纳米复合物通过静电作用与细胞膜结合,以胞吞方式进入细胞内。在质子化作用下纳米复合物逃离溶酶体,使携带的siRNA免受溶酶体酶降解,从而靶向作用于目的mRNA。对PEI纳米粒进行表面修饰后,可显著降低其细胞毒性和提高siRNA递送效率。因此,这种PEI纳米粒递送siRNA先进技术的有效利用对于肿瘤治疗有着广阔的应用前景。     

敲低Ube2w增加葡聚糖硫酸钠诱导的小鼠溃疡性结肠炎的易感性

目的 探讨泛素结合酶(E2)W(UBE2W)对葡聚糖硫酸钠(DSS)诱导的小鼠溃疡性结肠炎性反应的影响.方法 在小鼠成纤维细胞系L929中转染Ube2w siRNA(1#,2#,3#)不同干扰序列,Western blot检测UBE2W蛋白表达,选择干扰效果最佳的siRNA进行小鼠体内转染实验.给10只C57BL/6雄...     

组氨酸修饰聚酰胺-胺型树状高分子提高血清中基因转染效率的研究

组氨酸的咪唑环由于其独特的质子化机理而受到了广泛的关注。研究表明,组氨酸修饰的基因载体可对抗血清对基因转染的影响,提高载体在血清中的基因转染效率。本研究利用氨解反应将组氨酸接枝到聚酰胺-胺树枝状高分子(PAMAM)的表面,制备了一种新型的PAMAM衍生物—组氨酸修饰的聚酰胺-胺树枝状高分子(His-PAMAMG4)。利用1HNMR对His-PAMAM G4进行表征,证明一个PAMAM G4分子上接枝37个组氨酸分子。对His-PAMAM G4/DNA复合物进行结合DNA能力、粒径、表面电位、粒子形态等理化性能的表征,证明该新型聚合物对DNA分子具有良好的压缩能力。细胞活性检测结果表明,His-PAMAM G4对Bel 7402和Hela细胞的毒性均显著低于未经修饰的PAMAM G4。His-PAMAM G4在血清中的转染效率与PAMAM G4相比大幅度提高,也显著高于阳离子聚合物PEI 25k和市售商品阳离子脂质体Lipofectamine。因此,His-PAMAM G4有望成为一种高效、安全、可在体内应用的非病毒基因载体。     

RICTOR对类风湿关节炎成纤维样滑膜细胞活力的影响

 目的：通过siRNA介导的RNA干扰技术沉默类风湿关节炎（RA）成纤维样滑膜细胞（FLS）mTORC2的特异组成蛋白RICTOR的表达,观察其对细胞活力的影响。方法：组织块法培养RA-FLS。应用阳离子脂质体转染的方法,把化学合成的特异性RICTOR siRNA转染RA-FLS,并以转染非特异性siRNA作为阴性对照。利用荧光定量PCR法分析转染24 h后细胞RICTOR mRNA表达水平的变化;Western blotting法分析转染48和72 h后细胞RICTOR蛋白表达水平的变化;以噻唑蓝（MTT）比色法检测转染成纤维样滑膜细胞不同时间（24、48和72 h）RICTOR siRNA对细胞活力的影响。结果：荧光定量PCR结果显示特异性RICTOR siRNA转染组与对照组相比,细胞中RICTOR的mRNA表达水平显著下调,24 h干扰效率达78.3%±63.71%（P<0.01）。Western blotting结果显示与对照组相比,RICTOR siRNA转染组48 h和72 h后RICTOR蛋白表达水平明显降低,沉默效率分别为92.48%±6.14%和98.57%±1.40%（均P<0.01）。MTT结果显示,早期（24和48 h）RICTOR siRNA转染组与阴性对照组细胞存活率比较无显著差异;72 h后,RICTOR siRNA转染组与阴性对照相比,细胞活力明显降低,抑制率为90.14%±1.90%（P<0.01）。结论：转染特异性RICTOR siRNA可降低RA-FLS的活力,提示mTORC2可能与RA-FLS的生长有关。     

非病毒载体聚乙烯亚胺转基因因素的优化

聚乙烯亚胺属于阳离子多聚物,可浓缩DNA作为转基因非病毒载体。但其转染影响变量较多,如果不能充分控制将不能得到重复的、良好的结果。这里测定了聚乙烯亚胺转基因效率的影响因素,为合成更复杂的人工转基因载体创造条件。我们通过聚乙烯亚胺转染编码β-半乳糖苷酶的pSVβ质粒到CO S-7和N IH 3T 3细胞中,测定质粒因素、血清、细胞密度、操作方式以及转染复合物的保存因素对聚乙烯亚胺转基因效率的影响。结果表明:质粒中生物活性抑制剂明显降低转染效率,通过截流分子量3000或10000的超滤可以除去生活性抑制剂;断裂的质粒降低转染效率;培养液中的血清、白蛋白降低转染效率;细胞密度影响转染效率;PE I/DNA复合物与细胞作用8h后吸去,转染效果最优。冻存显著降低PE I/DNA转染复合物转染效率。聚乙烯亚胺可以作为合成新型非病毒载体的骨架,但必须控制有关因素才能发挥最佳的和可重复性的转染结果。     

靶向CⅡTA基因siRNA对大鼠角膜基质细胞MHCⅡ基因表达影响的研究

目的：探讨靶向主要组织相容性复合物(MHC)Ⅱ类抗原转录激活因子（MHCⅡtransactivator CⅡTA）的化学合成小干扰RNA(small interfering RNA)抑制大鼠角膜基质细胞表面的MHCⅡ类抗原表达的可行性。 方法： 设计并合成靶向CⅡTA基因的5条siRNA，分离并培养大鼠角膜基质细胞，经重组大鼠IFN-γ刺激后，在阳离子脂质体的介导下，将siRNA转染大鼠角膜基质细胞。于转染后24 h收集细胞,用荧光定量PCR方法检测CⅡTA和MHCⅡ mRNA水平的变化;流式细胞仪检测MHCⅡ抗原表达的变化。 结果： 大鼠角膜基质细胞经重组大鼠IFN-γ刺激后，CⅡTA和MHCⅡ的表达大幅增强。荧光定量PCR检测显示化学合成的5条siRNA通过脂质体转染大鼠角膜基质细胞后,均能不同程度地抑制CⅡTA和MHCⅡ的表达，与对照组具有显著差异(P<0.01)。其中siRNA-4组的抑制作用最明显，对CⅡTA、MHCⅡ基因的mRNA抑制率分别为95.10%±1.25%、82.70%±1.95%。流式细胞仪检测显示siRNA-4组对MHCⅡ抗原分子表达抑制率为81.90%±1.23%。 结论： 在大鼠角膜基质细胞中，靶向CⅡTA siRNA抑制了自身mRNA表达,并阻止其调控的MHCⅡ类分子的相应表达。从而为进一步研究利用siRNA技术抑制MHCⅡ的表达防治角膜缘移植排斥反应提供了实验依据。     

Structural contributions of blocked or grafted poly(2-dimethylaminoethyl methacrylate) on PEGylated polycaprolactone nanoparticles in siRNA delivery

The multiformity in polymer structure and conformation design provides a great potential in improving the gene silencing efficiency of siRNA by polymer vectors. In order to provide information on the polymer design for siRNA delivery, the structural contributions of blocked or grafted poly(2-dimethylaminoethyl methacrylate) on PEGylated polycaprolactone nanoparticles (NPs) in siRNA delivery were studied. Herein, two kinds of self-assembly nanoparticles (NPs) formed by amphiphilic cationic polymers, methoxy poly(ethylene glycol)-block-polycaprolactone-block-poly(2-dimethylaminoethyl methacrylate) (mPEG-PCL-b-PDMAEMA, PECbD) and methoxy poly(ethylene glycol)-block-(polycaprolactone-graft-poly(2-dimethylaminoethyl methacrylate)) (mPEG-PCL-g-PDMAEMA, PECgD), were used to deliver siRNA for in vitro and in vivo studies. The physiochemical properties including size and zeta potential of PECbD NPs/siRNA and PECgD NPs/siRNA complexes were characterized. In vitro cytotoxicity, cellular uptake and siRNA knockdown efficiency were evaluated in HeLa-Luc cells. The endosome escape and intracellular distribution of PECbD NPs/siRNA and PECgD NPs/siRNA in HeLa-Luc cells were also observed. In vivo polymer mediated siRNA delivery and the complexes distribution in isolated organs were studied using mice and tumor-bearing mice. At the same total degree of polymerization (DP) of DMAEMA, PECgD NPs/siRNA complexes possessed higher zeta potentials than PECbD NPs/siRNA complexes (at the same N/P ratio), which may be the reason that PECgD NPs/siRNA complexes can deliver more siRNA into the cytoplasm and lead to higher in vitro luciferase and lamin A/C silencing efficiency than PECbD NPs/siRNA complexes. The in vivo imaging measurement and histochemical analysis also confirmed that siRNA could be delivered to lungs, livers, pancreas and HeLa-Luc tumors more efficiently by PECgD NPs than PECbD NPs. Meanwhile, the PDMAEMA chains of PECgD could be shortened which provides benefits for clearing. Therefore, PECgD NPs have great potential to be used as efficient non-viral carriers for in vivo siRNA delivery.     

The effect of serum in culture on RNAi efficacy through modulation of polyplexes size

Serum in the culture medium is one crucial factor that compromises RNAi efficiency of non-viral vectors. However, mechanistic roles of serum in siRNA delivery remain unknown. In this work, we took one cationic polymer, pullulan chemically modified by spermine (termed as pullulan-spermine, Ps), as a siRNA carrier model to investigate the effects of serum on key steps in siRNA delivery including formation of Ps and siRNA polyplexes (Ps-siRNA), cellular uptake, lysosomal escape, and cytotoxicity. We demonstrate that low serum concentration (1.25% and 2.5%) in culture medium results in large particles of Ps-siRNA, while high serum concentration (10%–40%) leads to small particles of Ps-siRNA. The larger particles initiated the internalization of siRNA more effectively in comparison to the smaller ones. The engulfed Ps-siRNA particles mainly locate in lysosomes. The large particles exhibited stronger abilities of destabilizing lysosomes than that of the small particles as large Ps-siRNA particles contain more amines and subsequently elicit a stronger proton sponge effect which results in more effective lysosomal escape of siRNA. Despite the lower RNAi efficiency, the small particle of Ps-siRNA in the high serum medium generates much lower cytotoxicity. These findings explain why serum significantly affects RNAi and also propose a strategy for improving RNAi efficiency and safety by modulating serum concentration and enhancing lysosomal destabilization.     

Multifunctional,self-assembling anionic peptide-lipid nanocomplexes for targeted siRNA delivery

Formulations of cationic liposomes and polymers readily self-assemble by electrostatic interactions with siRNA to form cationic nanoparticles which achieve efficient transfection and silencing in vitro. However, the utility of cationic formulations in vivo is limited due to rapid clearance from the circulation, due to their association with serum proteins, as well as systemic and cellular toxicity. These problems may be overcome with anionic formulations but they provide challenges of self-assembly and transfection efficiency. We have developed anionic, siRNA nanocomplexes utilizing anionic PEGylated liposomes and cationic targeting peptides that overcome these problems. Biophysical measurements indicated that at optimal ratios of components, anionic PEGylated nanocomplexes formed spherical particles and that, unlike cationic nanocomplexes, were resistant to aggregation in the presence of serum, and achieved significant gene silencing although their non-PEGylated anionic counterparts were less efficient. We have evaluated the utility of anionic nanoparticles for the treatment of neuronal diseases by administration to rat brains of siRNA to BACE1, a key enzyme involved in the formation of amyloid plaques. Silencing of BACE1 was achieved in vivo following a single injection of anionic nanoparticles by convection enhanced delivery and specificity of RNA interference verified by 5′ RACE-PCR and Western blot analysis of protein.     

The effect of RAFT-derived cationic block copolymer structure on gene silencing efficiency

In this work a series of ABA tri-block copolymers was prepared from oligo(ethylene glycol) methyl ether methacrylate (OEGMA(475)) and N,N-dimethylaminoethyl methacrylate (DMAEMA) to investigate the effect of polymer composition on cell viability, siRNA uptake, serum stability and gene silencing. Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization was used as the method of polymer synthesis as this technique allows the preparation of well-defined block copolymers with low polydispersity. Eight block copolymers were prepared by systematically varying the central cationic block (DMAEMA) length from 38 to 192 monomer units and the outer hydrophilic block (OEGMA(475)) from 7 to 69 units. The polymers were characterized using size exclusion chromatography and (1)H NMR. Chinese Hamster Ovary-GFP and Human Embryonic Kidney 293 cells were used to assay cell viability while the efficiency of block copolymers to complex with siRNA was evaluated by agarose gel electrophoresis. The ability of the polymer-siRNA complexes to enter into cells and to silence the targeted reporter gene enhanced green fluorescent protein (EGFP) was measured by using a CHO-GFP silencing assay. The length of the central cationic block appears to be the key structural parameter that has a significant effect on cell viability and gene silencing efficiency with block lengths of 110-120 monomer units being the optimum. The ABA block copolymer architecture is also critical with the outer hydrophilic blocks contributing to serum stability and overall efficiency of the polymer as a delivery system.     

Interactions between self-assembled polyelectrolyte shells and tumor cells

Layer-by-layer self-assembled polyelectrolyte shells are a new class of micro/nanocapsules with unique physicochemical properties for potential applications in drug/gene delivery. The objective of this study was to investigate the interactions of polyelectrolyte shells ( approximately 1 mum in diameter) with MCF-7 breast cancer cells and identify key parameters that affect such interactions. Tailoring of surface properties of polyelectrolyte shells was achieved by choosing different outermost layer materials, including cationic polymers, anionic polymers, and lipid bilayers. Different surface compositions led to a wide range of electrostatic potentials from -46 to +47 mV in phophate-buffered saline buffer. Confocal microscopy studies showed that the polyelectrolyte shells were internalized into the cell cytoplasm, but not into the nuclei. Correlation of cell uptake with shell surface compositions was complicated by the adsorption of serum proteins on the surface of polyelectrolyte shells, particularly polycation-coated shells. To prevent protein adsorption, poly(ethylene glycol) (PEG) grafted poly(ethyleneimine) (PEI) copolymers (1:1, 1:5, 1:10 graft ratios) were synthesized and introduced on the shell surface. Shells coated with PEI-PEG copolymers effectively reduced protein adsorption whereas PEI-PEG copolymers with lower graft ratios achieved higher cell uptake efficiency after 24 h of incubation with MCF-7 cells.     

The hydrolysis of cationic polycarboxybetaine esters to zwitterionic polycarboxybetaines with controlled properties

In this work, we report a new class of materials, cationic polycarboxybetaine esters, which have unique properties when they interact with proteins, DNAs, and bacteria. These cationic polymers can be converted to nontoxic and nonfouling zwitterionic polymers upon their hydrolysis. Due to their unique properties, they are very promising for a wide range of applications, such as highly effective gene delivery carriers and environmentally friendly antimicrobial coatings. Three positively charged polyacrylamides, of which the pedant groups bear carboxybetaine ester groups, were synthesized. These three polymers have different spacer groups between the quaternary ammonium and the ester groups. Their hydrolysis behaviors were studied using proton NMR under different NaOH concentrations. Their interactions with biomolecules and microorganisms before and after hydrolysis were demonstrated by protein adsorption/resistance, DNA condensation/release, and antimicrobial properties. The polymers were grafted onto a gold-coated surface covered with initiators using surface-initiated atom transfer radical polymerization (ATRP). Fibrinogen adsorption was measured by surface plasmon resonance (SPR) sensors. While the polymer-grafted surfaces have high protein adsorption, the surfaces became nonfouling after hydrolysis. Linear polymers were also synthesized and DNA/polymer complexes were evaluated. Agarose gel electrophoresis shows that DNA can be condensed into nanoparticles by the cationic polymers before hydrolysis and released from the DNA/polymer complexes upon the hydrolysis of the cationic polymers into zwitterionic polymers. The complexes formed were characterized by dynamic light scattering measurements. In addition, the interactions of linear polymers with bacteria were also evaluated. The polycarboxybetaine ester with a pentene spacer exhibits evident antimicrobial properties when they are incubated with Gram negative bacteria (Escherichia Coli). The polymer can be converted to a nontoxic polycarboxybetaine after hydrolysis. This work shows that the biological properties of polycarboxybetaine esters can be dramatically changed via controlled hydrolysis.     

Blood compatibility of novel water soluble hyperbranched polyglycerol-based multivalent cationic polymers and their interaction with DNA

A novel class of hyperbranched polymers based on polyglycerol (PG) and poly(ethylene glycol) (PEG) are synthesized by multibranching anionic ring opening polymerization. Multivalent cationic sites are added to these polymers by a post-amination and quarternization reactions. Blood compatibility studies using these polymers at different concentrations showed insignificant effects on complement activation, platelet activation, coagulation, erythrocyte aggregation and hemolysis compared to branched cationic polyethyleneimine (PEI). The degree of quarternization does not have large influence on the blood compatibility of the new polymers. Cytotoxicity of these polymers is significantly lower than that of PEI and is a function of quarternized nitrogen present in the polymer. Also, these polymers bind DNA in the nanomolar range and are able to condense DNA to highly compact, stable, water soluble nanoparticles in the range of 60-80 nm. Gel electrophoresis studies showed that they form electroneutral complexes with DNA around N/P ratio 1 irrespective of the percentage of quarternization under the conditions studied.     

KDR为靶的siRNA抑制乳腺癌细胞增殖的体内外研究

目的:采用化学修饰的小干扰RNA(siRNA)在体内外抑制含激酶插入区受体(KDR)基因表达,探讨化学修饰的siRNA介导的RNA干扰(RNAi)技术在乳腺癌基因治疗的可行性和特异性.方法:采用阳离子脂质体Lipofectamine2000TM作为转染试剂将针对人KDR基因的siRNA转染人类乳腺细胞株MCF-7,诱导RNAi,采用四甲基偶氮唑蓝(MTT)法,RT-PCR,Western blot试验等检测KDR基因和蛋白表达及细胞增殖变化.采用阳离子聚合物纳米粒In vivo jetPEITM为转染试剂将siRNA直接注射进裸鼠移植瘤,监测肿瘤生长变化,RT-PCR,免疫组化方法等监测KDR基因和蛋白表达变化.结果:靶向KDR基因siRNA转染MCF-7后,细胞增殖被抑制,KDRmRNA和蛋白的表达明显降低;裸鼠体内实验显示siRNA治疗组瘤组织的增长受到明显抑制;RT-PCR,免疫组化结果同时表明治疗组KDR表达下调.各对照组指标无明显变化.结论:化学修饰的siRNA介导的RNAi在体内外均能成功抑制靶基因的表达和MCF-7细胞增殖,是潜在的肿瘤治疗新方法,而KDR亦可作为肿瘤治疗的新靶点.     

Self-crosslinked human serum albumin nanocarriers for systemic delivery of polymerized siRNA to tumors

The safe and effective systemic delivery of siRNA is a prerequisite for the successful development of siRNA-based cancer therapeutics. For the enhanced delivery of siRNA, cationic lipids and polymers have been widely used as siRNA carriers to form electrolyte complexes with anionic siRNA. However, the considerable toxicity of strong cationic-charged molecules hampers their clinical use. In this study, we utilized human serum albumin (HSA), which is the most abundant of the plasma proteins, as a siRNA carrier for systemic tumor-targeted siRNA delivery. Both HSA and siRNA molecules were thiol-introduced to improve the binding affinity for each other. The resulting thiolated HSA (tHSA) and polymerized siRNA (psi) formed stable nanosized complexes (psi–tHSAs) by chemical crosslinking and self-crosslinking. After internalization, the psi–tHSAs showed target gene silencing activity in vitro comparable to conventional Lipofectamine™-siRNA complexes, without remarkable cytotoxicity. After intravenous injection in tumor-bearing mice, psi–tHSAs accumulated specifically at the tumor sites, leading to efficient gene silencing in the tumors in a sequential manner. The therapeutic VEGF siRNA was loaded into psi–tHSAs, which significantly inhibited tumor-related angiogenesis in PC-3 tumor xenografts and resulted in retarding the growth of PC-3 tumors. The results showed that self-crosslinked psi–tHSA nanocarriers might provide a promising approach for the systemic siRNA therapy of various human cancers.