pSP偶联低分子量壳聚糖介导siRNA对靶基因的沉默

放射性标记针对荧光素酶报告基因（Luc）的siRNA,与不同分子量的壳聚糖（CS）制备成CS/siRNA复合物,转染可稳定表达Luc基因的MDA-MB-231/Luc人乳腺癌细胞系.与 较大分子量壳聚糖相比,低分子量壳聚糖（LMWC）与siRNA形成的复合物具有更小的粒径及更强的siRNA转染能力,但是对靶基因的沉默效应却不高. 其原因归咎于低分子量壳聚糖（Mr为2 000或5 000,LMWC）与siRNA间强烈的电荷引力限制了siRNA在细胞内的释放.合成基序为LLLRRRDNEY*FY*VRRLL的可磷酸化短肽(pSP)与LMWC相偶联,合成pSP-LMWC.分别对siRNA及pSP-LMWC进行FAM及Dabcyl标记,利用FRET技术检测细胞内pSP-LMWC与siRNA的解离.结果表明,pSP修饰可大幅度增加siRNA与LMWC在细胞内的解离,成为有功能的游离形式,从而显著下调靶基因的表达.本文的结果表明,低分子量壳聚糖具有良好的siRNA递送能力,促进其与siRNA在细胞内的解离可有效提高siRNA对靶基因的沉默效应.     

可磷酸化短肽偶联壳聚糖介导IGF-1和IL-1RA双基因联合治疗兔关节软骨损伤

非病毒基因转移载体--壳聚糖被广泛用于基因转染,然而相对较低的转染效率限制了其在基因治疗中的应用.本课题组曾经报告可磷酸化短肽修饰壳聚糖(hosphorylatable short peptide coupled chitosan,pSP-CS)可增加体外培养细胞的DNA转染效率.本研究中采用pSP-CS作为基因载体介导人白细胞介素-1受体拮抗剂基因(interleukin-1 receptor antagonist gene, IL-1RA)和人胰岛素样生长因子-1基因(insulin-like growth factor 1 gene, IGF-1) 局部转染, 联合治疗兔关节软骨损伤.将pSP-CS与单基因表达质粒pBudCE4.1-IGF-1、pBudCE4.1-IL-1RA和共表达质粒pBudCE4.1-IGF-1+IL-1RA制成pSP-CS/pDNA复合物,制备股骨外侧髁全层软骨损伤模型,pSP-CS/pDNA 复合物关节腔内注射4周. ELISA分析发现,转基因组关节腔灌洗液中含有大量外源蛋白IGF-1和IL-1RA. 定量PCR检测mRNA显示, 各转基因组明显下调基质金属蛋白酶-3(matrix metallo-proteinase-3, Mmp-3)基因表达; 上调基质金属蛋白酶抑制剂-1(matrix metallo-proteinase inhibitor-1, Timp-1)和二型胶原(Collagen II) 基因表达(P < 0.05);双基因转染组作用明显优于单基因转染组(P< 0.05). HE及Collagen II免疫组化染色显示, 各转基因组软骨损伤处出现不同程度的软骨性修复,以双基因转染组作用最优. 本研究表明,pSP-CS可以携带外源基因进入软骨组织并局部大量表达,IGF-1与IL-1RA协同作用明显促进损伤软骨修复,为今后临床多基因治疗软骨损伤提供了实验基础.     

可磷酸化短肽偶联壳聚糖介导IL-1RA与IGF-1共转染对兔关节软骨细胞的作用

探讨可磷酸化短肽偶联壳聚糖(phosphorylatable short peptide coupled chitosan,pSP-CS),介导人白细胞介素 1受体拮抗剂基因(interleukin-1 receptor antagonist protein,IL-1RA)和人胰岛素样生长因子1基因(insulin like growth factor-1,IGF-1) 共转染,对体外培养的兔关节软骨细胞的作用. 将pSP-CS 与共表达质粒pBudCE4.1-IL-1RA+IGF-1、单基因表达质粒pBudCE4.1-IL-1RA、pBudCE4.1-IGF-1和空质粒pBudCE4.1制成pSP-CS/pDNA复合物,转染体外分离培养的正常兔原代关节软骨细胞. ELISA 法检测IL-1RA和IGF-1的表达,以表征pSP CS转染效率;Cell Counting Kit-8 (CCK-8) 法分析软骨细胞的增殖活力;流式细胞仪检测软骨细胞的凋亡;定量PCR检测软骨细胞中基质金属蛋白酶抑制剂-1(matrix metallo proteinase inhibitor-1, Timp-1)、基质金属蛋白酶-3(matrix metalloproteinase-3, Mmp-3)、聚集蛋白聚糖 (Aggrecan) 基因表达. 转基因组IL-1RA和IGF-1有较高的表达水平;各转基因组明显促进细胞增殖、抑制细胞凋亡、下调Mmp-3基因表达、上调Timp 1和Aggrecan基因表达,且双基因组作用明显优于单基因组(P<0.05). 结果表明,pSP-CS可以携带外源基因进入软骨细胞并大量表达, IGF-1与IL-1RA协同作用明显提高体外培养软骨细胞的生物活性, 为今后研究pSP-CS介导多基因体内治疗软骨损伤提供了基础.     

两种阳离子纳米基因载体及植物基因介导效果的研究

以阳离子聚乙烯亚胺(polyethylenimine, PEI)和壳聚糖(chitosan, CS)作为两种植物基因载体,分别制备了载基因PEI纳米粒(PEI/DNA)和壳聚糖-DNA纳米粒(CS/DNA),并对其形态、粒度分布、包封率、DNA结合的稳定性及纳米颗粒对DNA的保护等方面进行表征.并以GFP基因为报告基因进行植物细胞转染,比较两者转化效率.结果表明PEI/DNA纳米粒稳定性,对DNA的保护以及转染效率等方面均优于壳聚糖-DNA纳米粒.     

应用PAMAM dendrimers作为DNA运送载体的体外研究

Starburst^TM PAMAM dendrimers分子是一类新型的高分枝、辐射状对称的树状高分子,在生理条件下其表面具有高密度的正电荷,可以通过静电相互作用与核酸形成复合物后,介导遗传物质进入细胞.研究了G3, G3.5, G5, G7, G7.5, G9各代dendrimers分子与DNA结合后介导其转染细胞的能力,并初步评价这种复合物转染对细胞活力的影响.实验证实,全代的PAMAM dendrmers皆可与DNA结合,并可在体外培养的细胞中介导高效的DNA转染.PAMAM dendrimer/DNA复合物很稳定,在较大的pH值变化范围内(pH 2～10)不解离.PAMAM dendrimers可保护与之复合的DNA分子免受限制性内切酶的降解.在一定的电荷比范围内,高代数的dendrimers分子与DNA形成的复合物对培养细胞的转染效率高于低代数dendrimer分子,复合物所介导的转染效率在不同的细胞系之间也有差异.在有效作用浓度范围内（≤1.3×10^-1 g/L）,PAMAM dendrimers/DNA复合物对被转染细胞无毒性.但是,未与DNA复合的dendrimers分子在较低浓度时则表现出毒性,表明Starburst^TM PAMAM dendrimers分子可作为新型的低毒非病毒DNA载体,用于介导DNA对体外培养细胞的转染. 这些前期观察,为将纳米级高分子聚合物dendrimers分子作为基因转运载体应用于体内提供了初步的实验依据.     

聚乙烯亚胺转基因影响因素的测定及其优化

聚乙烯亚胺 (PEI)为阳离子多聚物 ,可浓缩DNA形成纳米级颗粒 ,作为基因释放载体转染真核细胞 .选用Mr2 5 0 0 0 ,分枝状的聚乙烯亚胺转染质粒 ,比较多种转基因效率的影响因素 .通过MTT法测定PEI对COS 7细胞的细胞毒性 .利用电泳阻滞实验测定PEI与DNA形成复合物时所需的比例 .通过PEI转染增强型绿色荧光蛋白的pEGFP质粒、编码β 半乳糖苷酶的pSVβ质粒 ,探索氯喹、白蛋白、血清、盐离子浓度、质粒剂量、细胞数量等对聚乙烯亚胺转基因效率的影响 .实验发现 ,PEI对细胞的毒性作用与剂量相关 .PEI DNA的N P比在 3 0以上方可完全结合DNA .溶酶体抑制剂氯喹可增加转染效率 .培养液中的白蛋白、血清会降低转染效率 .生理盐溶液作为配制PEI DNA复合物的溶媒 ,转染效率高于 5 %葡萄糖作为溶媒 .随着转染质粒剂量的增加 ,转染效率呈剂量依赖正效应 .聚乙烯亚胺是有效的体外真核细胞转染剂 ,可用于合成更复杂的基因释放载体 .     

阳离子脂质体介导RNA干扰的效果评价

考察自制的肽型阳离子脂质体CDO14作为RNA转染载体的细胞毒性及其运载si RNA进行RNA干扰的效果。通过MTT法检测脂质体对稳定表达荧光素酶的肺癌A549(Luc-A549)细胞的毒性。以脂质体为载体将荧光素酶si RNA(Luc-si RNA)转染至Luc-A549细胞内,用发光仪检测转染细胞内荧光素酶含量,BCA法检测细胞内总蛋白含量。在裸鼠腋下接种Luc-A549细胞,成瘤后尾静脉注射Luc-si RNA和脂质体的复合物,利用活体成像系统检测模型小鼠体内荧光素酶的表达量。细胞毒性实验表明,自制脂质体的毒性与商品脂质体DOTAP相近,低于商品脂质体Lipo2000;细胞转染实验表明自制脂质体作为基因转染载体的转染效率高于DOTAP;体内转染实验表明CDO14作为载体转染效果优于DOTAP。结果表明,肽型阳离子脂质体CDO14具有毒性小、转染效率高等优点,有望作为转染载体用于基因治疗。     

壳聚糖作为基因药物载体的研究进展

以壳聚糖及其衍生物作为基因的载体的转染效率受到许多因素的影响, 如复合物粒子大小、壳聚糖/DNA的比值、壳聚糖的分子量、脱乙酰度、转染过程中血清的浓度、介质的pH值等。对壳聚糖进行一定程度的修饰, 可以改变壳聚糖的转染效率。介绍了壳聚糖作为基因转移载体的转染条件, 转染效率和转染机制的研究情况及研究进展。     

脂质体介导法转染肿瘤细胞效率的优化

目的:研究优化影响脂质体转染效率的因素,以提高脂质体转染效率,为相关研究和应用提供参考.方法:以绿色荧光蛋白(GFP)作为报告基因,采用脂质体Lipofectamine 2000包裹pU6H1-GFP-FAK重组质粒转染Caco-2细胞,研究了细胞接种密度、DNA用量、脂质体与DNA的比例、脂质体-DNA复合物的形成时间、细胞与脂质体复合物的孵育时间、血清的有无及细胞的传代次数等因素对脂质体转染效率的影响.结果:2-5次细胞传代,2×105接种密度、4μg DNA用量、2.5:1的脂质体与DNA比例、30min脂质体-DNA复合物形成时间以及6h细胞与复合物孵育时间,转染效率最高.血清在本实验室条件下并不影响转染效率.结论:实验获得的优化条件可以明显提高脂质体对肿瘤细胞的转染效率,可作为有关研究或应用的参考.     

壳聚糖纳米粒介导基因转染的影响因素及改性修饰

壳聚糖带正电荷,可与带负电荷的DNA结合形成纳米级的多聚复合物(纳米粒)。作为一种基因载体,壳聚糖对DNA具有很好的结合和保护作用,对生物体无毒、相容性好,被广泛应用于基因转染及基因预防和治疗中。壳聚糖的主要缺点是转染效率较低,但对其进行改性或修饰后,有可能提高其转染效率。     

Detection of promoter activity by flow cytometric analysis of GFP reporter expression

Low efficiency of transfection is often the limiting factor for acquiring conclusive data in reporter assays. It is especially difficult to efficiently transfect and characterize promoters in primary human cells. To overcome this problem we have developed a system in which reporter gene expression is quantified by flow cytometry. In this system, green fluorescent protein (GFP) reporter constructs are co-transfected with a reference plasmid that codes for the mouse cell surface antigen Thy-1.1 and serves to determine transfection efficiency. Comparison of mean GFP expression of the total transfected cell population with the activity of an analogous luciferase reporter showed that the sensitivity of the two reporter systems is similar. However, because GFP expression can be analyzed at the single-cell level and in the same cells the expression of the reference plasmid can be monitored by two-color fluorescence, the GFP reporter system is in fact more sensitive, particularly in cells which can only be transfected with a low efficiency.     

Correct evaluation of reporter assays in different cell lines by direct determination of the introduced plasmid amount

Transfection efficiency in reporter gene assays is usually determined by cotransfection of a reference reporter gene under the control of a constitutively active strong promoter and determination of the reference enzyme activity. The SV40 promoter-driven beta-galactosidase reporter plasmid is frequently used as the reference reporter plasmid. Here we show that the beta-galactosidase expression in different cell lines does not correctly reflect the amount of plasmid taken up by cells and thus is not an accurate measure of transfection efficiency. The direct determination of introduced plasmid concentration in lysates of transfected cells is suitable for monitoring the transfection efficiency in reporter gene assays even if different cell lines are compared.     

新型聚乙烯亚胺衍生物在COS-7 细胞中转染和毒性的研究

目的:研究以乙二醛为连接剂的聚乙烯亚胺(Polyethyleneimine,PEI)衍生物Polyimine-PEI对非洲绿猴肾癌细胞COS-7的转染活性和细胞毒性的影响。方法:以荧光素酶质粒为报告基因,研究高分子与DNA的复合物在COS-7细胞的转染活性,用MTT方法研究高分子对COS-7细胞的毒性。结果:COS-7细胞实验显示,Polyimine-PEI具有很低细胞毒性,其毒性显著低于PEI25kDa,同时也具有高效输送质粒的能力。结论:Polyimine-PEI是一种新型的高效,低毒在基因治疗领域有相当前景的非病毒载体。     

新型聚乙烯亚胺衍生物在Hep G2 细胞中转染和毒性的研究

目的:研究以对苯二甲醛( Terephthalaldehyde)为连接剂的聚乙烯亚胺(Polyethyleneimine,PEI)衍生物PEI-Tp对肝癌细胞Hep G2的转染活性和细胞毒性的影响.方法:以荧光素酶质粒作为报告基因,研究高分子和DNA的复合物在Hep G2细胞中的转染活性,用MTT的方法研究高分子对Hep G2细胞的毒性.结果:Hep G2细胞转染结果显示构建的聚乙烯亚胺衍生物PEI-Tp具有高效输送质粒的能力;细胞毒性结果显示PEI-Tp随着浓度的增加,其毒性显著低于PEI25 kDa.结论:Hep G2细胞实验数据显示PEI-Tp是一种高效、低毒,在基因治疗领域有相当前景的非病毒载体.     

N-alkylated chitosan as a potential nonviral vector for gene transfection

Alkylated chitosans (ACSs) were prepared by modifying chitosan (CS) with alkyl bromide. The self-aggregation of ACSs in acetic acid solution was characterized by fluorescence spectroscopy and dynamic light scattering method. The results indicate that introducing alkyl side chains leads to the self-aggregation of ACSs, and CS with a 99% deacetylation degree shows no aggregation due to the electrostatic repulsion. The electrophoresis experiment demonstrates that the complex between CS and DNA was formed at a charge ratio (+/-) of 1/1; ACS/DNA complexes were formed at a lower charge ratio (+/-) of 1/4. A small amount of alkylated chitosans play the same shielding role as chitosan in protecting DNA from DNase hydrolysis. Differential scanning calorimetry (DSC) and atomic force microscopy (AFM) were employed separately to investigate the thermodynamic behavior of dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/CS and DPPC/ACS mixtures and the variation in topological structure of DPPC membrane induced by CS and ACS. It is shown that CS and ACS can cause the fusion of DPPC multilamellar vesicles as well as membrane destabilization. In contrast, the perturbation effect induced by ACS is more evident due to the hydrophobic interaction. CS and ACS were used to transfer plasmid-encoding CAT into C(2)C(12) cell lines. Upon elongating the alkyl side chain, the transfection efficiency is increased and levels off after the number of carbons in the side chain exceeds 8. It is proposed that the higher transfection efficiency of ACS is attributed to the increasing entry into cells facilitated by hydrophobic interactions and easier unpacking of DNA from ACS carriers due to the weakening of electrostatic attractions between DNA and ACS.     

新型聚乙烯亚胺衍生物在COS-7细胞中转染和毒性的研究

目的：研究以乙二醛为连接剂的聚乙烯亚胺（Polyethyleneimine,PEI）衍生物Polyimine-PEI对非洲绿猴肾癌细胞COS-7的转染活性和细胞毒性的影响。方法：以荧光素酶质粒为报告基因,研究高分子与DNA的复合物在COS-7细胞的转染活性,用MTT方法研究高分子对COS-7细胞的毒性。结果：COS-7细胞实验显示,Polyimine-PEI具有很低细胞毒性,其毒性显著低于PEI25kDa,同时也具有高效输送质粒的能力。结论：Polyimine-PEI是一种新型的高效,低毒在基因治疗领域有相当前景的非病毒载体。     

超正电荷绿荧光蛋白+36GFP作为核酸载体的细胞穿透性分析

旨在通过原核表达纯化超正电荷绿色荧光蛋白+36GFP,研究其与核酸的结合作用及作为核酸载体的细胞转导功能。将pET+36GFP-HA2质粒转化到大肠杆菌BL21(DE3)菌株中,然后表达纯化+36GFP蛋白。将得到的目的蛋白在特定浓度下分别转导293细胞、HepG2细胞、A549细胞和B16细胞,流式细胞仪检测+36GFP的转导效率;+36GFP蛋白(100 nmol/L)转导A549细胞,激光共聚焦显微镜观察结果;将+36GFP蛋白与质粒DNA按不同比例孵育,凝胶阻滞实验检测+36GFP与DNA的结合能力;激光共聚焦显微镜和流式细胞仪检测+36GFP蛋白携带质粒DNA转导细胞后报告基因的表达。结果显示,+36GFP蛋白具有较高的细胞转导效率,且随浓度升高转导效率增加,呈浓度依赖性。凝胶阻滞实验显示,+36GFP能够与质粒DNA结合,阻滞DNA在凝胶中迁移,且呈现一定的浓度依赖性。+36GFP包裹质粒转导细胞后,可高效携带质粒DNA转导进入细胞,使质粒报告基因得到表达。本研究成功表达纯化了+36GFP蛋白,证实该蛋白具有较高的细胞转导效率,可将外源核酸携带入细胞使外源基因得到表达。     

A physicochemical approach for predicting the effectiveness of peptide-based gene delivery systems for use in plasmid-based gene therapy.

Novel synthetic peptides, based on carrier peptide analogs (YKAKnWK) and an amphipathic peptide (GLFEALLELLESLWELLLEA), have been formulated with DNA plasmids to create peptide-based gene delivery systems. The carrier peptides are used to condense plasmids into nanoparticles with a hydrodynamic diameter (DH) ranging from 40 to 200 nm, which are sterically stable for over 100 h. Size and morphology of the carrier peptide/plasmid complex have been determined by photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM), respectively. The amphipathic peptide is used as a pH-sensitive lytic agent to facilitate release of the plasmid from endosomes after endocytosis of the peptide/plasmid complex. Hemolysis assays have shown that the amphipathic peptide destabilizes lipid bilayers at low pH, mimicking the properties of viral fusogenic peptides. However, circular dichroism studies show that unlike the viral fusion peptides, this amphipathic peptide loses some of its alpha-helical structure at low pH in the presence of liposomes. The peptide-based gene delivery systems were tested for transfection efficiency in a variety of cell lines, including 14-day C2C12 mouse myotubes, using gene expression systems containing the beta-galactosidase reporter gene. Transfection data demonstrate a correlation between in vitro transfection efficiency and the combination of several physical properties of the peptide/plasmid complexes, including 1) DNA dose, 2) the zeta potential of the particle, 3) the requirement of both lytic and carrier peptides, and 4) the number of lysine residues associated with the carrier peptide. Transfection data on 14-day C2C12 myotubes utilizing the therapeutic human growth hormone gene formulated in an optimal peptide gene delivery system show an increase in gene expression over time, with a maximum in protein levels at 96 h (approximately 18 ng/ml).