纳米粒子PLGA介导HIF-1α基因转染的实验研究

目的 研究纳米粒子PLGA携带特异性HIF-1α基因转染的可行性及其效率.方法 构建HIF-lα基因的真核表达质粒pcDNA3.1(+)的重组体.应用复乳溶剂挥发法制备pcDNA3.1(+)-HIF-1α的PLGA纳米粒,用正交设计法对纳米粒子的制备工艺进行优化,检测其包埋率、体外释放情况及粒度.观察纳米粒的形态、大小和粒度,研究体外释药特性、抗核酸酶降解性能.用PLGA-pcDNA3.1(+)-HIF-1α纳米粒转染U251细胞,Western blot检测PLGA纳米粒子中HIF-1α蛋白表达.结果 用正交设计法优化制备工艺成功构建pcDNAB.1(+)-HIF-1α的PICA纳米粒,形态圆整,大小均匀,平均粒径102 nm,含药量0.83%,包封率可达72%,有对抗核酸酶降解能力,体外释药缓慢.PLGA-pcDNA3.1(+)-HIF-1α纳米粒可持续高效表达HIF-1α蛋白.结论 PLGA-pcDNA3.1(+)-HIF-1α纳米粒可用于特异性基因的转染.     

阳离子脂质体介导肽核酸转染K562细胞的研究

目的 优化阳离子脂质体介导肽核酸(PNA)转染K562细胞的条件.方法 以阳离子脂质体lipofectamine 2000为载体,将标记有FITC荧光基团的PNA转染K562细胞,在荧光显微镜下观察并计算其转染效率,采用Cell Counting Kit(CCK-8)检测其细胞毒性.应用正交试验筛选出最佳的PNA和脂质体的用量及比例,并优化稀释用培养基血清浓度,以获得最优的转染效果.结果 以2.5× 105/mL～3×105/mL的细胞密度接种,100 μL/孔的培养基中加入PNA 6.25 pmol,PNA与脂质体的体积比为1∶3.5,稀释用培养基未加胎牛血清时,细胞转染效率和细胞毒性最佳,转染效率为87.2％,细胞存活率(RGR)为94.1％.结论 PNA可通过阳离子脂质体转染进入K562细胞,优化条件后得到的细胞转染效率和细胞存活率能够满足基因表达研究的实验要求.     

阴离子脂质体－阳离子脂质体复合物介导的基因转染

目的评价阴离子脂质体-阳离子脂质体复合物介导质粒转移至HepG2肝癌细胞中及其毒副作用。方法制备携载表达绿色荧光蛋白质粒的阳离子脂质体,与阴离子脂质体形成复合物。测定脂质体复合物的zeta电位,凝胶阻滞实验考察质粒包封情况,流式细胞仪测量各阴离子脂质体-阳离子脂质体复合物的转染效率,MTT法检测细胞毒性。结果复合物能完全包裹质粒,其zeta电位低于阳离子脂质体zeta电位;脂质体复合物介导的转染效率略低于阳离子脂质体,其细胞生存率高于阳离子脂质体。结论阴离子脂质体-阳离子脂质体复合物在降低细胞毒性的同时,可实现对HepG2细胞较高的转染效率。     

高强度聚焦超声联合脂质体介导p53基因转染胰腺癌细胞的实验研究

目的探讨高强度聚焦超声(HIFU)联合脂质体介导p53基因转染胰腺癌细胞的可行性以及转染后对细胞生物学行为的影响。方法实验分为5组:Ⅰ组为单纯细胞组,Ⅱ组为HIFU+质粒组,Ⅲ组为HIFU+脂质体+质粒组,Ⅳ组为脂质体+质粒组,Ⅴ组为单纯质粒组。每组予以相应处理后,采用反转录聚合酶链反应(RT-PCR)法检测各组细胞p53mRNA表达情况,四甲基偶氮唑盐(MTT)法及流式细胞术检测细胞增殖、凋亡的情况。结果荧光显微镜下见Ⅲ组有较多绿色荧光蛋白表达的细胞(转染率为10.6%),Ⅱ组及Ⅳ组有少量的荧光细胞(转染率分别为1.2%,4.8%);Ⅴ组仅有极少的荧光细胞(0.3%);Ⅰ组无荧光表达;Ⅲ组和Ⅳ组经RT-PCR法均检测到p53mRNA表达,且2组灰度值差异有统计学意义(P<0.05);MTT结果显示Ⅱ、Ⅲ、Ⅳ组细胞均有明显的生长抑制作用,而其中Ⅲ组的增殖抑制作用最为明显,与其他各组比较差异具有统计学意义(P<0.05);Ⅲ组细胞凋亡率为(14.5±1.6)%,与其他4组比较,凋亡率明显增高(P<0.05)。结论 HIFU联合脂质体可提高p53质粒转染率;p53质粒转染后促凋亡作用增强,肿瘤细胞生长进一步受到抑制。     

腺病毒及脂质体法介导脐静脉内皮细胞转染的实验研究

目的:利用腺病毒载体和脂质体转染脐静脉内皮细胞探讨转染脐静脉内皮细胞的理想条件.方法:分别使用腺病毒载体和Lipofectamine 2000脂质体介导VEGF165基因转染脐静脉内皮细胞.转染后通过倒置荧光显微镜检测转染效率,对比探讨转染脐静脉内皮细胞的理想方法.结果:采用脂质体法转染脐静脉内皮细胞效果不理想,转染率约11.60%.采用腺病毒载体转染率明显高于脂质体法,且随感染复数增加而增加.结论:采用腺病毒介导VEGF165转染脐静脉内皮细胞较脂质体法转染效率高,当M0I=50时可作为转染安全有效的条件.这为细胞转染技术在脐静脉内皮细胞的研究提供实验基础.     

抗凋亡基因bcl-2转染哺乳动物细胞的研究

目的：构建抗凋亡基因bcl-2的哺乳动物细胞表达型质粒并转染哺乳动物细胞293T细胞。方法：利用基因重组技术，将bcl-2全编码cDNA序列，导人pCMV-Tag1表达型质粒；应用磷酸钙共沉淀技术，将其转染293T细胞：并利用免疫沉淀和Westernblotting技术检测Bcl-2融合蛋白的表达。结果：bcl-2全编码cDNA序列成功导人pCMV-Tag1表达型质粒；在转染后的293T细胞成功地检测出bcl-2融合蛋白的表达。融合蛋白大小与预期完全一致。结论：该表达型质粒可以成功转染哺乳动物细胞，为对胰岛细胞的转染奠定了基础。     

聚乙烯亚胺介导基因转染的影响因素研究

目的研究非病毒基因载体聚乙烯亚胺（PEI）介导基因转染的影响因素。方法采用透射电镜观察PEI与DNA形成复合物的形态,分别考察质粒量、复合物形成时间、复合物在细胞上作用时间和氮磷比（N/P）对转染效率的影响,以筛选较优的转染条件。结果PEI可有效地与DNA形成复合物,当质粒量为每孔2μg、复合物形成时间为30min、复合物在细胞上的作用时间为4h且N/P为20时转染效率最高。结论PEI可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得到最佳的和可重复的转染结果。     

脂质体介导乙型肝炎病毒核心抗原基因转染人树突状细胞的效率

目的 观察脂质体介导HBcAg基因转染来源于人外周血单个核细胞(PBMC)树突状细胞(DC)的转染效率.方法 用HES离心沉淀法分离人外周血PBMC,经粒-巨细胞集落刺激因子(GM-CSF)、白细胞介素-4(IL-4)诱导培养DC,培养第5天以阳离子脂质体为载体将报告基因GFP(DNA:脂质体比例为1:3、1:4、1:5、1:6)及HBcAg基因(DNA:脂质体1:5)导入DC,于72h经流式细胞仪检测绿色荧光蛋白(GFP)的表达率,于48、72、96、120h流式细胞仪检测HBcAg的表达率.将基因转染后的DC 与自体淋巴细胞混合培养,检测其细胞内γ干扰素(IFN-γ)、IL-4表达水平.结果 倒置显微镜下观察脂质体转染后DC的的形态无明显变化.报告基因GFP与脂质体比例不同,其转染效率有差异,72h的表达率为37.12%(1:3)、48.55%(1:4)、52.13%(1:5)、50.75%(1:6).HBcAg基因转染DC后48、72、96、120h的HBcAg表达率分别为31.58%、55.80%、54.18%、56.99%.转染后DC与自体淋巴细胞混合培养后,淋巴细胞高表达IFN-γ,较少表达IL-4,呈现Th1为优势的免疫应答.结论 应用阳离子脂质体能有效的将HBcAg基因转染到人PBMC来源的DC,转染72h后抗原表达稳定;转染HBcAg基因的DC仍以诱导Th1免疫应答反应为主.     

PAMAM-D对异种移植用外源基因转染猪精子细胞介导作用的研究

目的:探讨新型纳米材料聚酰胺-胺型树枝状聚合物PAMAM-D对异种移植用外源基因人衰变加速因子(hDAF)转染猪精子细胞的介导作用.方法:制备PAMAM-D/hDAF cDNA复合物,分为0.2、0.4、0.6、0.8和1.0μg组,每组eDNA再按照氮磷比10:1、20:1、40:1分别加入相应剂量PAMAM-D及不加PAMAM-D,对复合物进行酶切和电泳;将复合物与猪精子细胞共孵育后,原位杂交法检测hDAF对猪精子细胞的转染效率.结果:PAMAM-D能阻止PAMAM-D/DNA复合物中DNA的降解;PAMAM-D可以促进猪精子细胞对外源性DNA的摄取,其中0.4和0.6μg组的转染效率高于其他3组,0.4 μg线状质粒加入PAMAM-D(氮磷比20:1)转染率为(47.5±0.2)%,是不加PAMAM-D的167%.结论:以PAMAM-D为载体可以提高外源基因对猪精子细胞的转染效率,有助于外源基因与精子细胞的稳定结合.     

两种阳离子脂质体介导基因转染的比较研究

In order to study the important factors involved in cationic liposome-mediated gene transfer,   Lipofectamine 2000 or DOTAP was evaluated using three types of cells (Hep-2, MCF-7 and SW-480) in vitro transfection efficiencies.  Different properties of the two reagents were analyzed and compared by DNA     arrearage assay and MTT assay.  Both Lipofectamine 2000 and DOTAP had strong capability to combine with DNA; Lipofectamine 2000 can get higher transfection efficiency of the three cells by using GFP as report gene, meanwhile, DOTAP can also get higher transfection efficiency against Hep-2 cell.  However, DOTAP showed lower transfection efficiency against MCF-7 and SW-480 cell.  On the other hand, the cytotoxicity assay showed that over 85% cell viability of MCF-7 cell could be achieved both by Lipofectamine 2000 and DOTAP under the optimal transfection condition.  Relatively speaking, Lipofectamine 2000 has very high transfection efficiency in a broad range of cell lines, but because of the special selectivity of cell type on liposome, DOTAP also has a broad application prospect.     

Polymeric Gene Transfection on Insulin-Secreting Cells: Sulfonylurea Receptor-Mediation and Transfection Medium Effect

Purpose In vitro transfection of secreting cells is regarded as one strategy for improved cell engineering/transplantation. Insulin-secreting insulinoma cell lines or pancreatic β-cells could be genetically engineered using designed polymeric vectors which are safer than viral vectors. This study investigates the effects of the constituents in transfection media on polymeric transfection.Methods Polyplexes conjugated with sulfonylurea (SU) were evaluated under different transfection conditions for gene transfection and their effects on cytotoxicity and insulin secretion. Several components in transfection media specifically associated with the insulin secretion pathway were amino acids, vitamins, Ca²⁺ and K⁺. The interactions of the polyplexes with insulin were monitored by surface charge and particle size to monitor how insulin as a protein influences transfection.Results For an insulin-secreting cell line (RINm5F), polyplexes in Ca²⁺-containing KRH medium (Ca²⁺(+)KRH) enhanced transfection and did not cause damage to biological functions. When adding amino acids, vitamins, or K⁺ or depleting Ca²⁺ from Ca²⁺(+)KRH, poly(l-lysine)/DNA complexes showed a greater reduction in transfection than SU receptor (SUR)-targeting polyplexes (SU-polyplex). Positively charged polyplexes interacted with insulin, developing a negative surface charge, and these interactions may cause a decrease in transfection.Conclusion The findings suggest that in vitro and ex vivo polymeric transfection of insulin-secreting cells can be modulated and enhanced by adjusting the transfection conditions.     

Transfection Efficiency and Cytotoxicity of Nonviral Gene Transfer Reagents in Human Smooth Muscle and Endothelial Cells

PURPOSE: Evaluation of a nonviral transfection reagent with respect to efficient gene transfer into primary human vascular cells. METHODS: Complexes consisting of seven commercially available transfection reagents (DAC-30, DC-30, Lipofectin, LipofectAMINE PLUS, Effectene, FuGene 6 and Superfect) and EGFP encoding plasmid DNA were studied. The in vitro transfection efficiency and cytotoxicity in human aorta smooth muscle cells (HASMCs) and endothelial cells (HAECs) and rat smooth muscle cells (A-10 SMCs) were assayed in the presence of serum using flow cytometric analysis and ATP-quantitation assay, respectively. RESULTS: Human primary cells were transfected less efficiently compared to the rat smooth muscle cell line. Transfection efficiency depended on the type of reagent, the reagent/DNA ratio, and, most importantly, on the cell type used. Determination of cytotoxicity showed that the effects of transfection on cell viability did not significantly differ from one another depending on the cell type. The exception to this was Superfect, which obviously reduced cell viability in all cell types. CONCLUSIONS: Our experiments showed that DAC-30 is the preferred transfection reagent for HASMCs and HAECs, exhibiting an improved efficiency combined with an acceptable cytotoxicity. Therefore, it might offer a therapeutic option for the treatment of cardiovascular disease and prove suitable for further drug development.     

Novel Biocompatible Cationic Copolymers Based on Polyaspartylhydrazide Being Potent as Gene Vector on Tumor Cells

Introduction The reaction between α,β-poly(aspartylhydrazide) (PAHy), a water soluble synthetic polymer and 3-(carboxypropyl)trimethyl-ammonium chloride (CPTACl) produced copolymers bearing permanent positive charges (PAHy–CPTA) with molecular weight of 10 kDa and PAHy–CPTA copolymers differing in positive charge amount (18–58%) were chosen for biological investigations. Materials and methods Biophysical properties of DNA/PAHy–CPTA polyplexes were evaluated in terms of DNA condensation, zeta potential and size distribution. Cytotoxicity studies on Neuro2A murine neuroblastoma cells evidenced absence of toxicity of these copolymers up to 300 μg/ml unlike linear polyethylenimine (LPEI) that was highly toxic already at 20 μg/ml. Results and Discussion PAHy–CPTA copolymers did not induce any erythrocyte aggregation up to 1 mg/ml. Cellular interaction studies of PAHy–CPTA polyplexes evidenced a faster binding of these polyplexes with cells compared to DNA/LPEI polyplexes. The in vitro transfection ability of PAHy–CPTA polyplexes was strongly affected by experimental conditions reaching about 10% of the transfection efficiency of optimized LPEI polyplexes. Conclusions Finally, in vivo application studies confirmed the biocompatibility of PAHy–CPTA copolymers. With LPEI, clear signs of microvesicular fatty liver were observed and with LPEI polyplexes significant weight loss. In strong contrast, PAHy–CPTA did not induce histopathological changes or weight loss.     

Novel Reticular Cyclen‐Based Polymer as Gene Vector in DNA Transfection

This study provided an experimental evidence for the use of cyclen (1, 4, 7, 10‐tetraazacyclododecane)‐based polymer for gene delivery. The interesting interaction of the polymer with plasmid DNA was studied by using fluorescence titration, circular dichroism spectra, agarose gel electrophoresis and atomic force microscopy. It was found that polyplex was formed between the polycation and plasmid DNA. The results demonstrated that the cyclen‐based polymer could act as non‐viral gene vector with relatively low cytotoxicity.     

Effective Transfection of Rabies DNA Vaccine in Cell Culture Using an Artificial Lipoprotein Carrier System

PURPOSE: To evaluate the transfection efficiency in cell culture of rabies plasmid DNA vaccine carried by a novel artificial lipoprotein system. METHODS: Phospholipid nanoemulsions resembling the lipid core of natural lipoproteins were prepared. The artificial lipoprotein carrier system for DNA was constructed by assembling of the nanoemulsion (NE)-palmitoyl-poly-L-lysine (p-PLL)-rabies DNA complex. Agarose gel electrophoresis, zeta potential, and mobility measurement were conducted to determine the surface charge balance in various complex compositions. Transfection and transfection efficiency were examined by fluorescence microscopy and flow cytometry, respectively. RESULTS: The artificial lipoprotein system was successfully constructed and the rabies DNA vaccine was effectively transfected in glioma cell line SF-767. The amount of p-PLL incorporated into the artificial lipoprotein formulations had a significant effect on transfection efficiency. The new system also proved to be more efficient in cellular transfection of rabies DNA vaccine than the commercial lipofectamine formulation. CONCLUSIONS: Effective transfection of rabies DNA vaccine in cell culture can be achieved using the novel artificial lipoprotein carrier system, and the charge balance of the NE-p-PLL-DNA complex appears an important factor.     

Cationic Cholesterol Promotes Gene Transfection Using the Nuclear Localization Signal in Protamine

Purpose. The purpose of this study was to evaluate protamine-mediated gene transfection by liposomes with a novel cationic cholesterol derivative (I) compared to those with DC-Chol or DOTMA (Lipofectin). Methods. Plasmid pGL3 DNA was complexed to the cationic liposomes with the derivative (I), DC-Chol, or DOTMA in SFM101(Nissui) at room temperature for 15 min, and thereafter the complex was incubated with target cells (NIH3T3) for 4 h at 37°C. The cells then were washed and cultured for another 40 h in the growth medium at 37°C before luciferase assay. Results. The transfection efficiency by the liposomes with the derivative (I) was much higher than that by the liposomes with DC-Chol or DOTMA. In addition, its transfection efficiency was enhanced greatly by the addition of protamine. Atomic force microscopy showed clearly how the size of the DNA-liposome complex was changed by protamine. Furthermore, fluorescence microscopic images showed that Cy5-labeled antisense DNAs were transferred quicker into the nucleus of the target cells by the liposomes with the derivative I in the presence of protamine. Conclusion. Although there exist several possible mechanisms, such as improved protection of DNA intracellularly by derivative (I), one possibility is that the DNA-protamine-liposome complex with the derivative (I) promoted gene transfection more significantly into the nucleus of the target cells using the nuclear localization signal of protamine.     

慢病毒载体介导apelin基因转染人脐带间充质干细胞的实验研究

【摘要】目的构建带有荧光标记基因增强型绿色荧光蛋白（EGFP）和apelin基因的重组质粒pUbi-apelinFLAG-pSV40-EGFP并进行慢病毒包装,探讨其转染人脐带间充质干细胞的最佳感染复数（MOI）值及目的基因表达情况。方法化学合成目的基因片段并扩增。采用In-Fusion技术将酶切后的目的基因片段与线性质粒载体连接,转化入感受态DH5α细胞中后筛选阳性克隆并进行测序。重组质粒慢病毒载体转染293T细胞,包装慢病毒并测定滴度。将重组质粒慢病毒按不同MOI值转染人脐带间充质干细胞,根据转染效率得到最佳MOI值,并采用Real-timePCR及Western blot方法检测目的基因表达情况。结果通过PCR扩增获得酶切位点碱基修饰后的大小约284bp的目的基因片段,与慢病毒质粒载体连接后形成pUbi-apelin-FLAG-pSV40-EGFP重组质粒,测序结果与预期完全符合,并成功包装慢病毒颗粒。最佳MOI值为20,转染效率为（90.32±3.61）%。慢病毒载体能高效转染人脐带间充质干细胞且2周内持续稳定上调目的基因mRNA及蛋白的表达。结论重组质粒慢病毒载体pUbi-apelin-FLAGpSV40-EGFP可有效转染人脐带间充质干细胞,并可持续高表达apelin基因     

In Vitro Gene Transfection in Human Glioma Cells Using a Novel and Less Cytotoxic Artificial Lipoprotein Delivery System

Purpose. To develop and evaluate a novel artificial lipoprotein delivery system for in vitro gene transfection in human glioma cells. Method. Nanoemulsion was formulated with similar lipid compositions present in natural lipoproteins. The oil phase of nanoemulsion was composed of triolein (70%), egg phosphatidylcholine (22.7%), lysophosphatidylcholine (2.3%), cholesterol oleate (3.0%), and cholesterol (2.0%). To replace the surface protein as in natural lipoprotein, poly-L-lysine was modified to add palmitoyl chains at a basic condition and was incorporated onto the nanoemulsion particles through hydrophobic interaction. A model plasmid DNA, pSV--Gal containing a reporter gene for -galactosidase was carried by the nanoemulsion/poly-L-lysine particles. The charge variation of so-formed complex was examined by agarose gel electrophoresis and zeta potential measurement. In vitro transfection was conducted on human SF-767 glioma cell line using this new system. After standard X-Gal staining, transfected cells were observed under light microscope. The effect of chloroquine on the transfection was examined and, finally, the cytotoxicity of this new system was evaluated in comparison with commercial Lipofectamine gene transfection system. Results. The plasmid DNA was effectively carried by this artificial lipoprotein delivery system and the reporter gene was expressed in the glioma cells. Transfection efficiency was significantly increased by the treatment of chloroquine, indicating that endocytosis possibly was the major cellular uptake pathway. Compared to Lipofectamine system, this new delivery system demonstrated similar transfection efficiency but a much lower cytotoxicity. In the experiment, the cell viability showed up to 75% using this system compared to only 24% using Lipofectamine system. Conclusion. A new artificial lipoprotein delivery system was developed for in vitro gene transfection in tumor cells. The new system showed similar transfection efficiency but a much lower cytotoxicity compared with commercial Lipofectamine system.