转染癌胚抗原基因的人树突状细胞疫苗表达的研究

目的　探索使用质粒转导制备转基因DCs疫苗的方法。方法　采用细胞因子rhIL 4和rhGM CSF诱导培养法制备人外周血树突状细胞 ,用Lipofectine向人DCs转染含人CEA真核表达质粒。结果　转染组DCs培养上清CEA含量明显高于未转染组培养上清CEA含量。结论　人CEA真核表达质粒转染DCs可内源性表达CEA     

人乳头瘤病毒16 E6转染冻融人外周血树突状细胞疫苗体外抗宫颈癌作用

目的：制备来源于人外周血并转染人乳头瘤病毒（HPV）16 E6基因的冻融树突状细胞（DC）疫苗，检测其细胞形态。转染情况及体外诱导的免疫效应。方法：细胞因子扩增人外周血DC，低温-70℃冻存；Lipofectamine转染HPV16 E6制备DC疫苗。动态形态学观察，免疫细胞化学检测E6分子表达，体外诱导并测定细胞毒性T淋巴细胞活性。结果：转染E6基因的人外周血冻融DC呈形态迥异的多突起状，免疫细胞化学染色显示E6蛋白表达阳性；MTT法检测人宫颈癌细胞株（CaSKIi细胞）的杀伤活性明显高于对照组（P〈0．01），与新鲜DC疫苗比无统计学意义（P〉0．05）。结论：转染历基因的人外周血冻融DC疫苗保持了功能成熟DC的形态特征，且内源性表达E6蛋白，并在体外诱导出高效特异性的抗宫颈癌免疫反应，为宫颈癌的主动特异性免疫治疗提供了理想的疫苗和实验依据。     

Limitations and caveats of magnetic cell labeling using transfection agent complexed iron oxide nanoparticles

Cell labeling with various types of nanomaterial, such as FDA‐approved iron oxide nanoparticles (IONPs) has become common practice in biomedical research. The low uptake of IONPs stimulates the use of transfection agents (TA), but the effect on stability of the IONPs and their cellular interactions has received minimal attention. In the present study, we evaluated the use of Lipofectamine as a commonly used TA and tested different ratios of TA and IONPs. While the TA–IONP complexes are stable in saline, at a high ratio of TA over IONP, substantial aggregation occurred in serum‐containing media. Even for the highest ratio, TA was unable to completely cover the IONPs, resulting in a net negative charge of all complexes. At high TA–IONP ratios, more complexes remained surface‐associated without internalization, resulting in cell death, while at lower TA–IONP ratios, complexes were more avidly taken up through fluid‐phase pinocytosis and clathrin‐mediated endocytosis. At later time points, the endocytosed complexes accumulated within the lysosomes and affected the appearance of lysosomal structures. The data indicate that TAs should be used with care as, depending on the ratio of TA and IONP, the complexes may aggregate, inducing cell death and preventing internalization. Copyright © 2012 John Wiley & Sons, Ltd.     

pSecTag-EGFP真核表达载体的构建及其转染鸡胚胎成纤维细胞条件的优化

目的构建含报告基因增强绿色荧光蛋白(EGFP)的分泌型真核表达载体pSecTag-EGFP,用以研究pSecTag/HygroB转染鸡胚胎成纤维细胞的转染效率,为目的基因的成功表达奠定基础。方法用PCR方法克隆得到EGFP基因,通过酶切、连接、转化构建pSecTag-EGFP分泌型真核表达载体,设计优化实验,通过脂质体介导的方法转染鸡胚胎成纤维细胞,在荧光显微镜下直接观察该基因的表达。用B radford法检测不同组合的细胞培养液中的EGFP蛋白的相对含量。结果脂质体介导的方法能有效转染鸡胚胎成纤维细胞,实验中EGFP蛋白相对表达量随质粒量的增加而明显升高(P<0.01)。其中脂质体2μl、质粒1.0μg时目的蛋白相对表达量最高。结论pSecTag-EGFP质粒构建成功,并成功优化了转染条件。     

聚乙烯亚胺介导的报告基因在体外转染效率的研究

目的　探讨阳离子聚合体载体PEI2 5 (分枝状 ,2 5KDa)在体外的转染效率。方法　PEI2 5和LipofectamineTM2 0 0 0作为转染试剂 ,瞬时转染CHO细胞系 ,通过检测荧光素酶来评估PEI和LipofectamineTM2 0 0 0的转染效率。结果　当N P =5 ,6 ,7时 ,PEI2 5的转染效率与LipofectamineTM2 0 0 0相比无显著性差异 (P >0 .0 5 )。结论　经济实用的PEI适用于体外的瞬时转染。     

重组腺病毒介导mIL-12 基因转染的 EL-4细胞生物学特性的研究

目的：观察 ｍ Ｉ Ｌ１２ 基因转染的小鼠 Ｅ Ｌ４ 细胞（ Ｃ５７ Ｂ Ｌ／６ 小鼠 Ｔ 淋巴瘤细胞株）体外生物学特性的改变。方法：阳离子脂质体协同 Ｉ Ｌ１２ 重组腺病毒感染 Ｅ Ｌ４ 细胞。结果及结论：基因转染的 Ｅ Ｌ４ 细胞有 ｍ Ｉ Ｌ１２ 的 ｍ Ｒ Ｎ Ａ 表达,培养上清中可以检测到 ｍ Ｉ Ｌ１２ 的生物学活性。与野生型 Ｅ Ｌ４ 细胞相比,ｍ Ｉ Ｌ１２ 重组腺病毒感染的 Ｅ Ｌ４ 细胞形态及体外增殖能力无明显改变, 转染细胞的成瘤性降低。此外,研究结果还表明,阳离子脂质体能显著提高腺病毒的感染效率。     

阳离子脂质体对重组腺病毒转染效率的影响

目的　观察不同因素对重组腺病毒转染效率的影响。方法　以重组腺病毒去感染小鼠T淋巴瘤细胞株EL 4,加入不同的刺激因子和lipofectamine,通过计数X gal染色阳性细胞数测定转染效率。结果　rmIL 2 ,植物血凝素均不能改善重组腺病毒对EL 4的感染效率 ,而阳离子脂质体可以明显提高重组腺病毒对EL 4的感染效率。结论　可以利用阳离子脂质体来提高重组腺病毒的转染效率。     

High loading efficiency and sustained release of siRNA encapsulated in PLGA nanoparticles: Quality by design optimization and characterization

Poly(dl-lactide-co-glycolide acid) (PLGA) is an attractive polymer for delivery of biopharmaceuticals owing to its biocompatibility, biodegradability and outstanding controlled release characteristics. The purpose of this study was to understand and define optimal parameters for preparation of small interfering RNA (siRNA)-loaded PLGA nanoparticles by the double emulsion solvent evaporation method and characterize their properties. The experiments were performed according to a 2⁵⁻¹ fractional factorial design based on five independent variables: The volume ratio between the inner water phase and the oil phase, the PLGA concentration, the sonication time, the siRNA load and the amount of acetylated bovine serum albumin (Ac-BSA) in the inner water phase added to stabilize the primary emulsion. The effects on the siRNA encapsulation efficiency and the particle size were investigated. The most important factors for obtaining an encapsulation efficiency as high as 70% were the PLGA concentration and the volume ratio whereas the size was mainly affected by the PLGA concentration. The viscosity of the oil phase was increased at high PLGA concentration, which explains the improved encapsulation by stabilization of the primary emulsion and reduction of siRNA leakage to the outer water phase. Addition of Ac-BSA increased the encapsulation efficiency at low PLGA concentrations. The PLGA matrix protected siRNA against nuclease degradation, provided a burst release of surface-localized siRNA followed by a triphasic sustained release for two months. These results enable careful understanding and definition of optimal process parameters for preparation of PLGA nanoparticles encapsulating high amounts of siRNA with immediate and long-term sustained release properties.     

Polyethyleneimine-mediated gene delivery into rat pheochromocytoma PC-12 cells

In this study, we examined the use of polyethyleneimine (PEI) as a non-viral gene carrier and lipofectamine(trade mark) 2000 as control for rat pheochromocytoma PC-12 cells. The complex formation of PEI and DNA or lipofectamine and DNA was characterized by gel electrophoresis and measurement of particle size and surface charge. A gradual increase in surface charge (from 0.7 to 43 mV) and a gradual decrease in particle size (from 900 to 130 nm) was observed in the PEI-DNA complex with higher PEI concentrations. The cytotoxicity of PC-12 cells for lipofectamine-DNA complex was similar to PEI-DNA complex at N:P charge ratios of 4 and 8. Transfection efficiency was 14% for lipofectamine and 15% for PEI. At low N:P ratio, DNA condenses poorly, so the particle size tends to be large and polydispersed, resulting in poor transfection efficiency. Meanwhile, a high N:P ratio results in high transfection efficiency and cytotoxicity. Transfected PC-12 cells showed the generation of neurites from transfected PC-12 cells in the presence of NGF, indicating the differentiation of PC-12 cells. NGF-differentiated PC-12 cells were transfected by PEI-DNA complex of N:P charge ratio 8. From real-time imaging for transfection, the enhanced green fluorescent protein (EGFP) started to localize in the nuclei of PC-12 cells at 5 h and localized in the cytoplasm from 15 h. Our study demonstrates that PEI or lipofectamine may be applied as an effective gene carrier for PC-12 cells.