FuGENE 6 Transfection Reagent及pEGFP-N1-HSF1转染JEG-3细胞条件的优化

目的:优化FuGENE~?6 Transfection Reagent(FuGENE)转染质粒pEGFP-N1-HSF1到人绒毛膜癌细胞JEG-3的条件。方法:利用DNA延滞实验考察pEGFP-N1-HSF1质粒与FuGENE试剂的结合力,用不同剂量的FuGENE试剂和质粒转染JEG-3细胞,通过倒置荧光显微镜和血球计数法计算其转染效率,筛选出最佳的转染条件并进行转染,通过qRT-PCR检测HSF1 mRNA的水平。结果:DNA延滞实验显示FuGENE和pEGFP-N1-HSF1质粒具有较高的结合力,当质粒量一定时,随着FuGENE量的增加,其转染效率先增加后降低;当FuGENE和质粒比例为2∶1(μL∶μg)时,转染效率最高,达(39.05±1.19)%;当固定转染比例时,随着转染剂量的增加,其转染效率先增加后降低,当转染剂量为250μL时,转染效率最高,达(42.98±2.56)%;qRT-PCR结果显示,转染组HSF1 mRNA的水平较对照组有显著上调。结论:在转染比例为2∶1,转染剂量为250μL时,对JEG-3细胞有最佳的转染效率。

氨基葡甘聚糖的质粒DNA转染载体作用的研究

目的 :发展一种新的半人工合成的质粒DNA转染载体。方法 :用氨基化方法将纯化的葡甘聚糖阳离子化 ,用凝胶阻滞检测法 (gelretardationassayforcomplexformation)观察氨基葡甘聚糖 DNA复合物的形成 ,以及用氨基葡甘聚糖作报告基因质粒pEGFP Cl的载体 ,转染HEK2 93细胞 ,观察转染效率。结果 :葡甘聚糖氨基化后溶液离子强度增大 2 0倍左右 ,氨基葡甘聚糖可与质粒DNA形成复合物 ,形成的复合物转染HEK2 93细胞后 ,报告基因pEGFP Cl获得阳性表达。氨基葡甘聚糖最大至 1 0 %仍未显示细胞毒性。结论 :氨基葡甘聚糖可发展为DNA载体系统 ,在HEK2

脂质体介导质粒DNA细胞转染的优化研究(英文)

Objective:The development of gene carriers for efficient gene delivery into cells has attracted growing attention in recent years.The aim of this study was to achieve a better outcome of AAV-293 cells transfection by plasmid DNA.Methods:We studied the optimal condition for higher efficiency of cationic lipid-mediated cell transfection.Four experimental groups were set.Plasmid DNA and liposome were mixed in each groups at different ratios(μg:μL),1:2.5,1:3.5,1:4.0 and 1:5.0,respectively.LacZ gene functioned as reporter gene,measuring the transfection efficiency of the four groups using the method of X-gal staining.Results:When the ratio was 1:3.5,the cell transfection rate was the highest.While the ratio of 1:2.5 recommended by product manual achieve the lowest transfection rate.Their difference had statistical significance.Conclusion:In order to obtain a higher transfection efficiency,optimization on conditions of the ratio of plasmid DNA to liposome is necessary in cell transfection.

磷酸钙转染方法的优化

通过比较293T细胞的接种密度、质粒DNA的量以及CO2浓度对磷酸钙转染方法的影响,确定最佳的转染条件.以各单因素试验的最适条件为依据,设计三因素三水平的正交试验.结果表明,磷酸钙介导质粒DNA转染,当293T细胞的接种密度为5×105/mL、质粒DNA的量为30μg、细胞培养环境CO2浓度为3%时,转染效果最佳.

bcl-2基因克隆、转染及其在PC12细胞中的表达

【目的】研究bcl 2基因 (bcl 2 )在PC12细胞中的表达。【方法】构建真核表达载体pcDNA3 bcl 2 ,采用脂质体介导将重组质粒导入PC12细胞 ,Westernblot和原位免疫组化检测外源基因表达。【结果】本实验成功构建了真核表达载体pcDNA3 bcl 2 ,并用脂质体介导的方法获得了稳定表达bcl 2的细胞克隆 ;Westernblot显示有 2 6ku的蛋白质表达 ,bcl 2单克隆抗体免疫组化染色呈阳性反应。【结论】重组质粒 pcDNA3 bcl 2经转染能够在PC12细胞中有效表达 ,为进一步研究 bcl 2对PC12细胞的生物学功能奠定了基础。

Experimental and clinic-opathologic study on the relationship between transcription factor Egr-1 and esophageal carcinoma

AIM To observe the growth suppression effect of exogenous introduction of early growth response gene-1 (Egr-1 gene) on esophageal carcinoma tissue as well as on esophageal carcinoma cell line Eca109 and to explore the potential application of Egr-1 gene in gene therapy of tumor.METHODS Eukaryotic expression vector of PCMV-Egr-1 plasmid was introduced into Eca109 cell line which expressed no Egr-1 protein originally with lipofectamine transfection method. The introduction and expression of PCMV-Egr-1 plasmid into Eca109 cell line was confirmed by G418 selection culture, PCR amplification of neogene contained in the vector, Western blot analysis and immunocytochemical analysis. The cell growth curve,soft agar colony formation rate and tumorigenicity in SClD mice were examined to demonstrate the growth suppression effect of exogenous Egr-1 gene on Eca109 cell line. The Egr-1 mRNA and Egr-1 protein were also detected in 50 surgical specimens of esophageal carcinoma by in situ hybridization and immunohistochemistry.RESULTS Exogenous Egr-1 gene was introduced successfully into Eca109 cell line and expressed Egr-1 protein stably. The transfected Eca109 cell line grew more slowly than control Eca109 as shown by cell growth curves, the soft agar colony formation rate (4.0% vs 6.9%, P＜0.01) and the average growth rate of tumor in SCID mice (35.5 ± 7.6 vs 65.8 ± 7.6, P＜0.05). The expression level of Egr-1 mRNA and protein significantly increased in dysplastic epithelia adjacent to cancer rather than in cancer tissues (65.8% vs 20.0% by ISH and 57.9% vs 14.0% by IHC, P＜0.01).CONCLUSION Exogenous Egr-1 gene shows the strong effect of growth inhibition in Eca109 cell line. Egr-1 in the cancer tissue shows down-regulated expression that supports the inhibited function of Egr-1 in cancer growth and suggests Egr-1 may have an important role in gene therapy of esophageal carcinoma.

金属配合物作为核酸释放的载体

基因治疗是指利用一种载体将健康的基因载入细胞替换致病的基因.由基因缺陷导致的人类疾病达1200多种,最合理的选择是采用基因替换的方法进行治疗.基因治疗的关键问题是解决"使用何种载体才能安全有效地将治疗基因载入靶细胞".非病毒基因载体主要是一些有机阳离子物种,一直受到极大重视;近年来,磷酸钙、纳米粒子和金属配合物释放核酸的功能也开始受到关注.本文总结了金属配合物作为非病毒基因载体使用的研究进展,希望由此理解配合物释放核酸的优势和不足之处.

悬浮培养昆虫细胞SF9瞬时表达重组蛋白的条件优化(英文)

瞬时基因表达(TGE)是一种省去筛选高表达细胞株步骤,快速获得重组蛋白质的技术。昆虫细胞作为一种广泛使用的蛋白表达系统,在国内外关于它的瞬时基因表达的研究比较少。这篇文章进行了以昆虫SF9细胞在悬浮培养条件下进行瞬时表达eGFP和肿瘤坏死因子受体融合蛋白(TNFR-Fc)的条件优化,以期为外源蛋白在昆虫细胞中瞬时表达打下基础。经优化后,转染效率达到37%,重组TNFR-Fc达到700μg/mL。