【摘要】 目的:探讨聚乙烯亚胺(polyethylene im ine,PEI)作为生存素(survivin)反义核酸(anti-sense oligodeoxynuc leotides,ASODN)的载体投递肝癌SMMC-7721细胞的条件。方法:凝胶阻滞实验筛选PEI与survivin ASODN形成静电复合物的最佳质量比;W ST-8法检测PEI对肝癌细胞的毒性;流式细胞仪及W ST-8法筛选荧光标记的PEI与ASODN在不同质量比时的瞬时转染效率及48 h转染效率;流式细胞仪及荧光显微镜分别检测荧光标记的ASODN以及PEI-ASODN复合物进入细胞的情况;W ST-8法检测血清对PEI转染效率的影响。结果:m(PEI)∶m(ASODN)为0.625∶1~2.5∶1时可以形成电中性状态的静电复合物;PEI终质量浓度≤4μg/mL对肝癌细胞的毒性较小;m(PEI)∶m(ASODN)为0.75∶1时转染效率最高;血清存在对PEI转染效率无明显影响。结论:PEI是一种低毒的ASODN投递载体,其终质量浓度≤4μg/mL可作为肝癌细胞的转染载体,血清条件下m(PEI)∶m(ASODN)为0.75∶1时,PEI携带survivin ASODN投递肝癌SMMC-7721细胞的效率最高。更多还原

【Abstract】 Aim: To investigate the best condition of survivin antisense oligodeoxynucleotides(ASODN) mediated by polyethyleneimine on the proliferatory of hepatocelluar carcinoma SMMC-7721 cells.Methods: Gel electrophoresis retardation assays sieve the best qualitative ratio of PEI /ASODN.Cytotoxic effects of PEI were quantified by WST-8 test in vitro.The instantaneous and 48 h transfection efficiencies of PEI were determined by flow cytometry and WST-8 test in various ratios of PEI / ASODN.Similarly,the uptake rates of FAM labeled PEI-ASODN or ASODN on SMMC-7721 cells were tested by flow cytometry,while the morphological uptaking of FAM labeled PEI-ASODN or ASODN was observed under fluorescence microscope.The influence of bovine serum culture on PEI transfection efficiency was detected by WST-8 test.Results: PEI could retard ASODN migration when PEI/ASODN ratio varied from 0.625∶1 to 2.5∶1.When incubated with PEI for 48 hours,the 85 % survival rate of SMMC-7721 cells appeared at 4 μg/mL,and when the PEI/ASODN ratio is 0.75 μg/mL,the best transfection efficiency of PEI appeared.The bovine serum in the culture medium didn’t influence the transfection efficiency.Conclusion: It is certificated that PEI is low-toxicant as ASODN transfection vector,and can be used to transfer ASODN to hepatocelluar carcinoma when it is under 4 μg/mL.The best transfection efficiency of PEI-ASODN on hepatocelluar carcinoma SMMC-7721 cells is tested when the PEI/ASODN ratio is 0.75 μg/mL with the bovine serum culture medium in vitro.更多还原