摘要
探讨利用脂质体介导真核表达质粒转染体外培养的人角朊细胞的最佳转染条件。体外分离培养正常人角朊细胞 ,培养至 6 0 %、70 %、80 %、90 %及 10 0 %融合时 ,应用不同浓度的LipofectAMINE包被真核表达质粒pCMV·SPORT β gal,分别转染 6、8、10、12及 2 4h。细胞经转染后再培养 48h ,行 β 半乳糖苷酶原位染色 ,镜下观察并计算阳性转染率。被转染的阳性细胞 ,可见质粒 β 半乳糖苷酶基因的良好表达 ;当细胞融合率为 80 %、90 % ,以12 5 μL/ 10 0 μL的LipofectAMINE包被 1 5 μg/ 10 0 μL的pCMV·SPORT β gal,转染时间为 8h的转染率最高 ,可分别达到 (31 35± 1 35 ) %、(32 32± 2 4) %。该实验说明LipofectAMINE可有效地介导真核表达质粒转染培养的人角朊细胞 ;转染率与细胞生长状态、脂质体包被质粒的浓度比例、及转染时间直接相关。
To investigate the optimum transfection condition in transfecting human keratinocytes with plasmid liposome complexes in vitro , the cultured human keratinocytes at 60% ～ 100% confluences were exposed to the eukaryotic expression plasmid, pCMV·SPORT β gal, coated with LipofectAMINE in different DNA/liposome mixing concentration ratios After cultured for another 48 hours following the ends of 6 ～ 24 hours exposures to the DNA liposome complexes, the transfected human keratinocytes were visualized by β galactosidase staining Then, the transfection efficiency was determined by calculating the rate of β galactosidase staining positive cells β galactosidase expression was showed clearly in human keratinocytes transfected with the DNA liposome complexes The highest efficiency was achieved with cultured cells at 80% and 90% confluences, demonstrating by the transfection rates of (31 35±1 35)% and (32 32±2 47)% respectively Meanwhile, the essential transfection conditions for these efficiencies were in coating pCMV·SPORT β gal DNA of 1 5μg/100μL with LipofectAMINE of 12 5μL/100μL, and exposing the cells to the DNA liposome complexes for 8 hours These results indicate that LipofectAMINE could effectively transfer eukaryotic expression plasmid into human keratinocytes in vitro , for which the optimization of transfection conditions involve in cells growth state, DNA/liposome mixing concentration ratio, and exposure time of cells to DNA liposome complexes
出处
《基础医学与临床》
CSCD
北大核心
2001年第2期180-184,共5页
Basic and Clinical Medicine
基金
国家自然科学基金! (396 0 0 15 3)
关键词
基因转染
脂质体
角朊细胞
细胞培养
gene transfection
liposome
keratinocyte
cell culture
