为开展目标蛋白质在梅花鹿耳成纤维细胞的功能研究提供理论和技术依据，培养梅花鹿耳成纤维细胞，优化脂质体转染梅花鹿耳成纤维细胞的条件，以获得最优质粒转染参数。采用组织块贴壁法和差速贴壁法分离梅花鹿耳成纤维细胞，观察细胞基本形态特征变化，免疫荧光法鉴定波形蛋白(Vimentin)的表达;以增强型绿色荧光蛋白(EGFP)基因为标记基因，利用LipofectamineTM 3000介导外源DNA转染梅花鹿耳成纤维细胞，探讨质粒 DNA 与脂质体比例(1。0∶1。0，1。0∶1。5，1。0∶2。0，1。0∶2。5，1。0∶3。0)、质粒用量(0。5，1。0，1。5，2。0，2。5 μg)、细胞传代次数(第2～6代)、细胞初始接种密度(50％，60％，70％，80％，90％)、转染效果观测时间(12，24，36，48，60h)对转染效率的影响。结果表明:组织块贴壁法培养的梅花鹿耳成纤维细胞在倒置荧光显微镜下可见长梭形细胞生长，细胞免疫荧光检测波形蛋白(Vimentin)的表达结果为阳性;筛选的最佳转染条件:当质粒DNA与脂质体比例为1。0∶2。0，质粒DNA用量为1。5μg，细胞传代次数为第3代，细胞初始接种密度为70％，转染后48 h进行观测时，即可获得较佳的转染效率。研究成功分离培养梅花鹿耳成纤维细胞，筛选出了脂质体转染梅花鹿耳成纤维细胞的最佳条件，可为梅花鹿基因工程的相关研究提供参考依据。
In Vitro Culture and Optimization of Transfection Conditions of Sika Deer Ear Fibroblasts
This study aims to cultivate sika deer ear fibroblasts,optimize the conditions for liposome transfection of sika deer ear fibroblasts,and obtain the optimal plasmid transfection parameters,pro-viding theoretical and technical basis for conducting functional research on target proteins in sika deer ear fibroblasts.Sika deer ear fibroblasts were separated by tissue block attachment method and differential attachment method,basic morphological characteristics of the cells were observed,and expression of Vimentin was identified by immunofluorescence.Using enhanced green fluorescent pro-tein(EGFP)gene as marker gene,exogenous DNA transfection was mediated into sika deer ear fibro-blasts by LipofectamineTM3000,to explore the effects of ratio of plasmid DNA to liposomes(1.0:1.0,1.0∶1.5,1.0∶2.0,1.0∶2.5,1.0∶3.0),plasmid dosage(0.5,1.0,1.5,2.0,2.5 μg),number of cell passages(2nd-6th),initial cell seeding density(50％,60％,70％,80％,90％),and observation time of transfection effect(12,24,36,48,60 h)on transfection efficiency.The results showed that the growth of long spindle-shaped cells of sika deer ear fibroblasts cultured with tissue block adherence method could be observed under inverted fluorescence microscope,and the result of immunofluorescence detection of Vimentin expression was positive.The optimal transfection conditions for screening:When ratio of plasmid DNA to liposomes was 1.0:2.0,amount of plasmid DNA was 1.5 μg,number of cell passages was the third generation,and initial cell inoculation density was 70％,and when observed 48 h after transfection,better transfection efficiency could be achieved.In this study,sika deer ear fibroblasts were successfully isolated and cultured,and the optimal conditions for liposome transfection of sika deer ear fibroblasts were screened,which can provide important reference for related research on sika deer genetic engineering.
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