慢病毒-基质细胞衍生因子-1α-绿色荧光蛋白载体转染大鼠心肌成纤维细胞

发布时间：2018-03-04 21:20

本文选题：心肌成纤维细胞　切入点：慢病毒-基质细胞衍生因子-α绿色荧光蛋白载体　出处：《解剖学报》2017年01期 　论文类型：期刊论文

【摘要】：目的探讨慢病毒-基质细胞衍生因子-1α-绿色荧光蛋白(LV-SDF-1α-GFP)对心肌成纤维细胞影响,最佳感染条件及目的蛋白表达和分泌特点。方法差速贴壁法分离培养乳大鼠心肌成纤维细胞,观察和免疫荧光鉴定。不同滴度及条件的LV-SDF-1α-GFP转染心肌成纤维细胞,观察荧光表达,探索最佳转染条件。LV-SDF-1α-GFP目的基因病毒以及阴性对照CON145病毒的转染心肌成纤维细胞,绘制生长曲线,探索转染对心肌成纤维细胞的增殖有无明显影响。利用最佳转染剂量组转染心肌成纤维细胞,斑点印迹法(Dot blotting)检测SDF-1α目的蛋白分泌。计量资料进行统计学分析。结果心肌成纤维细胞具很高活性,传至4代纯度高,折光度好,无自发性搏动。LV-SDF-1α-GFP最佳转染感染复数(MOI)值为10,添加感染增强液,聚凝胺组感染效果最好,效率达80%。LV-SDF-1α-GFP组以及阴性对照CON145组对心肌成纤维细胞毒性作用小,细胞形态无明显变化,与非转染组生长曲线相似,差异无统计学意义(P0.05)。转染LV-SDF-1α-GFP的心肌成纤维细胞培养至第4天SDF-1α蛋白分泌量达最高值,差异具有统计学意义(P0.05)。结论 LV-SDF-1α-GFP载体对心肌成纤维细胞具很高的转染效率,细胞毒性小,并能够分泌SDF-1α目的蛋白。
[Abstract]:Objective to investigate the effects of lentivirus-stromal cell derived factor-1 伪 -green fluorescent protein (LV-SDF-1 伪 -GFPFP) on myocardial fibroblasts, the optimal infection conditions and the characteristics of protein expression and secretion. Observation and immunofluorescence identification. Myocardial fibroblasts were transfected with LV-SDF-1 伪 -GFP with different titers and conditions. The fluorescence expression was observed and the optimal transfection conditions were explored. The target gene virus of LV-SDF-1 伪 -GFP and the negative control CON145 virus were transfected into myocardial fibroblasts. The growth curve was drawn to explore whether transfection had a significant effect on the proliferation of myocardial fibroblasts. The best transfection dose group was used to transfect myocardial fibroblasts. Dot blotting was used to detect the secretion of SDF-1 伪 target protein. The quantitative data were analyzed statistically. Results Myocardial fibroblasts had high activity, high purity and good diopter. The optimal transfection number moi value of non-spontaneous pulsatile. LV-SDF-1 伪 -GFP was 10. The infection effect was the best in the polycoagulant group, the infection efficiency was 80. LV-SDF-1 伪 -GFP group and the negative CON145 group had little toxic effect on myocardial fibroblast, but the cell morphology had no obvious change. Similar to the growth curve of non-transfection group, the difference was not statistically significant (P 0.05). The expression of SDF-1 伪 protein in LV-SDF-1 伪 -GFP transfected myocardial fibroblasts reached the highest level on the 4th day. Conclusion the LV-SDF-1 伪 -GFP vector has high transfection efficiency, low cytotoxicity and secretion of SDF-1 伪 protein in myocardial fibroblasts.
【作者单位】：新乡医学院医用组织再生重点实验室;河南省平顶山市第二人民医院心内科;新乡医学院第一附属医院感染科;新乡医学院第一附属医院心内科;
【基金】：新乡医学院科研项目培育基金(2013ZD121) 新乡市重点科技攻关计划项目(ZG15015)
【分类号】：R54

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