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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(P>0.05)。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(P<0.05)。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。
Objective To investigate the effect of lentivirus-derived stromal cell-derived factor-1α-green fluorescent protein (LV-SDF-1α-GFP) on cardiac fibroblasts, the optimal infection conditions and the expression and secretion of the target protein. Methods Myocardial fibroblasts isolated from cultured rat hearts were differentiated and cultured with differential adhesion method. LV-SDF-1α-GFP with different titer and conditions were transfected into cardiac fibroblasts, and the fluorescence expression was observed to explore the optimal transfection conditions. LV-SDF-1α-GFP gene and the negative control CON145 were transfected into cardiac fibroblasts, and the growth curve was drawn to explore whether transfection had significant effect on the proliferation of cardiac fibroblasts. The optimal transfection dose group was used to transfect cardiac fibroblasts and the secretion of SDF-1α protein was detected by dot blotting. Measurement data for statistical analysis. Results Myocardial fibroblasts with high activity, passed to the fourth generation of high purity, good refractive index, no spontaneous beating. The optimal MOI of LV-SDF-1α-GFP transfection was 10, and the infection was enhanced with polyferase. The infection rate was 80%. The LV-SDF-1α-GFP group and the negative control group CON145 had little effect on cardiac fibroblasts with no significant change in cell morphology, which was similar to that of non-transfected group (P> 0.05). SDF-1αprotein secretion reached the highest value on day 4 after transfection with LV-SDF-1α-GFP in cardiac fibroblasts, the difference was statistically significant (P <0.05). Conclusion LV-SDF-1α-GFP vector has high transfection efficiency to cardiac fibroblasts with low cytotoxicity and can secrete SDF-1αprotein.