解剖学报

解剖学报 ›› 2017, Vol. 48 ›› Issue (1): 43-47.doi: 10.16098/j.issn.0529-1356.2017.01.008

• 细胞和分子生物学 • 上一篇    下一篇

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

朱占占1 侯宾2 孙雯雯3 院慧芳1 张永春4 蔡新华1*   

  1. 1. 新乡医学院医用组织再生重点实验室， 河南 新乡 453003; 2. 河南省平顶山市第二人民医院心内科，河南 平顶山 467000; 3. 新乡医学院第一附属医院感染科， 河南 卫辉 453003; 4. 新乡医学院第一附属医院心内科， 河南 卫辉 453003

  • 收稿日期:2016-06-14 修回日期:2016-09-28 出版日期:2017-02-06 发布日期:2017-02-06
  • 通讯作者: 蔡新华 E-mail:cxh@xxmu.edu.cn
  • 基金资助:

    新乡医学院科研项目培育基金;新乡市重点科技攻关计划项目合同

Cardiac fibroblasts of the rat transfected by lenti virus-stromal cell-derived factor-1α-green fluorescent protein vector

ZHU Zhan-zhan1 HOU Bin2 SUN Wen-wen3 YUAN Hui-fang1 ZHANG Yong-chun4 CAI Xin-hua 1*   

  1. 1. Key Laboratory for Tissue Regeneration of He’nan Province,Ｘinxiang Medical University, He’nan Xinxiang 453003, China; 2. Department of Cardiology, Pingdingshan Second People’s Hospital, He’nan Pingdingshan 467000, China; 3. Department of Infectious Diseases, 4. Department of Cardiology, the First Affiliated Hospital of Xinxiang Medical College, He’nan Weihui 453003, China

  • Received:2016-06-14 Revised:2016-09-28 Online:2017-02-06 Published:2017-02-06
  • Contact: CAI Xin-hua E-mail:cxh@xxmu.edu.cn

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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（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。

关键词: 心肌成纤维细胞；慢病毒-基质细胞衍生因子-1&alpha, 绿色荧光蛋白载体；转染；斑点印迹法；乳大鼠

Abstract:

Objective To investigate the effect of lentivirus-stromal cell-derived factor-1α-green fluorescent protein(LV-SDF-1α-GFP) on the cardiac fibroblasts, the optimum conditions of infection, the expression and secretion of the target protein. Methods The cardiac fibroblasts of neonatal rats were primarily isolated and cultured by differential adherence methods, and were observed and identifi with immunofluorescence. LV-SDF-1α-GFP with different titers and conditions was transfected into cardiac fibroblasts. The expression of fluorescence and the optimal transfection conditions were observed. LV-SDF-1α-GFP target gene virus and negative control CON145 virus were transfected into cardiac fibroblasts. The growth curve was drawn, and the effect of transfection on the proliferation of cardiac fibroblasts was explored.The cardiac fibroblasts were transfected with the optimum transfection dose, and the expression of SDF-1α was detected by Dot-blotting. The measurement data underwent statistical analysis. Results There was no statistical difference between the cardiac fibroblasts with SDF-1α transfected lentivirus and without no-transfected SDF-1α lentivirus. The peak of the expression of SDF-1α appeared in culture day 4 and statistical analysis showed significantly difference (P<0.05). Conclusion The LV-SDF-1α-GFP vector is of higher transfection efficiency to cardiac fibroblasts with the both low cytotoxicity and ability of secreting SDF-1α protein.

Key words: Cardiac fibroblast, Lentivirus-stromal cell-derived factor-1α-green fluorescent protein vector, Transfection, Dot blotting, Neonatal rat