Zhang Peng, Zhang Hao, Hu Sheng-Shou, Chen Li-Guo, Wei Ying-Jie
Ministry of Health Key Laboratory of Cardiac Regeneration, Fuwai Hospital, CAMS and PUMC, Beijing 100037, China.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2005 Aug;27(4):504-8.
To investigate the feasibility of gene transfection of bone marrow mesenchymal stem cell (BMSC) by cationic lipid.
The relative optimal transfection condition was determined by scale transfection experiment in vitro and the transient transfection efficiency of enhanced green fluorescence protein (EGFP) gene for rat BMSC was determined with Lipofectamine2000 (LP2000). The relationship between cell cycle status and the expression of the gene was analyzed. The intensity and the ratio of EGFP gene expression versus time was determined by flow cytometry. In the in vivo study, the transgenic rat BMSC was injected into the myocardium of inbred rats, and the in vivo transcription of EGFP gene and the EGFP-expressing BMSC were traced in the myocardium after transplantation using reverse transcription-polymerase chain reaction and fluorescent microscopy, respectively.
EGFP gene transfection efficiency in BMSC was different under different transfection condition (DNA concentration and DNA: LP2000). Cationic lipid-mediated transfection demonstrated marked toxicity to BMSC. Cell cycle status restricted the expression efficiency of the gene introduced by cationic lipid. The EGFP expressing-BMSC and in vivo transcription of the EGFP gene could be detected in rat myocardium post implantation.
Cationic lipid is an effective carrier for gene-modified cell transplantation therapy.
探讨阳离子脂质体介导骨髓间充质干细胞（BMSC）基因转染的可行性。
通过体外批量转染实验确定相对最佳转染条件，采用Lipofectamine2000（LP2000）测定大鼠BMSC增强型绿色荧光蛋白（EGFP）基因的瞬时转染效率。分析细胞周期状态与该基因表达之间的关系。通过流式细胞术测定EGFP基因表达强度及表达量随时间的变化。在体内研究中，将转基因大鼠BMSC注射到近交系大鼠心肌内，分别采用逆转录-聚合酶链反应和荧光显微镜观察移植后心肌内EGFP基因的体内转录情况及表达EGFP的BMSC。
不同转染条件（DNA浓度及DNA∶LP2000）下BMSC中EGFP基因转染效率不同。阳离子脂质体介导的转染对BMSC显示出明显毒性。细胞周期状态限制了阳离子脂质体导入基因的表达效率。植入后大鼠心肌中可检测到表达EGFP的BMSC及EGFP基因的体内转录。
阳离子脂质体是基因修饰细胞移植治疗的有效载体。
