阳离子脂法介导质粒转染大鼠骨髓基质干细胞用于基因修饰的细胞移植治疗

Plasmid Transfection of Rat Bone Marrow Mesenchymal Stem Cells by Cationic Lipid for Gene-modified Cell Transplantation Therapy

目的探讨阳离子脂转染方法在骨髓基质干细胞(BMSC)基因转染中的应用。方法通过梯度试验确定相对最佳的转染条件并测定LipofectamineTM2000(LP2000)介导增强型绿色荧光蛋白(EGFP)基因转染大鼠BMSC的瞬时转染效率;分析细胞周期对导入基因表达的影响;以流式细胞仪测定转基因BMSC的EGFP表达强度和阳性比例随时间的变化;将EGFP基因转染的大鼠BMSC注射法移植入同源大鼠心肌内,以逆转录PCR法检测心肌组织内EGFP基因的转录,荧光显微镜法检测EGFP阳性的BMSC。结果在不同转染条件下(DNA浓度、DNA:LP2000),LP2000介导EGFP基因转染大鼠BMSC的瞬时转染效率不同;通过阳离子脂法导入基因的表达效果受细胞周期制约;细胞移植后,能在大鼠心肌组织中检测到EGFP基因的转录以及表达EGFP基因的BMSC。结论阳离子脂可以作为基因修饰的细胞移植治疗的有效载体。为了提高基因转染的瞬时效率和基因的表达效率,降低毒性、满足转基因细胞移植的要求,尚需进一步的研究。

Objective To investigate the feasibility of gene transfection of bone marrow mesenchymal stem cell （BMSC） by cationic lipid. Methods 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 Lipofectamine^TM2000 （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 myoeardium 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. Results 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 bedetected in rat myocardium post implantation. Conclusion Cationic lipid is an effective carrier for gene-modified cell transplantation therapy.