骨髓间充质干细胞基因工程改造方法的比较

Comparison in techniques of gene transfection for bone marrow derived mesenchymal stem cells

目的:对脂质体介导、反转录病毒载体介导、腺相关病毒载体介导这3种常用的基因转染方法进行比较,寻找一种适合于骨髓间充质干细胞的基因转移方法。方法:实验于2004-10/2005-06在首都医科大学北京神经科学研究所完成。①在脂质体介导下,将增强的绿色荧光蛋白基因导入骨髓间充质干细胞,然后通过G418抗性筛选,观察转染效率。②反转录病毒介导的基因转染,首先利用LipofectAMINETM 2000转染包装细胞系PT67,获得重组反转录病毒上清,然后用病毒上清直接感染骨髓间充质干细胞。转染后的细胞同样用G418筛选,得到阳性克隆后进行定点消化、扩增。③在腺相关病毒介导的基因转染过程中,首先通过磷酸钙沉淀法转染包装细胞系HEK293,得到重组AAV-LacZ病毒颗粒直接感染骨髓间充质干细胞,1周后进行β-gal染色,计算转染效率。结果:①脂质体介导的基因转染24h后在荧光显微镜下观察,可见少量细胞呈现绿色荧光蛋白阳性,阳性率大约为5%。抗生素筛选3周后细胞全部死亡,经过多次试验均未得到阳性细胞克隆。②反转录病毒感染后,在荧光显微镜下观察可见少量骨髓间充质干细胞表达增强的绿色荧光蛋白。当加入G418筛选后,大量细胞死亡,1周后仅有极少数细胞存活。继续培养3～4周后,细胞形成克隆,定点消化细胞克隆,扩增后95%的细胞表达绿色荧光蛋白。③腺相关病毒上清感染骨髓基质细胞1周后,用β-gal染色估计骨髓间充质干细胞转染效率大约为75%。但传代后阳性细胞所占比例明显下降,大约2%。结论:骨髓间充质干细胞易于接受外援基因。3种基因转移方法相比:脂质体转染法转染效率最低,不适合骨髓间充质干细胞;反转录病毒载体法感染效率最高,最适用于骨髓间充质干细胞;而腺相关病毒载体法感染效率较高,但该载体系统没有抗生素筛选,所以无法使阳性细胞得到纯化和扩增,其可能更适合于体内基因直接感染。

AIM： To compare 3 commonly used methods of gene transfection by LipofectAMINE-mediated, retrovirus vector mediated and adeno-associated virus （AAV） vector mediated respectively, so as to seek for an gene transfection method appropriate for mesenchymal stem cells （MSCs）. METHODS： The experiment was conducted in the Capital University of Medical Sciences, Beijing Institute for Neuroscience. （1） MSCs were transfected with enhanced green fluorescent protein （EGFP） gene under the mediation of LipofectAMINE, and G418 resistance was adopted for screen, so as to observe the transfection efficiency. （2）Retrovirus mediated gene transfection was carried out. First, the package cell line PT67 was transfected by lipofectAMINE ^TM2000, so as to obtain recombinant retrovirus supernatant, which was directly used in the transfection of MSCs. Cells after transfection were screened by the conditioned medium G418 to obtain positive cell clones, which were digested and amplified in fixed-point. （3） In the transfection of AAV mediated gene, calcium phosphate precipitation method was used for the transfection of package cell line HEK293, so as to obtain recombinant AAV-LacZ virus particles to directly transfect the MSCs. Transfection efficiency was estimated by 13-gal staining method at one week later. RESULTS： （1） Gene transfection mediated by LipofectAMINE at 24 hours later under the fluorescence microscope showed that little amount of ceils were positive in GFP with the positive rate of about 5%. Cells all died at 3 weeks after antiiotic screening, while positive cell clones were not gained even after many times of test. （2） After retrovirus transfection, there were little MSCs expressed EGFP under the fluorescence microscope, while apoptosis was found in large amounts of cells after G418 screen, and only little cells survived at one week later. After 3-4 weeks culture, cells were cloned, and digested cells in fixed point were cloned： 95% of cells after amplification expressed GFP. （3） One week after transfection of MSCs with AAV supernatant, the transfection efficiency of MSCs estimated by 13-gal staining was about 75%, while the positive ratio was decreased after passage, which was about 2%. CONCLUSION： MSCs are easily to be gene engineered and readily to be an useful vihecle for gene therapy. Comparison among three gene transfection methods： Of three methods, LipofectAMINE transfection is the lowest in transfection efficiency, which is not suitable for MSCs. The retrovirus vector mediated transfection method is the most appropriate for MSCs with the highest transfection efficiency. Although the tranfection efficiency with AAV vector transfection is much higher, there are no antibiotic screen in the system, so the positive cells can not be purified and amplified, while it may be more appropriate for direct gene transfection in vivo.