摘要
目的探讨以最近发展起来的核转染技术直接将编码绿色荧光蛋白DNA质粒转染到兔原代骨髓基质细胞的细胞核内进行基因修饰的可行性。方法从兔股骨抽取骨髓,密度梯度离心法获取原代骨髓基质细胞。以NucleofectorTM技术转染pEGFP-C2(EGFP组),以同期培养未转染的细胞作为对照组。测定细胞的活力、贴壁率、生长曲线以及转染的效率。结果在转染后24h成功发现EGFP的表达。两组细胞具有相似的形态学变化、贴壁率以及生长曲线。EGFP的表达逐渐增强,至第6天达到最高峰(47.8%),观察一个月未发现表达减弱。结论pEGFP-C2基因核转染对兔原代骨髓基质细胞的体外增殖无明显影响;EGFP可以作为兔骨髓基质细胞有效的基因表达标记;NucleofectorTM技术是一种简易而高效的转染兔原代骨髓基质细胞的方法。
Objective To explore the feasibility of recently developed nucleofection method in delivering plamid DNA directly into the nucleus for the introduction of a plasmid encoding enhanced green fluorescent protein （EGFP） into primary bone marrow stromal cells （BMSCs） of rabbit. Methods Rabbit BMSCs were harvested by means of density gradient eentrifugation following a thighbone puncture. The primary BMSCs were cultured and either transfected with pEGFP-C2 by nucleofector technology （as EGFP group） or uninfected （as control） in vitro. Compared with the control, the cellular viability, adhesive rates and the growth curves of the labeled cells were respectively analyzed. Transfection efficiencies were evaluated through the detection of EGFP expression. Results EGFP were successfully expressed 24 h after nucleofection. Similar morphological development, adhesive rates and growth curves were found in the 2 groups. The positive EGFP expression was enhanced gradually alone with the prolonged culture time, and showed the strongest 6 d after marked, with about 47.8% of EGFP-positive cells in the total BMSCs. The EGFP did not attenuate even 1 month after the marking. Conclusion Neuclofection of pEGFP-C2 shows no significant effect on the proliferation of rabbit BMSCs. EGFP plays an important role in stable gene marking of rabbit BMSCs. Nucleofection is an efficient nonviral gene transfer method for the introduction of genes into primary rabbit BMSCs.
出处
《中华神经医学杂志》
CAS
CSCD
2006年第3期226-229,共4页
Chinese Journal of Neuromedicine
基金
国家自然科学基金(3027049,30400464)
广东省自然科学基金项目[粤科基办2000(25),2004(08)]
广东省科技计划项目[粤财企2001(367),2003(209),粤科计字2004(112)]
全军医学科研"十五"计划重大课题(01Z054)
关键词
绿色荧光蛋白
骨髓基质细胞
核转染
兔
Green fluorescent protein
Bone marrow stromal cells
Nucleofection
Rabbit
