pDsRed1-N1基因核转染兔原代骨髓基质细胞的实验研究

目的探讨以最近发展起米的核转染技术直接将编码红色荧光蛋白DNA质粒转染到兔原代骨髓基质细胞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Nucleofectorw^TM技术转染pDsRed1—N1（DsRed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48h成功发现DsRed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。DsRed的表达逐渐增强，至第10天达到最高峰（54．2％），观察1个月未发现表达减弱。结论pDsRed1-N1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；DsRed可以作为兔骨髓基质细胞有效的基因表达标记；Nucleofector^TM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。

Transfer pDsRed1-N1 into primary rabbit bone marrow stromal cells by nucleofection

Objective To approach the feasibility of transfecti ng the DNA plasmid of encoding red fluorescent protein directly into the nucleus o f rabbit primary bone marrow stromal cell with recently developed nucleofection technique. Methods Rabbit primary bone marrow stromal cells (BM SCs) were harvested by means of density gradient centrifugation following a thig hbone puncture. The primary BMSCs were cultured and either transfected to pDsRed 1-N1 by nucleofector~TM technique (as DsRed group) or left uninfected(as c ontrol group) in vitro. The cellular viability, adhesive rate, the growth curves and the efficiency of transfection of both DsRed and control groups were analyz ed. Result DsRed were successfully expressed at 48h after nucle ofection. Similar morphology evolvement, adhesive rates and growth curves were o btained from the two groups. The positive DsRed expression enhanced gradually al one with a prolonged culturing time, and reached its peak value at the 10~th day after marked, with about 54.2% of DsRed-positive cells in the total BMS Cs. The DsRed did not attenuate even until 1 month following the mark. C onclusion Neuclofection of pDsRed1-N1 showed no significant effect on the proliferation of rabbit BMSCs. DsRed worked efficiently for the purpose of s table gene marking of rabbit BMSCs, and nucleofection is an efficient method for transferring genes into primary rabbit BMSCs.