CAJ | 학술논문

目的探讨以最近发展起来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NucleofectorTM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; 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.
목적탐토이최근발전기래적핵전염기술직접장편마홍색형광단백DNA질립전염도토원대골수기질세포세포핵내진행기인수식적가행성.방법종토고골추취골수,밀도제도리심법획취원대골수기질세포.이NucleofectorTM기술전염pDsRed1-N1(DsRed조),이동기배양미전염적세포작위대조조.측정세포적활력、첩벽솔、생장곡선이급전염적효솔.결과재전염후48h성공발현DsRed적표체.량조세포구유상사적형태학변화、첩벽솔이급생장곡선.DsRed적표체축점증강,지제10천체도최고봉(54.2%),관찰1개월미발현표체감약.결론 pDsRed1-N1기인핵전염대토원대골수기질세포적체외증식무명현영향; DsRed가이작위토골수기질세포유효적기인표체표기; NucleofectorTM기술시일충간역이고효적전염토원대골수기질세포적방법.
Objective To approach the feasibility of transfecting the DNA plasmid of encoding red fluorescent protein directly into the nucleus of rabbit primary bone marrow stromal cell with recently developed nucleofection technique. Methods Rabbit primary bone marrow stromal cells (BMSCs) were harvested by means of density gradient centrifugation following a thighbone puncture. The primary BMSCs were cultured and either transfected to pDsRed1-N1 by nucleofectorTM technique (as DsRed group) or left uninfected(as control group) in vitro. The cellular viability, adhesive rate, the growth curves and the efficiency of transfection of both DsRed and control groups were analyzed. Result DsRed were successfully expressed at 48h after nucleofection. Similar morphology evolvement, adhesive rates and growth curves were obtained from the two groups. The positive DsRed expression enhanced gradually alone with a prolonged culturing time, and reached its peak value at the 10th day after marked, with about 54.2% of DsRed-positive cells in the total BMSCs. The DsRed did not attenuate even until 1 month following the mark. Conclusion Neuclofection of pDsRed1-N1 showed no significant effect on the proliferation of rabbit BMSCs. DsRed worked efficiently for the purpose of stable gene marking of rabbit BMSCs, and nucleofection is an efficient method for transferring genes into primary rabbit BMSCs.