摘要：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Nucleofector^TM技术转染pDsRed1-N1，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48 h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。

    关键词：; 骨髓基质细胞; 核转染

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

    CHEN Zhen-zhou, XU Ru-xiang, JIANG Xiao-dan, TENG Xiao-hua, ZHOU Hu-tian

    Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510280, China

    Abstract: Objective To test the feasibility of nucleofection, a recently developed method for delivering plamid DNA directly into the nucleus, for introducing red fluorescent protein into primary rabbit bone marrow stromal cells(BMSCs). Methods Rabbit BMSCs were harvested by means of density gradient centrifugation following a thighbone puncture. The primary BMSCs were cultured and either transfected with pDsRed1-N1 using Nucleofector^TM (DsRed group) or left uninfected (control group) in vitro. The cell viability, adhesion rate, and the growth curves of the labeled cells were analyzed in comparison with the control cells, and the transfection efficiency was evaluated according to the intensity of DsRed expression. Results DsRed expression was observed 48 h after nucleofection of the BMSCs, which exhibited similar morphological evolvement and adhesion rate with identical growth curve with the control cells. Positive DsRed expression enhanced gradually as the cell culture was prolonged, reaching the peak level 10 days after the transfection when about 54.2% of the total BMSCs were DsRed-positive. DsRed expression did not attenuate even until 1 month after the labeling. Conclusions Neuclofection of pDsRed1-N1 shows no significant effect on the proliferation of rabbit BMSCs. DsRed works efficiently for the purpose of stable gene marking in rabbit BMSCs, and nucleofection is an efficient nonviral gene transfer method for introducing exogenous genes into primary rabbit BMSCs. ......