摘要
目的研究以最近发展起来的核转染技术转染增强型绿荧光蛋白（EGFP）质粒进行基因修饰对兔原代骨髓基质细胞向神经细胞方向分化的影响。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pEGFP—C2（EGFP组），以同期培养未转染的细胞作为对照组（control组）。以“CYTOKINE·神经干细胞培养基”＋10％胎牛血清诱导BMSCs向神经细胞方向分化。流式细胞仪检测转染效率。比较两组细胞的生长增殖以及Nestin、NSE、GFAP抗原的表达情况。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以及生长曲线。诱导18d，两组细胞NSE、GFAP的表达比例无统计学意义。结论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；EGFP可以作为兔骨髓基质细胞有效的基因表达标记；核转染pEGFP-C2对兔原代骨髓基质细胞体外增殖及向神经细胞方向分化无明显影响。
Objective To approach the effect of transfecting the plasmids encoding enhanced green fluorescent protein （EGFP） with recently developed nucleofection technique on the differentiation of rabbit primary bone marrow stromal cells （BMSCs） along neuron pathway. Methods Rabbit bone marrow stromal cells were harvested by means of density gradient centrifugation following a thigh bone puncture, The primary BMSCs were cultured and either transfected with pEGFP-C2 by nucleofector technology（ as EGFP group ） or uninfected（as control） in vitro. ＂CYTOKINE · NSCs medium＂ （prepared by our lab, Patent No, ZL02134314.4 ） supplemented with 10% fetal bovine serum （FBS） were used to induce BMSCs differentiated along neuron pathway. FACS was employed to assess the transfection efficient. Compared with the Control, the cellular growth curves and expression of Nestin, NSE, and GFAP of the labeled cells were analyzed, respectively. Results EGFP were successfully expressed at 24h after nucleofection. Similar morphology evolvement, growth curves were obtained in the two groups. There is no significant difference in the proportion of NSE or GFAP positive cells between the treated group and the control group. Conclusion Nucleofection is an efficient nonviral gene transfer method for the introduction of genes into primary rabbit BMSCs. EGFP played an important role in stable gene marking of rabbit BMSCs. Neuclofection of pEGFP- C2 shows no significant effect on the proliferation and differentiation along neuron pathway of rabbit BMSCs.
出处
《中华神经外科杂志》
CSCD
北大核心
2007年第4期305-308,共4页
Chinese Journal of Neurosurgery
基金
国家自然科学基金重点项目（U0632008）
关键词
绿色荧光蛋白
核转染
骨髓基质细胞
分化
兔
Enhanced green fluorescent protein
Nucleofection
Bone marrow stromal cells
Differentiation
Rabbit
