不同荧光蛋白标记技术对兔骨髓基质干细胞体外增殖的影响

Effects of different fluorescent protein labelings on proliferation of rabbit bone marrow stromal cells in vitro

目的探讨不同荧光蛋白标记方法对兔骨髓基质干细胞体外增殖能力的影响。方法从兔骨髓中分离培养骨髓基质干细胞,分别采用逆转录病毒和质粒转染两种方式进行增强型绿色荧光蛋白和红色荧光蛋白标记,G418筛选培养3周后,测定细胞生长曲线和贴壁率的变化。结果逆转录病毒载体pLEGFP-N1、真核表达载体pDsRed2-C1均可以成功标记骨髓基质干细胞,G418筛选培养后,细胞表达明显的荧光蛋白,其阳性率增加。pLEGFP-N1标记组细胞倍增时间为(34.9±1.2)h,pDsRed2-C1组为(36.1±1.4)h,未标记组为(33.8±0.5)h,3组数据间无统计学差异(P>0.05)。结论荧光蛋白标记对骨髓基质干细胞体外增殖无明显影响,合理应用不同荧光蛋白及其表达载体将成为深入研究组织工程种子细胞的有力工具。

Objective To observe the effects of different fluorescent protein labelings on the proliferation of cultured rabbit bone marrow stromal cells (BMSCs). Methods First of all we isolated and culture-expanded BMSCs from rabbit femoral marrow. Next BMSCs were transfected with the recombinant retrovirus containing enhanced green fluorescent protein (EGFP) or with the plasmid containing red fluorescent protein (RFP). Then G418 resistant clones were expanded for 3 weeks. At last the growth curves and adhesive rates of the 2 groups of labeled cells and the control were analyzed. Results EGFP and RFP were successfully expressed after BMSCs had been transfected with the retroviral vector pLEGFP-N1 or with the plasmid pDsRed2-C1. The percentage of labeled BMSCs was increased by selection of chemical drug (G418). The population doubling time was (34.9±1.2) h in the group of pLEGFP-N1, (36.1±1.4) h in the group of pDsRed2-C1, and (33.8±0.5) h in the group of unmarked BMSCs respectively, and there was no significant difference among the 3 groups (P >0.05). Conclusion Since the expression of EGFP or RFP shows no effect on the proliferation of rabbit BMSCs, sensible application of labeled BMSCs with different fluorescent proteins is of great help to the research on seeding cells in tissue engineering.