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目的观察不同荧光蛋白表达对体外培养的小鼠成纤维细胞系NIH3T3增殖能力的影响,为细胞示踪技术提供理论依据。方法将体外扩增培养的NIH3T3细胞随机分为对照组、pLEGFP-N1组、pEGFP-N1组、pDsRed2-C1组。对照组不作任何处理,其他3组分别采用逆转录病毒载体pLEGFP-N1和真核表达载体pEGFP-N1、pDsRed2-C1两种转染方式进行增强型绿色荧光蛋白(EGFP)和红色荧光蛋白(RFP)标记,经G418筛选培养后,观察各组细胞荧光蛋白表达情况,并计算其阳性表达率;观察各组细胞贴壁率,绘制生长曲线并测定倍增时间。结果对照组NIH3T3细胞未见荧光蛋白表达;pLEGFP-N1、pEGFP-N1组均表达EFGP,pDsRed2-C1组表达RFP,而pLEGFP-N1组阳性表达率高于另外两组(P<0.01).各组细胞均有较高的贴壁率。pEGFP-N1组细胞倍增时间为(39.6±0.6)h,pDsRed2-C1组(40.3±0.7)h,pLEGFP-N1组(36.5±0.7)h,均明显晚于对照组(27.9±0.6)h(P<0.01).结论荧光蛋白表达对NIH3T3细胞体外增殖有一定程度的影响,但逆转录病毒载体抑制作用低于普通真核表达载体,可作为细胞移植时荧光蛋白标记的较好选择。
Objective To observe the effect of different fluorescent proteins on the proliferation of mouse fibroblast cell line NIH3T3 cultured in vitro and to provide a theoretical basis for cell tracing technology. Methods NIH3T3 cells cultured in vitro were randomly divided into control group, pLEGFP-N1 group, pEGFP-N1 group and pDsRed2-C1 group. The other three groups were treated with retroviral vector pLEGFP-N1 and eukaryotic expression vectors pEGFP-N1 and pDsRed2-C1 respectively for EGFP and RFP ). After G418 screening, the expression of fluorescent protein in each group was observed and the positive expression rate was calculated. The cell attachment rate was observed and the growth curve was drawn and the doubling time was determined. Results There was no expression of fluorescent protein in NIH3T3 cells in control group, EFGP in pLEGFP-N1 and pEGFP-N1 groups, RFP in pDsRed2-C1 group, and pLEGFP-N1 group (P <0.01) Group cells have a higher rate of adherent. The cell doubling time was (39.6 ± 0.6) h in pEGFP-N1 group, 40.3 ± 0.7 h in pDsRed2-C1 group and 36.5 ± 0.7 h in pLEGFP-N1 group, which were significantly later than that in control group (27.9 ± 0.6) h P <0.01) .Conclusion The expression of fluorescent protein has some effects on the proliferation of NIH3T3 cells in vitro, but the inhibitory effect of retroviral vector is lower than that of normal eukaryotic expression vector, which may be a good choice for fluorescent protein labeling in cell transplantation.