稳定表达绿色荧光蛋白的B16细胞株的建立及其生物学特性研究

Researches on a Stable EGFP-B16 Cell Line and Its Characterization

目的建立稳定表达绿色荧光蛋白的B16黑色素瘤细胞株,并研究其生物学特性。方法转染增强型绿色荧光蛋白基因入B16细胞,经G418联合单克隆培养,筛选阳性细胞。激光共聚焦荧光显微镜观察EGFP阳性细胞形态;流式细胞仪检测细胞内EGFP基因的蛋白表达;RT-PCR检测EGFP基因的mRNA表达;皮下接种及MTT法评价细胞在体内外的生长特性。结果流式细胞仪检测表达绿色荧光蛋白的细胞阳性率为99.81%,RT-PCR检测到EGFP基因的mRNA阳性表达。生长曲线显示其体外增殖趋势与B16相似,EGFP-B16体内成瘤速度较B16慢(p<0.05)。结论稳定表达绿色荧光蛋白的EGFP-B16细胞株成功建立,可作为进一步研究恶性黑色素瘤的材料。

Objective To establish a stable EGFP-B16 melanoma cell line, and study its characteristics. Methods B16 melanoma cell line is transfected with EGFP （Enhanced Green Fluorescent Protein） gene. Positive B16 cells are sorted out by G418 combine with monoclonal selection and observed by laser confocal fluorescence microscopy （ LCFM ）. The protein expression of EGFP gene is analysed by flow cytometry （FC）. The mRNA of EGFP gene is detected by RT-PCR （ Reverse Transcription Polymerase Chain Reaction）. The cell proliferation is assessed by MTY in vitro and cell inoculation in vivo. Results The rate of positive cells examed by flow cytometry is 99.81% ; the EGFP mRNA positive expression is detected by RT-PCR. Original and transfected cells are compared cytomorphologically and biologically. There are no significant changes in cells morphology and growth rhythm before and after transfeetion. EGFP-B16 grows a little more slowly than B16 both in vivo（p 〈 0. 05 ）. Conclusion The EGFP-B16 ceil line stably expresses green fluorescent protein, which can be used for malignant melanoma further study is successfully established,is expressed stably by EGFP- B16 cell line.