稳定表达GFP的骨髓增生异常综合征转白血病细胞株的建立  

Establishment of A Myelodysplastic Syndrome Transformed to Leukemia Cell Line Stably Expressing Green Fluorescent Protein

作　　者：庄琳 马燕 王倩 许小平 张晶 朱晨 顾静文 秦文娇 

机构地区：[1]复旦大学附属华山医院血液科,上海200040

出　　处：《中国实验血液学杂志》2017年第5期1487-1492,共6页Journal of Experimental Hematology

基　　金：国家自然科学基金:81070404;81100340;上海市第四轮公共卫生三年行动计划重点学科建设慢病流行病学15GWZK0801

摘　　要：目的:建立稳定表达绿色荧光蛋白(green fluorescent protein,GFP)的骨髓增生异常综合征转白血病的细胞株,评价其生物学特性及其应用。方法:以人骨髓增生异常综合征转化为白血病细胞系SKM-1为母系细胞,通过携带GFP基因的慢病毒进行转染,以有限稀释法获得GFP阳性的单细胞克隆,并将扩大培养后的细胞通过皮下注射的方式进一步在小鼠体内筛选,致瘤后将瘤块分离,继续进行培养,获得SKM-1/GFP细胞株。通过荧光倒置显微镜观察细胞形态,应用流式细胞术检测细胞荧光表达水平,应用CCK-8法检测细胞生长特性,将扩大培养后的SKM-1/GFP细胞接种在免疫缺陷小鼠的皮下,致瘤后观察瘤块以及各个脏器组织的浸润情况。结果:SKM-1/GFP细胞形态和生长特性与SKM-1母系细胞无差异,流式细胞术检测GFP的表达率为100%,体外CCK-8法检测生长曲线显示,SKM-1/GFP细胞与母系SKM-1细胞无明显差异。将SKM-1/GFP细胞接种于NOD/SCID小鼠皮下,14d时可见瘤块形成。30 d时,在荧光显微镜下观察荷瘤小鼠,可见瘤块自发荧光。在荧光显微镜下观察肿块冰冻切片,可见较为均一的GFP阳性细胞。冰冻切片显示,小鼠心、肝、胃、肾均有肿瘤细胞浸润。结论:成功建立了稳定表达GFP的骨髓增生异常综合征转白细胞株SKM-1/GFP,该细胞株可以灵敏地追踪肿瘤细胞,应用于微小残留病的研究,为研究骨髓增生异常综合征转化的急性白血病提供一个新的实验平台。Objective： To establish a myelodysplastic syndrome transformed to leukemia cell line stably expressing green fluorescent protein（GFP）,and to evaluate its biological characteristics and applications. Methods： SKM-1 cells were transfected by lentiviral particles with vector of GFP. The GFP positive single cell clone was isolated by limiting dilution and continued being cultured. The cells were injected into mice subcutaneously and were screened in vivo. Then SKM-1/GFP cells were obtained after tumour plaque was separated and cultivated. The cell morphology was observed by fluorescence microscopy. The GFP expression was further detected by flow cytometry. The cell proliferation was analysed by CCK-8 assay. SKM-1/GFP cells were inoculated to subcutaneous tissue of the immunodeficiency mice. The growth and invasion of the tumour were observed after tumour formation. Results： No differences in cell morphology and growth characteristics were observed between SKM-1 cells and SKM-1/GFP cells. The rate of GFP expression was100%. No differences in cell proliferation were observed between SKM-1 cells and SKM-1/GFP cells. The tumour mass was observed after 14 days of subcutaneous vaccination in NOD/SCID mice. Spontaneous fluorescence from plaque was observed by living fluorescence microscopy at 30 th day after vaccination. Homogenous GFP positive cells were observed by fluorescence microscopy in the frozen section of tumour mass. The invasion of SKM-1/GFP cells was also detected in heart,liver,stomach and kidney of mice. Conclusion： A myelody-splastic syndrome transformed to leukemia cell line stably expressing green fluorescent protein has been established successfully,which can track tumor cell sensitively and can be applied to the research of minimal residual leukemia. The establishment of SKM-1/GFP cells may serve as a powerful means for studing myelodysplastic syndrome transformation.

关 键 词：绿色荧光蛋白 骨髓增生异常综合征 白血病细胞株 

分 类 号：R551.3[医药卫生—血液循环系统疾病] R733.7[医药卫生—内科学]