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

发布时间：2018-04-13 16:12

本文选题：绿色荧光蛋白 + 骨髓增生异常综合征　；参考：《中国实验血液学杂志》2017年05期

【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。
[Abstract]:Aim: to establish a stable cell line of myelodysplastic syndromes (MDS) transformed into leukemia and evaluate its biological characteristics and application.Methods: human myelodysplastic syndrome (MDS) was transformed into leukemia cell line SKM-1 as matrilineal cell line. Lentivirus carrying GFP gene was transfected to obtain GFP positive single cell clone by limited dilution method.The expanded cultured cells were further screened in vivo by subcutaneous injection, the tumor blocks were separated after tumorigenesis, and the SKM-1/GFP cell lines were obtained.Cell morphology was observed by fluorescence inverted microscope, fluorescence expression level was detected by flow cytometry, cell growth characteristics were detected by CCK-8 method, and expanded cultured SKM-1/GFP cells were inoculated subcutaneously in immunodeficient mice.The tumor mass and the infiltration of various organs were observed after tumorigenesis.Results there was no significant difference in morphology and growth characteristics between the cell lines of 1% SKM-1 / GFP and that of SKM-1 matrilineal cells. The expression rate of GFP was 100 by flow cytometry. The growth curve of SKM-1 / GFP cells was not significantly different from that of matrilineal SKM-1 cells by CCK-8 assay in vitro.When SKM-1/GFP cells were inoculated into NOD/SCID mice subcutaneously for 14 days, tumor blocks formed. 30 days after inoculation, the tumor-bearing mice were observed under fluorescence microscope, and the tumor blocks were observed to be autofluorescence.GFP positive cells were observed under fluorescence microscope in frozen sections of the tumor.Frozen sections showed that tumor cells infiltrated the heart, liver, stomach and kidney of mice.Conclusion: a stable myelodysplastic syndrome transformed leukocyte strain SKM-1 / GFPwas successfully established, which can be used to trace tumor cells sensitively and be used in the study of minimal residual disease.To provide a new experimental platform for the study of acute leukemia transformed by myelodysplastic syndrome.
【作者单位】：复旦大学附属华山医院血液科;
【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
【分类号】：R551.3

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