通过体内传代和流式分选建立稳定转染MUC1 VNTR的B16细胞株

Establishment of B16 cell line expressing MUC1 VNTR by in vivo passage and sorting

目的获得具有较好致瘤性、在动物体内保留表达外源基因MUC1 VNTR(mucin1,variable number tandemrepeats)能力的稳定转染细胞株B16-GFP-MUC1,供MUC1靶向药物研究中体内抑瘤实验所用到的肿瘤模型。方法用脂质体转染的方法和有限稀释法获得单克隆细胞株;将该细胞株进行体内传代后,流式分选GFP阳性细胞群,并对分选后细胞用有限稀释法进行单克隆化。用流式细胞仪和共聚焦荧光显微镜观察各时期细胞克隆的GFP表达情况。结果脂质体转染方法得到的在G418压力选择下存在的细胞B16-1,其GFP阳性细胞比率最高为80%左右;致瘤性较差,75d内仅87.5%小鼠出瘤;体外的稳定性观察中,在无抗生素的培养液中观察5周,其阳性率降至20%左右;经过体内接种后仅有10%左右的肿瘤细胞保留了外源基因的表达。而经过体内传代和流式分选后得到的单克隆细胞群B16-39,其GFP阳性细胞比率>90%;致瘤性为20d内100%小鼠出瘤;体外培养8周,GFP阳性的细胞比率>90%;体内剥离后的肿瘤细胞的GFP阳性细胞比率均>90%。结论对于用于体内动物模型的稳定转染细胞,通过体内传代,流式分选并单克隆化,是获得致瘤性好,体内体外较为稳定的稳定转染细胞株的一个可行方案。

Objective To establish one B16 cell fine stably expressing MUC1 VNTR （mucin 1, variable number tandem repeats） B16- GFP-MUCI which had good tumorigenesis and could maintain the exogenous gene expression when inoculating on animal model used as tumor model for tumor suppression study of MUCI targeted drugs. Methods Transfected cell clone was estabhshed by liposome method and limited dilution assay, and then passaged in vivo. The GFP positive cells were isolated from tumor burden mice by flow cytometry （FCM） sorting, and then monocloned by limited dilution assay. GFP expression levels of different cell clones were observed by FCM and confocal fluorescent micros- copy. Results The GFP positive cell proportion in BI6-1 cell line obtained by liposome transfection and G418 pressure selection was about 80%. The tumorigenesis of GPF positive cells was not homogeneous; the tumor incidence was 87.5% in 75 days; only one tumor cell has 10% GFP positive proportion. While about the cell line B16-39 obtained after in vivo passage, FCM sorting, and monoclone, the GFP positive proportion was over than 90% ; the tumorigenesis was more homogeneous, the tumor incidence of mice got 100% in 20 days; the GFP positive proportion maintained over than 90% after subculture for 8 weeks in vitro ; all of the GFP positive proportion of cells isolated from tumor burden mouse was more than 90%. Condusion In vivo passage, FCM sorting, and monoclone are practical methods for establishing one stable transfected cell line used on in vivo aninal model.