反转录病毒介导的Luciferase基因稳定转染细胞株的建立及其生物发光成像检测

目的 建立反转录病毒介导的Luciferase基因(Luc2)稳定转染的细胞株,通过体内及体外实验探讨Luc2阳性细胞数与生物发光成像系统光通量之间的关系.方法 采用分子克隆方法构建pMX-Luc2质粒,将其与pMD.G质粒共转染293T gag-pol细胞,获得表达Luc2的反转录病毒.将该反转录病毒感染小鼠结肠癌CT26、人小细胞肺癌NCI-H446、人结肠癌HT-29、人卵巢癌SKOV3、人肝癌sMMC-7721细胞系,建立并筛选Luc2阳性表达的细胞株.分别接种10～1×104个SKOV3-Luc2细胞于培养皿中,在4只裸鼠背侧皮下16个位点分别接种200μl不同浓度(1×107、5×106、2.5×106、1 × 106、5×105、2.5 × 10s、1×105、5 × 104/ml)的SKOV3-Luc2细胞,在另外4只裸鼠尾静脉中分别注射1 ×106和3×106个SKOV3-Luc2细胞.采用生物发光成像系统检测体外、体内接种细胞的光通量值.结果 成功获得表达Luc2的反转录病毒,感染肿瘤细胞系CT26、NCI-H446、HT-29、SKOV3、SMMC-7721,筛选阳性细胞株,均可稳定表达Luc2.体外生物发光成像系统检测结果表明,光通量值与培养皿中接种的细胞数之间存在显著的线性关系(R2=0.944,β=0.972,P＜0.01)；活体检测结果表明,光通量值与裸鼠尾静脉注射和皮下接种的细胞数之间均存在显著的线性关系(R2=0.991,β=0.996,P＜0.01; R2=0.351,β=0.628,P＜0.01).结论 采用表达Luc2的反转录病毒感染肿瘤细胞系是快速建立Luc2阳性细胞株的一种可行方法.Luc2阳性细胞数与生物发光成像系统中光通量值具有显著的线性关系.

Establishment of cell strains stably-transfected with luciferase gene mediated by retrovirus and their detection with bioluminescence imaging system

Objective To establish cell strains stably transfected with Luciferase gene (Luc2), which was mediated by retrovirus,and explore the relationship between the number of Luc2-positive cells and light flux from bioluminescence imaging system by experiments in vitro and in vivo.Methods We co-transfected pMX-Luc2 plasmid and pMD.G plasmid into 293T gag-pol cells to get retrovirus expressing Luc2 gene.Stable Luc2 positive cell lines were generated and screened by transduction of RetroLuc2 in mouse colon cancer cell line CT26,human non-small cell lung cancer cell line NCI-H446,human colon cancer cell line HT-29,human ovarian carcinoma cell line SKOV3 and human hepatocellular carcinoma cell line SMMC-7721,all of them were identified by bioluminescence imaging system.Different numbers of SKOV3-Luc2 cells ranging from 10 to 10000 were plated onto culture dishes.Two xenograft models of ovarian cancer were reproduced by subcutaneous injection of 200μl SKOV3-Luc2 cell suspension with different concentrations(1×107,5×106,2.5×106,1×106,5×105,2.5×105,1×105 and 5×104/ml) into 16 sites on the back of 4 nude mice,or intravenous injection of 1×106 or 3 ×106 SKOV3-Luc2 cells into the tail vein.Light flux value of SKOV3-Luc2 cells in dishes and in mice was measured by bioluminescence imaging system.Results Retro-Luc2 was constructed successfully and expressed Luc2 stably in transduced CT26,NCI-H446,HT-29,SKOV3 and SMMC-7721 cell lines.Light flux was correlated in a linear manner with the number of Luc2-positive cells in dishes and in mice(R2=0.944,β=0.972;R2=0.991,β=0.996;R2=0.351, β=0.628; P<0.01). Conclusion Luc2-positive cell lines could be established rapidly and accurately by infecting tumor cells with retrovirus expressing Luc2. The number of Luc2 positive cells is significantly related in a linear manner to light flux from bioluminescence imaging system in vitro and in vivo.