表达bcr-abl基因片段的可移植小鼠细胞系的生物学特性

Establishment of a Mouse Tumor Cell Line Stably Expressing bcr-abl Fusion Fragment and Translatable in Mice

目的建立稳定表达bcr-abl融合基因的、可移植的小鼠肿瘤细胞系,解决慢性粒细胞白血病疫苗研究的瓶颈问题。方法从重组克隆载体pGEMbcr-abl中酶切出bcr-abl融合基因片段,并将其亚克隆进反转录病毒载体pLXSN中。脂质体介导重组反转录病毒载体pLXSNbcr-abl转染包装细胞PT67,G418筛选后获得稳定产病毒的包装细胞。收集病毒感染NIH/3T3细胞,加G418筛选后进行反转录病毒滴度测定,计算病毒效价为2×107CFU/mL。收集病毒上清感染SP2/0细胞(H-2d),经G418筛选获得稳定表达bcr-abl融合基因片段的SP2/0细胞株。然后用SP2/0/bcr-abl细胞攻击同品系BALB/c小鼠,观察SP2/0/bcr-abl移植瘤生长的情况。结果经特异性PCR扩增和RT-PCR反应扩增,从基因组整合和基因表达水平证实获得了能稳定表达bcr-abl融合基因片段的鼠SP2/0细胞系。SP2/0/bcr-abl细胞能够在同品系小鼠体内形成移植肿瘤。结论该表达bcr-abl融合基因片段的小鼠肿瘤细胞系将作为研究bcr-abl基因疫苗的有效实验工具,为检验bcr-abl基因疫苗激发的小鼠CTL应答研究奠定物质基础。

Objective To establish a mouse tumor cell line which stably expresses ber-abl fusion fragment and can be translated in mice. Methods The bcr-abl fusion gene fragment was subeloned into retroviral vector pLXSN from pGEMbcr-abl. The recombinant retroviral vector pLXSNbcr-abl was transfected into PT67 packaging cells with lipofectamine. The positive clones were selected out and cultured after G418 selection. Then viral supernatant was collected and the viral titer was determined to be 2 × 10^7CFU/mL. The SP2/0 cells were infected with the collected viral supernatant. SP2/0/bcr-abl cells expressing the fragment of bcrabl fiuion gene were selected. SP2/0/bcr-abl cells were inoculated subcutaneously into the abdomen of BALB/c mice. Results After C,418 selection, it was found that the bcr-abl fusion gene fragment was integrated into the chromosome of SP2/0 cells infected with recombinant retrovirus stably confirmed by PCR, and expressed in SP2/0 cells confirmed by RT-PCR, respectively. SP2/0/ bcr-abl cells could be transplanted subcutaneously into the abdomen of BALB/c mice. Conclusion A mouse tumor cell line exressing the fragment of bcr-abl fusion protein has been established and could be used as an experimental cell model for anfi-CML immunotherapy research.