逆转录病毒载体介导大鼠乳腺癌细胞共表达MIP-1α和B7-1

Retroviral vector mediated co-expression of MIP-lα and B7-1 in rat breast cancer cells

目的:建立共表达小鼠MIP-1α和B7-1基因的大鼠乳腺癌细胞株,并进行体外活性检测。方法:应用含不同选择标记的重组逆转录病毒载体,经1mg/mlG418和2μg/ml puromycin双药物筛选后获得MIP-1α+B7-1基因共表达的大鼠乳腺癌细胞株。RT-PCR、免疫组化检测mMIP-1α的表达,RT-PCR、流式细胞术检测mB7-1的表达;将共表达MIP-1α和B7-1的肿瘤细胞与大鼠脾淋巴细胞混合培养后,MTT法检测淋巴细胞的增殖指数、琼脂糖打孔法检测共表达MIP-1α和B7-1肿瘤细胞对单个核细胞的趋化活性。结果:经脂质体转染包装细胞产生的重组逆转录病毒上清病毒滴度达4.6×107CFU/L。细胞生长曲线显示,重组逆转录病毒感染对乳腺癌细胞增殖无明显影响。重组逆转录病毒感染的大鼠乳腺癌细胞株SHZ-88/mMIP-1α+mB7-1有B7-1和MIP-1α mRNA及蛋白的表达。淋巴细胞增殖指数PI值SHZ-88/PLXSN组为(0.76±0.25),SHZ-88/mMIP-1α+mB7-1组为(1.95±0.31),后者体外刺激淋巴细胞增殖能力增强(P<0.01),SHZ-88/pBabe puro组的趋化指数为(0.99±0.19),mMIP-1α+mB7-1组的为(3.88±0.33),后者显著增强了趋化活性。结论:通过逆转录病毒载体介导建立MIP-1α和B7-1基因共表达的大鼠乳腺癌细胞株具有招引单个核细胞,并将其激活的体外生物学活性。

Objective： To establish a rat breast cancer cell line stably co-expressing mouse MIP-1α and B7-1 ,and to assay its in vitro biological activity. Methods： mMIP-1αcDNA was cloned into retrovirus vector pBabe puro to construct pBahe puro/mMIP-1α, then pBabe puro/mMIP-1α was used to transfect packaging cells and the anti-puromycin PA-317 packaging cells were proliferated. Meanwhile, pLXSN/mB7-1 was constructed and the anti-G418 ceils were proliferated. Finally, the two supernatants were used to infect SHZ-88 together and the co-transfected cells were selected with 1 mg/ml G418 and 2 μg/ml puromycin together. Expression of mMIP-1α mRNA and protein in SHZ-88 and SHZ-88/mB7-1 ＋ mMIP-1α cells were analyzed by RT-PCR and immunocytochemistry, respectively. Expression of mB7-1mRNA and protein was analyzed by RT-PCR and flow cytometry, respectively. Lymphocyte proliferation activity of SHZ-88/B7-1 ＋ mMIP-1α was detected by MTT assay; chemotactic activity of MIP-1α was measured by chemotaxis assay. Resuits：A titer of 4.6 × 10^7 CFU/L was obtained after transfection with recombinant retroviral vector. The growth curve of cells showed that the recombinant retroviral had no effect on the growth of rat breast cancer cells. There was expression of B7-1 and MIP-1αmRNA/protein in SHZ-88/mMIP-1α ＋ mB7-1 ceils. The proliferation indices（PI） in mMIP-1α ＋ mB7-1 group （ 1.95 ±0. 31 ） was significantly higher than that in SHZ-88/PLXSN group （0.76 ±0. 25 ） （ P 〈0.01 ）. Chemotaxis assay showed that chemotactic activity of lymphocytes in mMIP-1α ＋ mB7-1 group （3.88 ±0. 33 ） was significantly higher than that in SHZ-88/pBabe puro group （0.99 ±0.19） （ P 〈0. 001 ）. Conclusion： A new rat breast cancer cell line SHZ-88/ mMIP-1α ＋ mB7-1 has been established, which can stably co-express MIP-1α and B7-1 gene and possesses biologic activity in vitro.