建立共表达小鼠MIP1α和B71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1 mg/ml G418和2 μg/ml puromycin双药物筛选后获得MIP1α+B71基因共表达的大鼠乳腺癌细胞株。RTPCR、免疫组化检测mMIP1α的表达，RTPCR、流式细胞术检测mB71的表达；将共表达MIP1α和B71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MIP1α和B71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SHZ88/mMIP1α+mB71有B71和MIP1α mRNA及蛋白的表达。淋巴细胞增殖指数PI值SHZ88/PLXSN组为(0.76±0.25)，SHZ88/mMIP1α+mB71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)，SHZ88/pBabe puro组的趋化指数为(0.99±0.19)，mMIP1α+mB71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MIP1α和B71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。

To establish a rat breast cancer cell line stably coexpressing mouse MIP1α and B71，and to assay its in vitro biological activity．Methods: mMIP1αcDNA was cloned into retrovirus vector pBabe puro to construct pBahe puro/mMIP1α, then pBabe puro/mMIP1α was used to transfect packaging cells and the antipuromycin PA317 packaging cells were proliferated. Meanwhile, pLXSN/mB71 was constructed and the antiG418 cells were proliferated. Finally, the two supernatants were used to infect SHZ88 together and the cotransfected cells were selected with 1 mg/ml G418 and 2 μg/ml puromycin together. Expression of mM1Plα mRNA and protein in SHZ88 and SHZ88/mB71+mM1P1α cells were analyzed by RTPCR and immunocytochemistry, respectively. Expression of mB71mRNA and protein was analyzed by RTPCR and flow cytometry, respectively. Lymphocyte proliferation activity of SHZ88/B71+mM1P1α was detected by MTT assay; chemotactic activity of MIP1α was measured by chemotaxis assay. Results：A titer of 4.6×107 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 B71 and MIP1αmRNA/protein in SHZ88/mMIP1α+mB71 cells. The proliferation indices(PI) in mMIP1α+mB71 group (1.95±0.31) was significantly higher than that in SHZ88/PLXSN group (0.76±0.25)(P<0.01). Chemotaxis assay showed that chemotactic activity of lymphocytes in mMIP1α+mB71 group (3.88±0.33) was significantly higher than that in SHZ88/pBabe puro group (0.99±0.19)(P<0.001).Conclusion: A new rat breast cancer cel1 line SHZ88/mMIP1α+mB71 has been established, which can stably coexpress MIP1α and B71 gene and possesses biologic activity in vitro.

国家自然科学基金资助项目(No.30471994);南京军区医学科研“十五”计划项目（No.02MA008)