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目的:探讨B7-1基因转染骨肉瘤细胞能否诱导抗骨肉瘤主动免疫作用。方法:利用脂质体将B7-1真核表达载体pcDNA3-B7-1和空载体pcDNA3分别导入骨肉瘤细胞株LM8中,G418筛选出阳性克隆(分别命名为LM8/B7-1和LM8/pcDNA3),通过RT-PCR、Western blot和流式细胞仪检测B7-1基因与蛋白的表达;通过软琼脂克隆形成实验观察转基因细胞株LM8/B7-1的体外增殖能力;用LM8、LM8/B7-1和LM8/pcDNA3分别经腹腔免疫小鼠,得到腹腔浸润淋巴细胞和致敏脾细胞,MTT法检测其体外杀伤活力;将LM8、LM8/B7-1和LM8/pcDNA3细胞分别接种到C3H雄性小鼠前腋下,观察其致瘤能力;观察LM8/B7-1致敏的小鼠对骨肉瘤细胞LM8是否具有免疫保护作用。结果:B7-1基因在转基因细胞LM8/B7-1中能得到高表达;LM8/B7-1体外增殖能力与LM8和LM8/pcDNA3无明显差异(P>0.05);LM8/B7-1诱导的CTL的杀伤活力显著高于LM8和LM8/pcDNA3诱导的CTL对相同靶细胞的杀伤活力,LM8/B7-1诱导的CTL对LM8/B7-1的杀伤活力也明显高于对LM8和LM8/pcDNA3的杀伤活力(P<0.05);LM8/B7-1致瘤能力较LM8和LM8/pcDNA3细胞明显下降(P<0.01);LM8/B7-1致敏的小鼠对骨肉瘤细胞LM8具有免疫保护作用。结论:B7-1基因转染骨肉瘤细胞能诱导抗骨肉瘤主动免疫作用,为利用B7-1基因进行骨肉瘤免疫基因治疗提供了实验依据。
Objective: To investigate whether B7-1 gene transfection of osteosarcoma cells can induce the active immunostimulation of osteosarcoma. METHODS: B7-1 eukaryotic expression vector pcDNA3-B7-1 and empty vector pcDNA3 were respectively introduced into osteosarcoma cell line LM8 by lipofectamine. Positive clones were screened by G418 (named as LM8 / B7-1 and LM8 / pcDNA3 ). The expression of B7-1 gene and protein was detected by RT-PCR, Western blot and flow cytometry. The proliferation of LM8 / B7-1 cells was observed by soft agar colony formation assay. 1 and LM8 / pcDNA3 were intraperitoneally immunized mice to obtain peritoneal infiltrating lymphocytes and sensitized spleen cells, MTT assay of its in vitro cytotoxicity; LM8, LM8 / B7-1 and LM8 / pcDNA3 cells were inoculated into C3H male Mice before underarm, to observe its tumorigenicity; to observe LM8 / B7-1 sensitized mice on osteosarcoma cells LM8 have immune protective effect. Results: The B7-1 gene was highly expressed in the transgenic cells LM8 / B7-1. The proliferation of LM8 / B7-1 was not significantly different from that of LM8 / LM8 / pcDNA3 (P> 0.05) The cytotoxic activity of CTL was significantly higher than that of LM8 and LM8 / pcDNA3-induced CTL. The killing activity of LM8 / B7-1 induced by CTL on LM8 / B7-1 was also significantly higher than that of LM8 and LM8 / pcDNA3 (P <0.05). The tumorigenicity of LM8 / B7-1 was significantly lower than that of LM8 and LM8 / pcDNA3 cells (P <0.01). The LM8 / B7-1 sensitized mice had protective immunity against osteosarcoma cell line LM8 effect. CONCLUSION: Transfection of B7-1 gene into osteosarcoma cells can induce the active immune response to osteosarcoma, providing an experimental basis for the gene therapy of osteosarcoma by B7-1 gene.