摘要
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 explore whether transfection of osteosarcoma cells with/37-1 gene can induce anti-osteosarcoma active immune response. Methods： Eukaryotic expression vector pcDNA3-/37-1 and empty vector pcDNA3 were separately transfected into osteosarcoma LM8 cells by lipofectamine mediation. The positive clones were then selected by G418 and called LM8/ B7-1 and LM8/pcDNA3, respectively. RT-PCR, western blot, and flow cytometry analysis （FCMA） were used to detect B7-1 gene expression and soft sugar test was used to determine the proliferation of transgenic cells in vitro. Mice were intraperitoneally immunized with LM8, LM8/B7-1, and LM8/pcDNA3 cells to obtain the abdominal infiltrating lymphocytes and sensitized spleen cells. The in vitro cytotoxicity of these cytotoxic T lymphocytes （CTLs） against tumor cells was detected by MTT assay. LM8, LM8/B7-1, and LM8/pcDNA3 cells were inoculated into the fore armpits of C3H male mice subcutaneously to observe the tumorigenicity of these cells. Whether LM8/B7-1 cells-sensitized mice had the protective immune response with LM8 cells was observed. Results：B7-1 gene was highly expressed in LM8/B7-1 cells. The proliferation capability of LM8/B7-1 cells had no significant difference with LM8 and LM8/pcDNA3 cells in vitro （P〉0. 05）. The cytotoxicity of CTLs induced by LM8/B7- 1 cells was significantly higher than that induced by LM8 and LMS/pcDNA3 cells against the same target cells （P〈0. 05）. The cytotoxicity of CTLs induced by LM8/B7-1 cells against LM8/B7-1 cells was also significantly higher than that against LM8 and LM8/pcDNA3 cells （P〈0.05）. The tumorigenicity of LM8/B7-1 cells in vivo significantly decreased compared with LM8 and LM8/pcDNA3 cells （P〈0. 01）. The sensitized mice had obvious protective immune response with LM8 cells. Conclusion：The transfection of osteosarcoma cells with/37-1 gene induced anti-osteosacoma active immune response. This provided experimental basis for/37-1 gene-induced immunotherapy of osteosarcoma.
出处
《肿瘤》
CAS
CSCD
北大核心
2006年第9期827-831,共5页
Tumor
基金
武汉市青年科技晨光计划(编号:20025001028)
作者简介
潘海涛（1979-），男（汉族），博士研究生
Corresponding author. Tei：027-85726439 E-mail ： zheng-qx@21cn. com
