白细胞介素12转染树突状细胞后与新建卵巢上皮性癌细胞融合的免疫原性研究

Primary study on fusions of ovarian carcinoma cells to dendritic cell transfected with interleukin-12 gene in vitro

目的研究白细胞介素（IL）12转染入树突状细胞（DC）后，与新建卵巢上皮性癌细胞融合形成融合细胞，分析融合细胞的体外免疫杀伤效应。方法以新鲜卵巢上皮性癌组织建立卵巢上皮性癌细胞系，并进行鉴定。体外诱导产生人脐血DC。将IL-12与pcDNA3．1（＋）质粒连接后，分别通过脂质体法和电穿孔法将IL-12-pcDNA3．1（＋）质粒转染DC，再使用聚乙二酸法将新建卵巢上皮性癌细胞与转染IL-12的DC融合，用四甲基偶氮唑蓝法检测融合细胞的体外免疫杀伤效应。结果（1）形态学观察和免疫组化法检测结果均证实，新建卵巢上皮性癌细胞系为原代卵巢上皮性癌细胞。（2）人脐血培养7-10d，贴壁细胞出现大量毛刺状突起；免疫荧光染色显示，脐血DC主要组织相容性复合物Ⅱ类分子人白细胞抗原（HLA）-DR的阳性表达率为99％，共刺激分子B7-2的阳性表达率为50％。（3）RT—PCR技术检测结果证实IL—12转染DC成功。将转染IL—12的DC与新建卵巢上皮性癌细胞融合，RT—PCR技术和免疫荧光染色分析结果均证实融合细胞形成。（4）将转染IL—12的DC和新建卵巢上皮性癌的融合细胞、DC和新建卵巢上皮性癌的融合细胞分别与患者的外周血单个核细胞共同培养，激活T淋巴细胞，结果显示，两种活化后的T淋巴细胞均能杀伤新建卵巢上皮性癌细胞，且前者作用更强。结论转染IL—12后的DC与新建卵巢上皮性癌细胞融合后形成的融合细胞能激活T淋巴细胞，且其体外免疫杀伤效应更强。

Objective To investigate the immune response of the fusions of ovarian carcinoma cells to dendritic cell（ DC ） transfected with interleukin （IL）-12 gene in vitro. Methods Recombinant human granuloeyte macrophage colony stimulating factor and recombinant human tumor necrosis factor ot were used to generate DC from cord blood in vitro. IL-12 fusion gene was cloned into a eukaryotic vector-pcDNA3.1 （ ＋ ）. DC were transfected with IL-12-pcDNA3. 1 （ ＋ ） using lipofectamine transfection method and electric transfection method respectively. Then polyethylene glycol mediated the fusion of transfected DC and ovarian carcinoma cells, and 3-（ 4, 5-dimethylthiazol-2-yl ） 2, 5-diphenyl tetrazolium bromide （ MTT ） test was carded out to observe the immune response. Results DC were generated in vitro from cord blood and expressed high levels of costimulatory（50% ） and MHC Ⅱ molecules（99% ）. RT-PCR showed that IL-12- pcDNA3. 1 （ ＋ ） had been successfully transfected into DC. RT-PCR and immunofluorescence analysis showed that DC were fused with ovarian carcinoma cells successfully. Then the fusion cells of ovarian carcinoma cells to transfected DC, and the fusions of ovarian carcinoma cells to DC were cocultured with peripheral blood mononuclear cell respectively. MTT test showed that both fusions could induce cytolytie T cell activity and lysis of ovarian carcinoma cells, and the former effect was stronger. Conclusion The cytolytic T cell activity induced by the fusions of ovarian carcinoma cells to transfected DC is enhanced.