阳离子脂质体介导粒细胞/巨噬细胞集落刺激因子基因转染的黑色素瘤细胞作瘤苗的研究

Study of Melanoma Cells as Tumor Vaccine Transfected with Granulocyte-Macrophage Colony-Timulating Factor Gene Using Cationic Lipids as Gene Vector

目的本研究拟探讨采用阳离子脂质体介导的鼠粒细胞/巨噬细胞集落刺激因子基因转染的B16F1细胞作为瘤苗的抗肿瘤效果。方法阳离子脂质体复合mGMCSF DNA转染B16F1细胞,ELISA法检测mGMCSF在B16F1中的表达。[3HTdR]掺入法分析B16F1分泌的mGMCSF生物活性。γ射线照射灭活基因修饰的B16F1细胞皮下免疫小鼠,ELISA分析小鼠血清中的mGMCSF水平。7 d后免疫鼠皮下攻击未修饰的B16F1细胞以检测瘤苗在小鼠体内的抗肿瘤效果5。1Cr释放实验检测免疫鼠抗B16F1的特异CTL反应。结果阳离子脂质体介导mGMCSF基因转染B16F1细胞后,未经阳性克隆筛选,mGMCSF得到特异且具有生物学活性的表达。但表达逐步下降由第1天的(135.5±21.1)μg/L下降到第5d的(47.8±5.3)μg/L。随γ射线照射剂量的增加,mGMCSF的表达逐步降低,但仍具有生物学活性。γ射线照射的5×105基因修饰B16F1接种小鼠后,鼠血清中的mGMCSF水平在4 h时为(198.4±23.1)ng/L,攻击的B16F1细胞在接种瘤苗的小鼠体内的生长完全被抑制,51Cr释放实验结果显示该瘤苗在小鼠体内诱导了特异的CTL反应。结论本研究表明阳离子脂质体作为基因载体介导鼠粒细胞/巨噬细胞集落刺激因子基因转染的黑色素瘤B16F1细胞,作为瘤苗在小鼠体内可有效地激发抗肿瘤免疫。

Objective To evaluate the ability of modified cells as tumor vaccine to inhibit the tumor growth and induce the anti-tumor CTL in mice. Methods Cationic lipid mixed with mGMCSF DNA were transfected into B16F1 cells. ELISA was used to detect mGMCSF level secreted by the transfected B16F1 cells and in the serum of mice inoculated with mGMCSF-secreting B16F1. [3^HTdR] incorporation assay was used to analyze the bioactivity of the secreted mGMCSF. 51^Cr release assay was used to analyze the specific CTL against B16F1. Gene modified B16F1 cells inactivated by γ irradiation immunized s. c. mice. The growth of challenged viable B16F1 cells were measured to reflect in vivo anti-tumor effects of tumor vaccine. Results mGMCSF with bioactivity was produced specifically by mGMCSF gene modified B16F1 cells without positive selection of clone for at least 5 days, using cationic lipids as gene deliver vector. The level decreased gradually from （135.5±21.1）μg/L on day 1 to （47. 8±5.3）μg/L on day 5. The level of mGMCSF with bioactivity from modified B16F1 cells was decreased by γ irradiation. Serum mGMCSF level was （198. 4±23. 1）ng/L at 4 h in the mice treated s. c. with 5 × 10^5 modified B16F1. The growth of challenged B16F1 cells was inhibited completely in the mice vaccinated with tumor vaccine that could induce the specific CTL response. Conclusion Murine granulocyte/macrophage colony-stimulating factor gene modified B16F1 cells could trigger the anti-tumor specific immune response efficiently in vivo when cationic lipid was used as gene transfer vector to modify B16F1 cells rapidly.